Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp. I. Functional Coupling of Cyclic A
http://www.100md.com
《内分泌学杂志》
Department of Zoology, University of Hong Kong, Hong Kong
Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, has been recently proposed to be the ancestral GH-releasing factor. Using grass carp as a model for bony fish, we examined the mechanisms for PACAP regulation of GH synthesis and secretion at the pituitary level. Nerve fibers with PACAP immunoreactivity were identified in the grass carp pituitary overlapping with the distribution of somatotrophs. At the somatotroph level, PACAP was shown to induce cAMP synthesis and Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC). In carp pituitary cells, PACAP but not vasoactive intestinal polypeptide increased GH release, GH content, total GH production, and steady-state GH mRNA levels. PACAP also enhanced GH mRNA stability, GH promoter activity, and nuclear expression of GH primary transcripts. Increasing cAMP levels, induction of Ca2+ entry, and activation of VSCC were all effective in elevating GH secretion and GH mRNA levels. PACAP-induced GH secretion and GH mRNA expression, however, were abolished by inhibiting adenylate cyclase and protein kinase A, removing extracellular Ca2+ or VSCC blockade, or inactivating calmodulin (CaM)-dependent protein kinase II (CaM kinase II). Similar sensitivity to VSCC and CaM kinase II blockade was also observed by activating cAMP production as a trigger for GH release and GH gene expression. These results suggest that PACAP stimulates GH synthesis and secretion in grass carp pituitary cells through PAC1 receptors. These stimulatory actions probably are mediated by the adenylate cyclase/cAMP/protein kinase A pathway coupled to Ca2+ entry via VSCC and subsequent activation of CaM/CaM kinase II cascades.
Introduction
PITUITARY ADENYLATE cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, is a pleiotropic neuropeptide known to regulate pituitary functions, neurotransmission, neuron survival, vasodilation, and gut motility as well as secretory activities in the pancreas and adrenal gland (1). It was first identified in ovine hypothalamus by its stimulatory effect on cAMP production in rat pituitary cells (2). In mammals, two forms of PACAP, PACAP38 and PACAP27, have been reported, and both of them are produced from the same precursor by posttranslational proteolysis and alternative -amidation (3). The biological actions of PACAP are mediated by three receptor subtypes, namely PAC1, VPAC1, and VPAC2 receptors (4). PAC1 receptors are specific for PACAP with little or no binding affinity for vasoactive intestinal polypeptide (VIP), a neuropeptide structurally related to PACAP38. Multiple variants of PAC1 receptors produced by alternative splicing of the hip or hop1/hop2 cassettes in the third intracellular loop have been reported (5). These isoforms exhibit differential coupling to the adenylate cyclase (AC)/cAMP and phospholipase C (PLC)/inositol trisphosphate (IP3) cascades (6). A novel form of PAC1 receptors, namely PACAPR TM4, with sequence modifications in transmenbrane domain II and IV has been identified in the rat cerebellum (7). This PAC1 receptor does not activate AC or PLC but can induce Ca2+ entry via L-type voltage-sensitive Ca2+ channels (VSCC). VPAC receptors, unlike PAC1 receptors, can bind PACAP and VIP with equal affinity. Besides, VPAC1 and VPAC2 receptors can be further differentiated by their binding affinity for the lizard venom helodermin (8). VPAC receptors in general do not activate PLC or IP3 production and are functionally coupled to the cAMP-dependent pathway (9).
The molecular structure of PACAP is highly conserved. The amino acid sequence of PACAP38 is identical among mammalian species. When comparing the amino acid sequence of PACAP38 from vertebrates ranging from fish to mammals, only minor substitutions can be noted (10). In fish, e.g. sockeye salmon, molecular cloning of PACAP has revealed that GHRH and PACAP38 are encoded in the same gene (11). This is at variance with the situation in mammals in which GHRH and PACAP are encoded in separate genes. Although GHRH and PACAP are evolved from the same ancestral gene (12), the GH-releasing action of GHRH is either weak or not observed in fish models, and PACAP has been proposed to be the ancestral GHRH in vertebrates (13). This idea is supported by the findings that PACAP can act as a potent GH secretagogue in representative species of bony fish (14) and amphibians (15). In mammals, the GH-releasing effect of PACAP is still controversial. In in vivo studies, PACAP elevates serum GH levels in the rat (16) and cattle (17) but not in humans (18) or sheep (19). In in vitro cultures of rat pituitary cells, PACAP induces GH release in some studies (20, 21) but not others (16, 22). Within the physiological dose range, PACAP-induced GH release can be noted in porcine (23) but not ovine pituitary cells (24). In the case of a stimulatory effect, PACAP’s action is mediated by AC/cAMP and/or PLC/IP3 cascades coupled to Ca2+ entry through VSCC (25, 26). To our knowledge, the downstream mechanisms that occur after Ca2+ entry have not been elucidated. Regarding the role of PACAP in GH gene expression, both stimulatory (27) and inhibitory actions (28) have been reported, and the molecular mechanisms involved are still unclear.
In this study, using grass carp as a model for modern-day bony fish, the direct actions of PACAP on GH synthesis and secretion at the pituitary level were examined. The functional relevance of PACAP as a hypophysiotropic factor in fish was evaluated by immunostaining of PACAP nerve fibers in grass carp pituitary sections. Column perifusion was used to study the effects of PACAP on the kinetics of GH secretion in grass carp pituitary cells. Using a static incubation approach, the effects of PACAP on GH production and GH gene expression were examined. To elucidate the postreceptor signaling mechanisms for PACAP regulation of GH release and GH synthesis, a pharmacological approach was used to test the hypothesis that the actions of PACAP were mediated by the AC/cAMP/protein kinase A (PKA) pathway coupled to Ca2+ entry via L-type VSCC and subsequent activation of calmodulin (CaM)/CaM kinase II-dependent mechanisms.
Materials and Methods
Animals
One-year-old grass carps (Ctenopharyngodon idellus) with body weight from 1.5–2.0 kg and gonadosomatic index less than 0.2% were purchased from local markets and kept in well-aerated 200-liter aquaria at 18 C for at least 3 d before experimentation. During the process of pituitary cell preparation, the fish were killed by anesthesia in 0.05% MS222 (Sigma Chemical Co., St. Louis, MO) followed by spinosectomy according to the regulations of animal use at the University of Hong Kong (Hong Kong).
Reagents and test substances
Ovine PACAP38 and PACAP27 were obtained from Phoenix Pharmaceuticals (Belmont, CA), and VIP was purchased from Bachem Fine Chemicals (La Jolla, CA). Forskolin, 3-isobutyl-1-methylxanthine (IBMX), 8-bromo-cAMP (8Br-cAMP), nifedipine, A23187, Bay K8644, H89, MDL 12330A, calmidazolium, KN62, KN93, and actinomycin D were acquired from Calbiochem (San Diego, CA). The Ca2+-sensitive dye Indo-1/AM was obtained from Molecular Probes (Eugene, OR), and the working stock of Indo-1/AM was prepared freshly in dimethylsulfoxide 15 min before dye loading. Other chemicals and materials used in this study were obtained from commercial sources with the highest quality available.
Measurement of GH release and GH production
Grass carp pituitary cells were prepared by a trypsin/DNase digestion method as described previously (29). For column perifusion, pituitary cells (2.5 x 106 cells per column) were cultured on Cytodex II beads (Sigma) and perifused with carp MEM (30) at a flow rate of 15 ml/hr in an ACUSYST-S perifusion system (Endotronics Inc., Minneapolis, MN). Test substances were introduced from a drug reservoir into individual columns through a three-way stopcock. Perifusate was collected in 5-min fractions, and GH content in these samples was measured using a RIA for grass carp GH (29). For static incubation experiments, pituitary cells (2.5 x 106 cells/ml per well) were cultured in poly-D-lysine-coated (0.1 μg/ml) 24-well plates (Corning Inc., Corning, NY). The duration of drug treatment was routinely fixed at 48 h unless stated otherwise. After incubation with test substances, culture medium was harvested for the measurement of GH secretion. Pituitary cells were lysed in double-distilled deionized water by three cycles of freezing and thawing, and the lysate obtained was used for the measurement of cellular GH content. In these experiments, GH production was defined as the sum of GH release and cellular GH content.
Measurement of steady-state GH mRNA levels
After drug treatment, pituitary cells were dissolved in Trizol (GIBCO, Gaithersburg, MD), and total RNA was prepared according to the instructions by the manufacturer. These RNA samples were dissolved, heat denatured at 70 C, and vacuum blotted onto a positively charged nylon membrane using a Bio-Dot slot-blot apparatus (Bio-Rad, Hercules, CA). After hybridization with a digoxigenin (DIG)-labeled GH cDNA probe, GH mRNA signals were quantified using a DIG luminescent detection kit (Roche, Stockholm, Sweden) (31). In this study, Northern blot was also conducted to test whether PACAP treatment could alter the form(s) and/or size of GH transcripts. In this case, total RNA was resolved in 1% gel with 0.22 M formaldehyde and transblotted onto a nylon membrane for GH mRNA measurement according to the standard procedures in our laboratory (32). In these experiments, parallel probing of -actin mRNA or 18S RNA was used as an internal control.
Ribonuclease protection assay for GH primary transcripts
Pituitary cells (5 x 106 cells/2 ml per well) were cultured in poly-D-lysine-precoated 12-well plates (Corning) for simultaneous measurement of cytosolic mature GH mRNA and nuclear GH primary transcripts. After drug treatment, cytosolic and nuclear RNA were isolated by sucrose gradient centrifugation followed by proteinase K and DNase I digestion. These RNA samples were then subjected to ribonuclease protection assays (RPA) previously validated for grass carp mature GH mRNA and GH primary transcripts (31). A DNA fragment covering the junction between exon II and intron II of grass carp GH gene (GenBank no. X60419) was used to produce the 32P-labeled antisense riboprobe by in vitro transcription. Sense-strand cRNAs were also prepared to serve as RPA standards. In these assays, mature GH mRNA (in the cytosol) and GH primary transcripts (in the nucleus) consistently produced protected fragments of 124 and 207 bp, respectively.
Measurement of GH promoter activity
Decreasing lengths of the grass carp GH promoter (–446 to –155) were PCR isolated and subcloned into pBL.CAT to generate pGH.CAT constructs for transfection studies in GH4C1 cells. GH4C1 cells, a rat pituitary cell line, were maintained in Ham’s F-10 medium (GIBCO) with 10% fetal bovine serum at a seeding density of 0.25 x 106 cells/3 ml per dish. After overnight incubation, transfection was conducted in 300 μl OPTI-MEM (GIBCO) for 6 h with 0.25 μg/dish pGH.CAT constructs, 0.5 μg/dish pEGFP-N1 (Clontech, Palo Alto, CA), and 7 μl lipofectamine (GIBCO). The green fluorescent protein (GFP) expression vector pEGFP-N1 was included to serve as an internal control for potential variations in transfection efficiency between dishes. After transfection, GH4C1 cells were cultured in Ham’s F-10 medium with 10% fetal bovine serum for 7 h before the addition of test substances. The duration of drug treatment was routinely fixed at 15 h. After that, cell lysate was prepared for chloramphenicol acetyltransferase (CAT) activity measurement using a two-phase fluor diffusion assay (33). GFP expression levels in these samples were quantified by fluorescence measurement using a Cytofluor 4000 plate reader (Perspective Biosystems, Framingham, MA).
Immunohistochemical staining of grass carp pituitary sections
Carp pituitaries were fixed in Bouin’s fixative, embedded in paraffin wax, and sectioned into 5-μm-thick sections according to standard procedures. Immunohistochemical staining was conducted using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Antisera for GH (1:50,000) and prolactin (PRL, 1:100,000) are gifts from Dr. R. E. Peter (University of Alberta, Alberta, Canada) and have been previously validated for immunostaining in the grass carp (29). The antiserum for human neurofilament (1:500; UpState, Milford, MA) was used to probe the neuronal structures/nerve fibers in the carp pituitary. PACAP antiserum (1:2000; Peninsula Laboratories, Belmont, CA) for immunostaining of PACAP nerve fibers was raised against PACAP38 and had no cross-reactivity with PACAP27. For immunostaining of somatotrophs, cytospin preparations of pituitary cells were prepared using an Autosmear CF-10 centrifuge (Sakura Technical, Nagano, Japan). GH immunoreactivity was visualized according to the immunostaining procedures described previously (34).
Measurement of intracellular Ca2+ level
Enriched somatotrophs were prepared from grass carp pituitary cells by Percoll gradient centrifugation (34) and preloaded with Indo-1/AM (10 μM) in HEPES-buffered saline medium (31) with 0.1% BSA at 28 C for 30 min. After dye loading, the cells were resuspended at 1.5 x 106 cells/ml in BSA-free HEPES-buffered saline medium, and [Ca]i measurement was performed in a thermostatted (28 C) quartz cuvette using a F-4500 fluorescence spectrophotometer (Hitachi, Indianapolis, IN). Wavelengths for excitation and emission were fixed at 329 nm (5-nm slit width) and 405 nm (10-nm slit width), respectively. In this study, [Ca2+]i concentrations were calibrated using the cell lysis method as described previously (35).
Measurement of cAMP production
Enriched somatotrophs or mixed populations of carp pituitary cells were resuspended in carp MEM and seeded at a density of 2 x 106 cells/2 ml in poly-D-lysine-coated 35-mm petri dishes (Corning). After overnight incubation, carp MEM was replaced with 0.9 ml HHBSA medium (36) and incubated at 28 C for 15 min before adding 0.1 ml x10 stock solutions of test substances. The duration of drug treatment was routinely fixed at 15 min. After that, culture medium was harvested for the measurement of cAMP release and cellular cAMP was extracted by adding 1 ml ice-cold absolute ethanol. These cAMP samples were then freeze-dried and stored at –20 C until their cAMP content was quantified by a BioTrak [125I]cAMP RIA (Amersham Pharmacia Biotech, Piscataway, NJ). In these experiments, cAMP production was defined as the sum of cAMP release and cellular cAMP content.
Data transformation and statistics
For perifusion studies, GH data (in ng/ml) from individual columns were expressed as a percentage of the average GH content of the first six fractions collected at the beginning of the experiment before drug treatment (as percent basal). This transformation was used for pooling of data from separate columns without distorting the profile of GH release. GH response was quantified by calculating the net change in GH release after drug treatment (i.e. a net change in area under the curve). GH transcripts were measured in terms of arbitrary density unit and normalized against the amount of -actin mRNA or 18S RNA in the same sample. The half-life (t1/2) of GH mRNA was deduced based on the one-phase exponential decay model using GraphPad Prism 3.02 (GraphPad, San Diego, CA). CAT activity was normalized against GFP expression in the same sample. Given that the levels of internal controls for GH mRNA and GH promoter activity did not exhibit significant changes in these studies, the normalized data were simply transformed as a percentage of the mean value in the control group without drug treatment (as percent control). In the case of cAMP assays, cAMP levels were measured in terms of pmol cAMP/ml or pmol cAMP/106 cells, and the data were also transformed as a percentage of the control for statistical analysis. For [Ca2+]i measurement, fluorescence data (in arbitrary fluorescence units) were transformed into Ca2+ levels using the single-wavelength calibration equation: [Ca2+]i = Kdx (F – Fmin)/(Fmax – F), where Kd is the dissociation constant for Indo-1, Fmin is basal fluorescence without Ca2+, Fmax is the maximal fluorescence under saturating Ca2+, and F is the fluorescence level at any unknown Ca2+ concentration. Fmin and Fmax were determined empirically for individual dye loading according to the method described by Thomas and Delaville (35). Data presented were analyzed using Student’s t test or ANOVA followed by Fisher’s least significance difference (LSD) test. Differences were considered significant at P < 0.05.
Results
Immunohistochemical staining of PACAP in grass carp pituitary sections
The grass carp pituitary could be divided into the anterior pituitary, including the rostral pars distalis (RPD) and proximal pars distalis (PPD), and the posterior pituitary, generally referred to as the neurointermediate lobe (Fig. 1A). PRL immunoreactivity was located exclusively in the RPD (Fig. 1B), whereas GH immunoreactivity was detected only in the PPD (Fig. 1C). Using an antiserum raised against PACAP38, PACAP immunoreactivity could be noted in the pars distalis of the carp pituitary, including both RPD and PPD (Fig. 1D). These immunostaining signals were not observed with the use of a similar dilution of normal rabbit serum (data not shown) and could be blocked by preabsorbing PACAP antiserum (1:2,000) with PACAP38 (0.1 mM) for 24 h at 4 C (Fig. 1E). Apparently, PACAP immunoreactivity was widely expressed in the anterior pituitary. When examined under a higher magnification with phase-contrast microscopy (Fig. 1, F and G), PACAP immunoreactivity was located only in the nerve fibers/nerve tracts but not in pituitary cells. These nerve fibers/nerve tracts were also stained positively with an antiserum for the neuronal marker neurofilament (Fig. 1, F and G, insets). In parallel studies, no immunostaining signals could be detected in the grass carp pituitary sections using an antiserum raised against PACAP27 (data not shown).
Effects of PACAP and VIP on GH release in grass carp pituitary cells
Using a column perifusion approach, the direct actions of PACAP on GH secretion were tested at the pituitary cell level. Increasing concentrations (0.1–1000 nM) of PACAP38 (Fig. 2A) and PACAP27 (Fig. 2B) were effective in inducing GH release from grass carp pituitary cells in a dose-dependent manner. The kinetics of these GH responses was rapid, and the peak of GH secretion could be noted within 10 min after the initiation of drug treatment. After the 10-min pulses of PACAP stimulation, GH release gradually returned to basal levels. Similar doses of human VIP, however, did not alter basal levels of GH secretion (Fig. 2C). A similar lack of GH responses was also noted when substituting cod VIP for human VIP in these experiments (data not shown).
Effects of PACAP on [Ca2+]i and cAMP production in grass carp somatotrophs
Using density gradient centrifugation, enriched somatotrophs were prepared from mixed populations of grass carp pituitary cells (Fig. 3A). After loading with the Ca2+-sensitive dye Indo-1/AM, [Ca2+]i levels in this cell preparation was increased by treatment with PACAP38 (1 μM). This stimulatory effect, however, was blocked by removing [Ca2+]e using the Ca2+ chelator EGTA (4 mM) (Fig. 3B) or by inactivating L-type VSCC using the dihydropyridine inhibitor nifedipine (10 μM). In these studies, the vehicle for PACAP38 did not modify basal [Ca2+]i levels. Using a 15-min short-term incubation protocol, increasing levels of PACAP38 (0.01–1000 nM) also triggered a dose-dependent increase in cAMP release, cellular cAMP content, and total cAMP production in carp somatotrophs (Fig. 3C).
cAMP- and Ca2+-dependent mechanisms in PACAP-induced GH release
To test whether activation of cAMP production and/or Ca2+ entry can induce GH secretion at the pituitary cell level, a pharmacological approach was used. In this case, increasing levels of the AC activator forskolin (0.1–10 μM) (Fig. 4A) and phosphodiesterase inhibitor IBMX (0.1–100 μM) (Fig. 4B) consistently elevated cAMP release, cAMP content, and cAMP production in pituitary cells cultured under static incubation conditions. In parallel experiments using column perifusion, forskolin (0.1–10 μM) (Fig. 4C) and IBMX (0.01–100 μM) (Fig. 4D) were both effective in triggering a dose-dependent increase in GH release. Similarly, elevating the functional levels of cAMP using a membrane-permeable cAMP analog 8Br-cAMP (30 μM to 3 mM) also induced GH secretion (Fig. 5A), and this GH-releasing effect could be mimicked by the Ca2+ ionophore A23187 (0.3–30 μM) (Fig. 5B) and L-type VSCC activator Bay K8644 (1 nM to 10 μM) (Fig. 5C). To examine the possible involvement of cAMP- and Ca2+-dependent mechanisms in PACAP-induced GH secretion, the GH-releasing action of PACAP38 was tested with the simultaneous treatment of the inhibitors for the AC/cAMP/PKA and Ca2+/ CaM/CaM kinase II pathways. In these studies, the stimulatory effects of forskolin (3 μM) and 8Br-cAMP (3 mM) on GH release were suppressed in a dose-dependent manner by the AC inhibitor MDL 12330A (0.03–30 μM) (Fig. 6A) and PKA inhibitor H89 (1–30 μM) (Fig. 6B), respectively. These results indicate that these two inhibitors were both effective in blocking the AC/cAMP/PKA pathway in fish pituitary cells. In parallel experiments, MDL 12330A (30 μM) and H89 (30 μM) were also effective in inhibiting both basal and PACAP38-stimulated GH release (Fig. 6, C and D). Furthermore, the GH-releasing action of PACAP38 could be blocked by perifusion with a Ca2+-free medium (Fig. 7A) or by the L-type VSCC inhibitor nifedipine (10 μM) (Fig. 7B). Comparable results were also noted by replacing PACAP38 with forskolin (3 μM) as the stimulant for GH secretion (Fig. 7, C and D). Consistent with these findings, the GH-releasing effect of PACAP38 was similarly suppressed by cotreatment with the CaM antagonist calmidazolium (1 μM) (Fig. 8A) or by the CaM kinase II inhibitors KN62 (5 μM) (Fig. 8B) and KN93 (5 μM) (Fig. 8C). Again, these CaM/CaM kinase inhibitors were effective in attenuating the GH responses triggered by forskolin treatment (3 μM) (Table 1).
Effects of PACAP on GH production and GH gene expression
Using a static incubation approach, the direct actions of PACAP38 on GH synthesis were tested at the pituitary cell level. In this case, a 48-h incubation with PACAP38 (1 μM) and forskolin (3 μM) significantly increased GH release (Fig. 9A), cellular GH content (Fig. 9B), and total GH production in grass carp pituitary cells (Fig. 9C). Parallel treatments with human and cod VIP (1 μM) were not effective in these regards (data not shown). As revealed by Northern blot, the GH responses observed at the protein level induced by PACAP (1 μM) and forskolin treatment (3 μM) also occurred with a concurrent rise in steady-state GH mRNA levels. Apparently, these GH mRNA responses did not involve modifications in the form or size of GH transcripts. To increase the throughput of our studies, a slot-blot assay was used to monitor GH mRNA levels in grass carp pituitary cells. In this case, the stimulatory effects of PACAP38 (1 μM) and forskolin (10 μM) on GH mRNA expression were found to be time dependent (Fig. 10, A and B). In parallel experiments, GH mRNA levels were also accentuated in a dose-dependent manner by increasing concentrations of PACAP38 (1 nM to 10 μM) (Fig. 10C) and forskolin (0.1–30 μM) (Fig. 10D). Again, human VIP (1 μM) was not effective in altering basal levels of GH mRNA expression (Fig. 10C, inset).
Given that steady-state mRNA represents a dynamic balance between the synthesis and degradation of target transcripts, the role of GH mRNA stability and GH gene transcription in PACAP-induced GH gene expression was also examined. In pituitary cells pretreated with the transcription inhibitor actinomycin D (8 μM), the clearance curves for GH mRNA were shifted to the right with an increase in t1/2 from 10.2 to 21.8 h and from 9.8 to 17.6 h by treatment with PACAP38 (1 μM) (Fig. 11A) and forskolin (10 μM) (Fig. 11B), respectively. Besides, the levels of cytosolic mature GH mRNA and nuclear GH primary transcripts were found to be up-regulated in carp pituitary cells by a 48-h incubation with PACAP38 (0.3 and 3 μM) and forskolin (10 μM) (Fig. 11C). In GH4C1 cells transfected with CAT constructs with decreasing lengths of grass carp GH promoter (–446 to –155), similar drug treatments for 15 h were also effective in stimulating CAT activity expression (Fig. 11D). PACAP38 (1 μM) and forskolin (10 μM), however, did not alter the promoter activities in the control groups transfected with pBL.CAT and TK.CAT, respectively (data not shown). In this study, basal levels of CAT activity were also elevated by 5' truncation of the GH promoter from –221 to –115, suggesting that inhibitory cis-acting element(s) may be present in this region.
cAMP- and Ca2+-dependent mechanisms in PACAP-induced GH mRNA expression
To study the functional role of cAMP- and Ca2+-dependent cascades in PACAP-induced GH gene expression, the stimulatory action of PACAP38 on GH mRNA levels was tested in the presence of the inhibitors for AC/cAMP/PKA and Ca2+/CaM/CaM kinase II cascades. In pituitary cells cultured under static incubation conditions, basal levels of GH transcripts were consistently elevated by 48-h treatment with PACAP38 (1 μM) (Fig. 12A) and forskolin (10 μM) (Fig. 12B). These stimulatory effects, however, were blocked by simultaneous treatment with the AC inhibitor MDL 12330A (30 μM) and PKA inhibitor H89 (30 μM), and these two inhibitors were also effective in reducing basal levels of GH mRNA expression. Similar to PACAP38, the Ca2+ ionophore A23187 (0.1–100 nM) (Fig. 13A) and L-type VSCC activator Bay K8644 (1–1000 nM) (Fig. 13B) could elevate GH mRNA levels in a dose-dependent manner. However, both basal and PACAP38-stimulated (1 μM) GH mRNA expression were inhibited by incubation with a Ca2+-free medium or by the VSCC blocker nifedipine (10 μM) (Fig. 13C). Similar results were also noted by replacing PACAP38 with forskolin (10 μM) as the trigger for GH mRNA expression (Fig. 13C, inset). In parallel experiments, basal levels of CaM mRNA were up-regulated in carp pituitary cells in a dose-related fashion by increasing concentrations of PACAP38 (1–1000 μM), and this stimulatory effect occurred concurrently with a rise in GH transcripts (Fig. 14A). Furthermore, the CaM kinase II inhibitors KN62 (5–5000 nM) (Fig. 14B) and KN93 (5 μM) (Fig. 14C) were both effective in reducing basal and PACAP38-induced (1 μM) GH mRNA expression. Again, similar results were obtained using forskolin (10 μM) as the stimulant in these experiments (Fig. 14C, inset).
Discussion
In mammals, PACAP serves as a hypophysiotopic factor and plays an active role in the regulation/modulation of pituitary functions. This idea is supported by the findings that 1) PACAP neurons are located in the paraventricular and supraoptic nuclei of the hypothalamus (37), 2) PACAP nerve terminals are present in the external zone of the median eminence (38), 3) PACAP immunoreactivity can be detected in the hypophysial portal blood at a level higher than that of systemic circulation (39), and 4) PACAP, under certain conditions, can induce GH, PRL, LH, and ACTH secretion at the pituitary cell level (40). Although the GH-releasing effect of PACAP is still controversial, PACAP receptors have been identified in rat somatotrophs (41, 42), somatotroph cell lines (43), and GH-secreting pituitary adenomas (44, 45). In the cases of a stimulatory action, the GH-releasing effect of PACAP is either weak (40) or modest (46), susceptible to receptor desensitization (47), and mediated through VPAC2 receptors (48). In this study, nerve fibers/nerve tracts with PACAP38 immunoreactivity were located in the grass carp pituitary overlapping with the distribution of somatotrophs in the pars distalis. Using a perifusion approach, PACAP38 and PACAP27 but not VIP were effective in stimulating GH release from grass carp pituitary cells. These results are in agreement with our recent studies in the goldfish (30) and common carp (49), in which PACAP-induced GH release is mediated through pituitary receptors resembling the mammalian PAC1 subtypes. Given that 1) the pituitary of bony fish is under the direct innervation of the hypothalamus (50) and 2) the perikarya of PACAP neurons can be identified in the hypothalamus of fish species, e.g. European eel (51), it is tempting to speculate that PACAP is produced in the hypothalamus and delivered to the anterior pituitary to serve as a GH-releasing factor in fish models. Because the antiserum for PACAP27 was unable to pick up any immunostaining signals in the carp pituitary, it raises the possibility that PACAP38 but not PACAP27 is the major form of PACAP acting at the pituitary level. In the rat, mainly based on the studies using HPLC and RIA, the protein level of PACAP38 in the hypothalamus was found to be at least 10-fold higher than that of PACAP27 (52), indicating that PACAP38 is the dominant form of PACAP expressed in the hypothalamo-pituitary axis.
Using grass carp pituitary cells as a cell model, we have demonstrated that PACAP38 not only induced GH secretion but also increased GH content, GH production, and GH mRNA expression by acting directly at the pituitary level. Because the stimulatory action of PACAP on GH gene expression could be noted in the nanomolar dose range and was not mimicked by VIP, it would be logical to assume that PACAP by acting on pituitary PAC1 receptors can serve as a potent stimulator for GH synthesis in the carp species. Although PACAP induction of GH mRNA expression has been reported in rat (53) and porcine pituitary cells (27), these stimulatory effects were not dose dependent and no corresponding changes in GH content could be noted in these previous studies. Furthermore, in rat somatotroph cell lines, e.g. GH3 cells, PACAP is known to reduce GH mRNA levels as well as GH content (28). The reasons for these discrepancies are still unknown and may be related to the differences in research methodologies and/or variations in the physiological status of cell models. Using enriched somatotrophs prepared from mixed populations of grass carp pituitary cells, PACAP38 was shown to activate cAMP production and Ca2+ entry via L-type VSCC. In carp pituitary cells, the stimulatory actions of PACAP on GH release and GH mRNA expression were mimicked by increasing cAMP synthesis/functional levels, induction of [Ca2+]e entry, and activation of L-type VSCC. PACAP38-stimulated GH release and GH mRNA expression, however, were blocked by inhibiting AC and PKA, removal of [Ca2+]e, and inactivation of VSCC. Using forskolin instead of PACAP38 as the stimulant for GH secretion and GH gene expression, similar inhibitions could be observed after Ca2+-free treatment or VSCC blockade, suggesting that [Ca2+]e entry through VSCC is operating downstream of the AC/cAMP/PKA pathway. This idea is in agreement with the previous reports that 1) VSCC phosphorylation is required for channel opening in GH-secreting cell lines (56) and 2) the -subunit (57) and 2-subunit (58) of VSCC can be PKA phosphorylated in a cAMP-dependent manner. Because PACAP is also known to activate tetrodotoxin-sensitive Na+ channels in GH3 cells (59), we do not exclude the possibility that the actions of PACAP on VSCC may be indirect via PKA phosphorylation of voltage-sensitive Na+ channels (54, 55).
To elucidate the downstream signaling events after Ca2+ entry, the role of CaM and CaM kinase II in PACAP induction of GH synthesis and secretion were also examined. In carp pituitary cells, PACAP-induced GH gene expression also occurred with a rise in CaM mRNA levels, suggesting that CaM-dependent mechanisms might have been activated. This idea is consistent with the findings that the GH-releasing action of PACAP38 could be abolished by CaM antagonism and CaM kinase II inhibition. Because inactivating CaM kinase II was also effective in blocking the stimulatory effects of PACAP38 and forskolin on GH release and GH mRNA levels, it is conceivable that Ca2+ entry induced by PACAP through cAMP-dependent mechanisms can activate CaM/CaM kinase II cascades to trigger GH exocytosis and GH gene expression. Functional coupling of PACAP with CaM-dependent signaling cascades has been recently reported in mammals. In guinea pig, PACAP reduces somatostatin immunoreactivity in cardiac ganglia neurons, and this inhibitory action can be blocked by CaM inhibitors (60). In mouse cerebellar granule cells, PACAP-induced expression of the circadian clock gene is sensitive to the blockade by CaM antagonists and CaM kinase inhibitors (61). Because the total amount of CaM in living cells is much lower than that of its target proteins (62), the operation of CaM-dependent signaling pathways is determined largely by their effectiveness in the competition for the limiting pool of CaM (63). The present finding of PACAP-induced CaM mRNA expression in carp pituitary cells may suggest that PACAP can not only stimulate GH synthesis and secretion by functional coupling of the Ca2+/CaM/CaM kinase II pathway with the cAMP-dependent cascade but also enhance the functionality of CaM-dependent signaling mechanisms by up-regulation of CaM gene expression.
At present, no information is available regarding the molecular mechanisms for PACAP regulation of GH synthesis. To shed light on this area, we examined the role of GH transcript stability and GH gene transcription in PACAP-induced GH gene expression. In carp pituitary cells pretreated with actinomycin D, the t1/2 of GH mRNA was increased by treatment with PACAP38 and forskolin, indicating that PACAP can improve the transcript stability of GH via activation of the cAMP-dependent pathway. In the rat, glucocorticoid and thyroid hormone can induce GH synthesis by prolonging the t1/2 of GH mRNA, and these stimulatory actions are caused by increasing the length of the poly(A+) tail (64). Similar mechanisms, however, may not be applicable to PACAP-induced GH mRNA expression because the size of GH transcript (1.16 kb) was not modified by PACAP38 or forskolin as revealed by the results of Northern blot. In parallel studies, the levels of cytosolic mature GH mRNA and nuclear GH primary transcripts were both elevated in grass carp pituitary cells by treatment with PACAP38 and forskolin. Given that primary transcripts undergo intron splicing rapidly and translocate into the cytoplasm as mature mRNA, the levels of primary transcripts in general are taken as a faithful index for target gene transcription (65). Therefore, the parallel rises in GH primary transcripts and mature GH mRNA observed in the present study may indicate that GH gene transcription has been activated after PACAP38 stimulation. This idea is also supported by the results of our transfection studies in GH4C1 cells, in which the promoter activity of grass carp GH gene was up-regulated by PACAP38 and forskolin, and the cis-acting element(s) responsive to these drug treatments could be mapped to the promoter region downstream of position –115. These results, taken together, suggest that PACAP by acting through cAMP-dependent mechanisms can increase GH mRNA expression both at the transcriptional level (by activating GH gene transcription in the nucleus) and at the posttranscriptional level (by enhancing GH transcript stability in the cytoplasm). In mammals, the transcription factor cAMP response element binding protein (CREB) by phosphorylation at serine 133 (e.g. by PKA/CaM kinases) serves as a key mediator for Ca2+/cAMP-dependent gene expression (66), and CREB phosphorylation can be observed during PACAP stimulation of melatonin synthesis (67) and mPer1 gene expression (68, 69). Besides, the cis-acting element for CREB, namely CRE, can be located in the 5' promoter of the human GH gene and mediates cAMP inducibility of GH gene transcription (70). Similar findings have also been reported in fish, e.g. Chinook salmon (33), suggesting that the role of CRE in mediating cAMP induction of GH gene expression is conserved during vertebrate evolution. Because putative CRE sites have been reported in the 5' promoter of grass carp GH gene (71), it raises the possibility that PACAP-induced GH gene expression is mediated by CREB transactivation via CRE sites in the GH promoter.
In summary, we have demonstrated that nerve fibers with PACAP immunoreactivity are present in the anterior pituitary of grass carp and PACAP by acting directly at the pituitary cell level can stimulate GH secretion and GH gene expression through receptors resembling mammalian PAC1 receptors. These stimulatory effects probably are mediated through Ca2+ entry and cAMP production in grass carp somatotrophs and involve the sequential activation of the AC/cAMP/PKA and Ca2+/CaM/CaM kinase II signaling pathways. Using grass carp pituitary cells as a model (Fig. 15), we propose that PACAP released from nerve terminals of hypothalamic neurons stimulates PAC1 receptors on somatotrophs. Subsequent activation of the AC/cAMP/PKA pathway triggers Ca2+ entry through L-type VSCC, and the rise in [Ca2+]i then activates CaM/CaM kinase II cascades to induce GH exocytosis. Activation of the Ca2+-dependent signaling events secondarily coupled to the cAMP-dependent pathway also elevates GH production by increasing GH mRNA levels. This increase in GH mRNA levels probably are caused by 1) enhancement of GH mRNA stability in the cytoplasm and 2) activation of GH gene transcription in the nucleus. In carp somatotrophs, the concurrent activation of GH synthesis may be essential to replenish the intracellular GH stores after PACAP-induced GH exocytosis. In mammals, GHRH is known to stimulate GH release through Ca2+ entry via L-type VSCC after activation of the AC/cAMP/PKA pathway (26). The postreceptor signaling mechanisms coupled to GHRH receptors appear to be highly comparable with that of PACAP in the carp model. Because PACAP has been proposed to be the ancestral GHRH (13), it is tempting to speculate that the functional replacement of PACAP by GHRH may occur at the level of membrane receptors (i.e. from PAC1 receptors to GHRH receptors) and the postreceptor signaling events leading to GH exocytosis are largely conserved during the course of vertebrate evolution.
Acknowledgments
This series of papers on the role of PACAP as a GH-releasing factor in fish is dedicated to Drs. R. E. Peter and J. P. Chang (University of Alberta, Canada) for their genuine interest in the training of young scientists. Special thanks are given to Dr. R. E. Peter (University of Alberta, Canada) for the supply of protein standard and antiserum for GH RIA. We are also indebted to Dr. W. K. K. Ho (Chinese University of Hong Kong) for his support in setting up the assay systems for GH mRNA and GH primary transcripts.
Footnotes
The present study was sponsored by grants from the Research Grants Council (Hong Kong) and Committee on Research and Conference Grants (University of Hong Kong) (to A.O.L.W.). Financial support from the Department of Zoology (University of Hong Kong) (to C.Y.L., H.Z., and L.H.) in the form of postgraduate studentship is also acknowledged.
First Published Online August 25, 2005
1 W.L. and C.Y.L. contributed equally to this work.
Abbreviations: AC, Adenylate cyclase; 8Br-cAMP; 8-bromo-cAMP; [Ca2+]e, extracellular Ca2+; [Ca2+]i, intracellular Ca2+; CaM, calmodulin; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element binding protein; DIG, digoxigenin; GFP, green fluorescent protein; IBMX, 3-isobutyl-1-methylxanthine; IP3, inositol trisphosphate; LSD, least significant difference; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PLC, phospholipase C; PRL, prolactin; PPD, proximal pars distalis; RPD, rostral pars distalis; VIP, vasoactive intestinal polypeptide; VSCC, voltage-sensitive Ca2+ channels.
Accepted for publication August 17, 2005.
References
Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H 2000 Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 52:269–324
Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH 1989 Isolation of a novel 38-residue hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164:567–574
Arimura A 1998 Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Jpn J Physiol 48:301–331
Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA 1998 International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 50:265–270
Journot L, Spengler D, Pantaloni C, Dumuis A, Sebben M, Bockaert J 1994 The PACAP receptor: generation by alternative splicing of functional diversity among G protein-coupled receptors in nerve cells. Semin Cell Biol 5:263–272
Laburthe M, Couvineau A 2002 Molecular pharmacology and structure of VPAC receptors for VIP and PACAP. Regul Pept 108:165–173
Chatterjee TK, Sharma RV, Fisher RA 1996 Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels. J Biol Chem 271:32226–32232
Hezareh M, Journot L, Bepoldin L, Schlegel W, Rawlings SR 1996 PACAP/VIP receptor subtypes, signal transducers, and effectors in pituitary cells. Ann NY Acad Sci 805:315–327; discussion 327–328
Rawlings SR, Hezareh M 1996 Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland. Endocr Rev 17:4–29
Sherwood NM, Krueckl SL, McRory JE 2000 The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr Rev 21:619–670
Parker DB, Coe IR, Dixon GH, Sherwood NM 1993 Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagon superfamily. Eur J Biochem 215:439–448
Adams BA, Lescheid DW, Vickers ED, Crim LW, Sherwood NM 2002 Pituitary adenylate cyclase-activating polypeptide and growth hormone-releasing hormone-like peptide in sturgeon, whitefish, grayling, flounder and halibut: cDNA sequence, exon skipping and evolution. Regul Pept 109:27–37
Montero M, Yon L, Kikuyama S, Dufour S, Vaudry H 2000 Molecular evolution of the growth hormone-releasing hormone/pituitary adenylate cyclase-activating polypeptide gene family. Functional implication in the regulation of growth hormone secretion. J Mol Endocrinol 25:157–168
Wong AOL, Li WS, Lee EK, Leung MY, Tse LY, Chow BK, Lin HR, Chang JP 2000 Pituitary adenylate cyclase activating polypeptide as a novel hypophysiotropic factor in fish. Biochem Cell Biol 78:329–343
Yon L, Alexandre D, Montero M, Chartrel N, Jeandel L, Vallarino M, Conlon JM, Kikuyama S, Fournier A, Gracia-Navarro F, Roubos E, Chow B, Arimura A, Anouar Y, Vaudry H 2001 Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians. Microsc Res Tech 54:137–157
Jarry H, Leonhardt S, Schmidt WE, Creutzfeldt W, Wuttke W 1992 Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats. Life Sci 51:823–830
Radcliff RP, Lookingland KJ, Chapin LT, Tucker HA 2001 Pituitary adenylate cyclase-activating polypeptide induces secretion of growth hormone in cattle. Domest Anim Endocrinol 21:187–196
Chiodera P, Volpi R, Capretti L, Caffarri G, Magotti MG, Coiro V 1996 Effects of intravenously infused pituitary adenylate cyclase-activating polypeptide on adenohypophyseal hormone secretion in normal men. Neuroendocrinology 64:242–246
Sawangjaroen K, Curlewis JD 1994 Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on prolactin, luteinizing hormone and growth hormone secretion in the ewe. J Neuroendocrinol 6:549–555
Velkeniers B, Zheng L, Kazemzadeh M, Robberecht P, Vanhaelst L, Hooghe-Peters EL 1994 Effect of pituitary adenylate cyclase-activating polypeptide 38 on growth hormone and prolactin expression. J Endocrinol 143:1–11
Goth MI, Lyons CE, Canny BJ, Thorner MO 1992 Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors. Endocrinology 130:939–944
Culler MD, Paschall CS 1991 Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone. Endocrinology 129:2260–2262
Martinez-Fuentes AJ, Castano JP, Gracia-Navarro F, Malagon MM 1998 Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 activate common and distinct intracellular signaling pathways to stimulate growth hormone secretion from porcine somatotropes. Endocrinology 139:5116–5124
Sawangjaroen K, Anderson ST, Curlewis JD 1997 Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro. J Neuroendocrinol 9:279–286
Murakami Y, Koshimura K, Yamauchi K, Nishiki M, Tanaka J, Kato Y 2001 Roles and mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth hormone and prolactin secretion. Endocr J 48:123–132
Gracia-Navarro F, Castano JP, Malagon MM, Sanchez-Hormigo A, Luque RM, Hickey GJ, Peinado JR, Delgado E, Martinez-Fuentes AJ 2002 Research progress in the stimulatory inputs regulating growth hormone (GH) secretion. Comp Biochem Physiol B Biochem Mol Biol 132:141–150
Martinez-Fuentes AJ, Malagon MM, Castano JP, Garrido-Gracia JC, Gracia-Navarro F 1998 Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 differentially stimulate growth hormone release and mRNA accumulation in porcine somatotropes. Life Sci 62:2379–2390
Yonehara T, Kanasaki H, Yamamoto H, Fukunaga K, Miyazaki K, Miyamoto E 2001 Involvement of mitogen-activated protein kinase in cyclic adenosine 3',5'-monophosphate-induced hormone gene expression in rat pituitary GH(3) cells. Endocrinology 142:2811–2819
Wong AOL, Ng S, Lee EK, Leung RC, Ho WK 1998 Somatostatin inhibits (D-Arg6, Pro9-NEt) salmon gonadotropin-releasing hormone- and dopamine D1-stimulated growth hormone release from perifused pituitary cells of Chinese grass carp, Ctenopharyngodon idellus. Gen Comp Endocrinol 110:29–45
Wong AOL, Leung MY, Shea WL, Tse LY, Chang JP, Chow BK 1998 Hypophysiotropic action of pituitary adenylate cyclase-activating polypeptide (PACAP) in the goldfish: immunohistochemical demonstration of PACAP in the pituitary, PACAP stimulation of growth hormone release from pituitary cells, and molecular cloning of pituitary type I PACAP receptor. Endocrinology 139:3465–3479
Zhou H, Ko WK, Ho WK, Stojilkovic SS, Wong AOL 2004 Novel aspects of growth hormone (GH) autoregulation: GH-induced GH gene expression in grass carp pituitary cells through autocrine/paracrine mechanisms. Endocrinology 145:4615–4628
Huo L, Lee EK, Leung PC, Wong AOL 2004 Goldfish calmodulin: molecular cloning, tissue distribution, and regulation of transcript expression in goldfish pituitary cells. Endocrinology 145:5056–5067
Wong AOL, Le Drean Y, Liu D, Hu ZZ, Du SJ, Hew CL 1996 Induction of Chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1. Endocrinology 137:1775–1784
Van Goor F, Goldberg IG, Wong AOL, Jobin RM, Chang JP 1994 Morphological identification of live gonadotropin, growth-hormone, and prolactin cells in goldfish (Carasssius auratus) pituitary cell cultures. Cell Tissue Res 276:253–261
Thomas AP, Delaville F 1991 The use of fluorescent indicators for measurements of cytosolic free calcium concentration in cell populations and single cells. In: McCormack JG, Cobbold PH, eds. Cellular calcium: a practical approach. London: IRL Press; 1–54
Yunker WK, Lee EK, Wong AOL, Chang JP 2000 Norepinephrine regulation of growth hormone release from goldfish pituitary cells. II. Intracellular sites of action. J Neuroendocrinol 12:323–333
Takahashi K, Totsune K, Murakami O, Satoh F, Sone M, Ohneda M, Sasano H, Mouri T 1994 Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in human hypothalamus: co-localization with arginine vasopressin. Regul Pept 50:267–275
Koves K, Gorcs TJ, Kausz M, Arimura A 1994 Present status of knowledge about the distribution and colocalization of PACAP in the forebrain. Acta Biol Hung 45:297–321
Dow RC, Bennie J, Fink G 1994 Pituitary adenylate cyclase-activating peptide-38 (PACAP)-38 is released into hypophysial portal blood in the normal male and female rat. J Endocrinol 142:R1–R4
Hart GR, Gowing H, Burrin JM 1992 Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats. J Endocrinol 134:33–41
Bresson-Bepoldin L, Jacquot MC, Schlegel W, Rawlings SR 1998 Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells. J Mol Endocrinol 21:109–120
Vertongen P, Velkeniers B, Hooghe-Peters E, Robberecht P 1995 Differential alternative splicing of PACAP receptor in pituitary cell subpopulations. Mol Cell Endocrinol 113:131–135
Rawlings SR, Piuz I, Schlegel W, Bockaert J, Journot L 1995 Differential expression of pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptor subtypes in clonal pituitary somatotrophs and gonadotrophs. Endocrinology 136:2088–2098
Robberecht P, Vertongen P, Velkeniers B, de Neef P, Vergani P, Raftopoulos C, Brotchi J, Hooghe-Peters EL, Christophe J 1993 Receptors for pituitary adenylate cyclase activating peptides in human pituitary adenomas. J Clin Endocrinol Metab 77:1235–1239
Oka H, Jin L, Reubi JC, Qian X, Scheithauer BW, Fujii K, Kameya T, Lloyd RV 1998 Pituitary adenylate-cyclase-activating polypeptide (PACAP) binding sites and PACAP/vasoactive intestinal polypeptide receptor expression in human pituitary adenomas. Am J Pathol 153:1787–1796
Propato-Mussafiri R, Kanse SM, Ghatei MA, Bloom SR 1992 Pituitary adenylate cyclase-activating polypeptide releases 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3. J Endocrinol 132:107–113
Wei L, Chan WW, Butler B, Cheng K 1993 Pituitary adenylate cyclase activating polypeptide-induced desensitization on growth hormone release from rat primary pituitary cells. Biochem Biophys Res Commun 197:1396–1401
Murakami Y, Koshimura K, Yamauchi K, Nishiki M, Tanaka J, Furuya H, Miyake T, Kato Y 1995 Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates growth hormone release from GH3 cells through type II PACAP receptor. Regul Pept 56:35–40
Xiao D, Chu MM, Lee EK, Lin HR, Wong AOL 2002 Regulation of growth hormone release in common carp pituitary cells by pituitary adenylate cyclase-activating polypeptide: signal transduction involves cAMP- and calcium-dependent mechanisms. Neuroendocrinology 76:325–338
Peter RE, Yu KL, Marchant TA, Rosenblum PM 1990 Direct neural regulation of the teleost adenohypophysis. J Exp Zool 4(Suppl):84–98
Montero M, Yon L, Rousseau K, Arimura A, Fournier A, Dufour S, Vaudry H 1998 Distribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla. Endocrinology 139:4300–4310
Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy DH, Kitada C 1991 Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 129:2787–2799
Shuto Y, Somogyvari-Vigh A, Vigh S, Wakabayashi I, Arimura A 1996 Effect of pituitary adenylate cyclase-activating polypeptide on GH gene expression in rat pituitary cells. Ann NY Acad Sci 805:684–691
Alarcon P, Garcia-Sancho J 2000 Differential calcium responses to the pituitary adenylate cyclase-activating polypeptide (PACAP) in the five main cell types of rat anterior pituitary. Pflugers Arch 440:685–691
Martinez-Fuentes AJ, Castano JP, Malagon MM, Vazquez-Martinez R, Gracia-Navarro F 1998 Pituitary adenylate cyclase-activating polypeptides 38 and 27 increase cytosolic free Ca2+ concentration in porcine somatotropes through common and distinct mechanisms. Cell Calcium 23:369–378
Chad J, Kalman D, Armstrong D 1987 The role of cyclic AMP-dependent phosphorylation in the maintenance and modulation of voltage-activated calcium channels. Soc Gen Physiol Ser 42:167–186
Mikala G, Klockner U, Varadi M, Eisfeld J, Schwartz A, Varadi G 1998 cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels. Mol Cell Biochem 185:95–109
Bunemann M, Gerhardstein BL, Gao T, Hosey MM 1999 Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the 2 subunit. J Biol Chem 274:33851–33854
Koshimura K, Murakami Y, Mitsushima M, Hori T, Kato Y 1997 Activation of Na+ channels in GH3 cells and human pituitary adenoma cells by PACAP. Peptides 18:877–883
Braas KM, Rossignol TM, Girard BM, May V, Parsons RL 2004 Pituitary adenylate cyclase activating polypeptide (PACAP) decreases neuronal somatostatin immunoreactivity in cultured guinea-pig parasympathetic cardiac ganglia. Neuroscience 126:335–346
Akiyama M, Minami Y, Nakajima T, Moriya T, Shibata S 2001 Calcium and pituitary adenylate cyclase-activating polypeptide induced expression of circadian clock gene mPer1 in the mouse cerebellar granule cell culture. J Neurochem 78:499–508
Persechini A, Stemmer PM 2002 Calmodulin is a limiting factor in the cell. Trends Cardiovasc Med 12:32–37
Tran QK, Black DJ, Persechini A 2003 Intracellular coupling via limiting calmodulin. J Biol Chem 278:24247–24250
Nielsen DA, Shapiro DJ 1990 Insights into hormonal control of messenger RNA stability. Mol Endocrinol 4:953–957
Beyersmann D 2000 Regulation of mammalian gene expression. Exs 89:11–28
Impey S, Goodman RH 2001 CREB signaling: timing is everything. Sci STKE 82:PE1–PE4
Maronde E, Schomerus C, Stehle JH, Korf HW 1997 Control of CREB phosphorylation and its role for induction of melatonin synthesis in rat pinealocytes. Biol Cell 89:505–511
Minami Y, Furuno K, Akiyama M, Moriya T, Shibata S 2002 Pituitary adenylate cyclase-activating polypeptide produces a phase shift associated with induction of mPer expression in the mouse suprachiasmatic nucleus. Neuroscience 113:37–45
von Gall C, Duffield GE, Hastings MH, Kopp MD, Dehghani F, Korf HW, Stehle JH 1998 CREB in the mouse SCN: a molecular interface coding the phase-adjusting stimuli light, glutamate, PACAP, and melatonin for clockwork access. J Neurosci 18:10389–10397
Shepard AR, Zhang W, Eberhardt NL 1994 Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells. J Biol Chem 269:1804–1814
Ho WK, Wong MW, Chan AP 1991 Cloning and sequencing of the grass carp (Ctenopharyngodon idellus) growth hormone gene. Biochim Biophys Acta 1090:245–248(Anderson O. L. Wong, Wensheng Li1, Ching)
Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, has been recently proposed to be the ancestral GH-releasing factor. Using grass carp as a model for bony fish, we examined the mechanisms for PACAP regulation of GH synthesis and secretion at the pituitary level. Nerve fibers with PACAP immunoreactivity were identified in the grass carp pituitary overlapping with the distribution of somatotrophs. At the somatotroph level, PACAP was shown to induce cAMP synthesis and Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC). In carp pituitary cells, PACAP but not vasoactive intestinal polypeptide increased GH release, GH content, total GH production, and steady-state GH mRNA levels. PACAP also enhanced GH mRNA stability, GH promoter activity, and nuclear expression of GH primary transcripts. Increasing cAMP levels, induction of Ca2+ entry, and activation of VSCC were all effective in elevating GH secretion and GH mRNA levels. PACAP-induced GH secretion and GH mRNA expression, however, were abolished by inhibiting adenylate cyclase and protein kinase A, removing extracellular Ca2+ or VSCC blockade, or inactivating calmodulin (CaM)-dependent protein kinase II (CaM kinase II). Similar sensitivity to VSCC and CaM kinase II blockade was also observed by activating cAMP production as a trigger for GH release and GH gene expression. These results suggest that PACAP stimulates GH synthesis and secretion in grass carp pituitary cells through PAC1 receptors. These stimulatory actions probably are mediated by the adenylate cyclase/cAMP/protein kinase A pathway coupled to Ca2+ entry via VSCC and subsequent activation of CaM/CaM kinase II cascades.
Introduction
PITUITARY ADENYLATE cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, is a pleiotropic neuropeptide known to regulate pituitary functions, neurotransmission, neuron survival, vasodilation, and gut motility as well as secretory activities in the pancreas and adrenal gland (1). It was first identified in ovine hypothalamus by its stimulatory effect on cAMP production in rat pituitary cells (2). In mammals, two forms of PACAP, PACAP38 and PACAP27, have been reported, and both of them are produced from the same precursor by posttranslational proteolysis and alternative -amidation (3). The biological actions of PACAP are mediated by three receptor subtypes, namely PAC1, VPAC1, and VPAC2 receptors (4). PAC1 receptors are specific for PACAP with little or no binding affinity for vasoactive intestinal polypeptide (VIP), a neuropeptide structurally related to PACAP38. Multiple variants of PAC1 receptors produced by alternative splicing of the hip or hop1/hop2 cassettes in the third intracellular loop have been reported (5). These isoforms exhibit differential coupling to the adenylate cyclase (AC)/cAMP and phospholipase C (PLC)/inositol trisphosphate (IP3) cascades (6). A novel form of PAC1 receptors, namely PACAPR TM4, with sequence modifications in transmenbrane domain II and IV has been identified in the rat cerebellum (7). This PAC1 receptor does not activate AC or PLC but can induce Ca2+ entry via L-type voltage-sensitive Ca2+ channels (VSCC). VPAC receptors, unlike PAC1 receptors, can bind PACAP and VIP with equal affinity. Besides, VPAC1 and VPAC2 receptors can be further differentiated by their binding affinity for the lizard venom helodermin (8). VPAC receptors in general do not activate PLC or IP3 production and are functionally coupled to the cAMP-dependent pathway (9).
The molecular structure of PACAP is highly conserved. The amino acid sequence of PACAP38 is identical among mammalian species. When comparing the amino acid sequence of PACAP38 from vertebrates ranging from fish to mammals, only minor substitutions can be noted (10). In fish, e.g. sockeye salmon, molecular cloning of PACAP has revealed that GHRH and PACAP38 are encoded in the same gene (11). This is at variance with the situation in mammals in which GHRH and PACAP are encoded in separate genes. Although GHRH and PACAP are evolved from the same ancestral gene (12), the GH-releasing action of GHRH is either weak or not observed in fish models, and PACAP has been proposed to be the ancestral GHRH in vertebrates (13). This idea is supported by the findings that PACAP can act as a potent GH secretagogue in representative species of bony fish (14) and amphibians (15). In mammals, the GH-releasing effect of PACAP is still controversial. In in vivo studies, PACAP elevates serum GH levels in the rat (16) and cattle (17) but not in humans (18) or sheep (19). In in vitro cultures of rat pituitary cells, PACAP induces GH release in some studies (20, 21) but not others (16, 22). Within the physiological dose range, PACAP-induced GH release can be noted in porcine (23) but not ovine pituitary cells (24). In the case of a stimulatory effect, PACAP’s action is mediated by AC/cAMP and/or PLC/IP3 cascades coupled to Ca2+ entry through VSCC (25, 26). To our knowledge, the downstream mechanisms that occur after Ca2+ entry have not been elucidated. Regarding the role of PACAP in GH gene expression, both stimulatory (27) and inhibitory actions (28) have been reported, and the molecular mechanisms involved are still unclear.
In this study, using grass carp as a model for modern-day bony fish, the direct actions of PACAP on GH synthesis and secretion at the pituitary level were examined. The functional relevance of PACAP as a hypophysiotropic factor in fish was evaluated by immunostaining of PACAP nerve fibers in grass carp pituitary sections. Column perifusion was used to study the effects of PACAP on the kinetics of GH secretion in grass carp pituitary cells. Using a static incubation approach, the effects of PACAP on GH production and GH gene expression were examined. To elucidate the postreceptor signaling mechanisms for PACAP regulation of GH release and GH synthesis, a pharmacological approach was used to test the hypothesis that the actions of PACAP were mediated by the AC/cAMP/protein kinase A (PKA) pathway coupled to Ca2+ entry via L-type VSCC and subsequent activation of calmodulin (CaM)/CaM kinase II-dependent mechanisms.
Materials and Methods
Animals
One-year-old grass carps (Ctenopharyngodon idellus) with body weight from 1.5–2.0 kg and gonadosomatic index less than 0.2% were purchased from local markets and kept in well-aerated 200-liter aquaria at 18 C for at least 3 d before experimentation. During the process of pituitary cell preparation, the fish were killed by anesthesia in 0.05% MS222 (Sigma Chemical Co., St. Louis, MO) followed by spinosectomy according to the regulations of animal use at the University of Hong Kong (Hong Kong).
Reagents and test substances
Ovine PACAP38 and PACAP27 were obtained from Phoenix Pharmaceuticals (Belmont, CA), and VIP was purchased from Bachem Fine Chemicals (La Jolla, CA). Forskolin, 3-isobutyl-1-methylxanthine (IBMX), 8-bromo-cAMP (8Br-cAMP), nifedipine, A23187, Bay K8644, H89, MDL 12330A, calmidazolium, KN62, KN93, and actinomycin D were acquired from Calbiochem (San Diego, CA). The Ca2+-sensitive dye Indo-1/AM was obtained from Molecular Probes (Eugene, OR), and the working stock of Indo-1/AM was prepared freshly in dimethylsulfoxide 15 min before dye loading. Other chemicals and materials used in this study were obtained from commercial sources with the highest quality available.
Measurement of GH release and GH production
Grass carp pituitary cells were prepared by a trypsin/DNase digestion method as described previously (29). For column perifusion, pituitary cells (2.5 x 106 cells per column) were cultured on Cytodex II beads (Sigma) and perifused with carp MEM (30) at a flow rate of 15 ml/hr in an ACUSYST-S perifusion system (Endotronics Inc., Minneapolis, MN). Test substances were introduced from a drug reservoir into individual columns through a three-way stopcock. Perifusate was collected in 5-min fractions, and GH content in these samples was measured using a RIA for grass carp GH (29). For static incubation experiments, pituitary cells (2.5 x 106 cells/ml per well) were cultured in poly-D-lysine-coated (0.1 μg/ml) 24-well plates (Corning Inc., Corning, NY). The duration of drug treatment was routinely fixed at 48 h unless stated otherwise. After incubation with test substances, culture medium was harvested for the measurement of GH secretion. Pituitary cells were lysed in double-distilled deionized water by three cycles of freezing and thawing, and the lysate obtained was used for the measurement of cellular GH content. In these experiments, GH production was defined as the sum of GH release and cellular GH content.
Measurement of steady-state GH mRNA levels
After drug treatment, pituitary cells were dissolved in Trizol (GIBCO, Gaithersburg, MD), and total RNA was prepared according to the instructions by the manufacturer. These RNA samples were dissolved, heat denatured at 70 C, and vacuum blotted onto a positively charged nylon membrane using a Bio-Dot slot-blot apparatus (Bio-Rad, Hercules, CA). After hybridization with a digoxigenin (DIG)-labeled GH cDNA probe, GH mRNA signals were quantified using a DIG luminescent detection kit (Roche, Stockholm, Sweden) (31). In this study, Northern blot was also conducted to test whether PACAP treatment could alter the form(s) and/or size of GH transcripts. In this case, total RNA was resolved in 1% gel with 0.22 M formaldehyde and transblotted onto a nylon membrane for GH mRNA measurement according to the standard procedures in our laboratory (32). In these experiments, parallel probing of -actin mRNA or 18S RNA was used as an internal control.
Ribonuclease protection assay for GH primary transcripts
Pituitary cells (5 x 106 cells/2 ml per well) were cultured in poly-D-lysine-precoated 12-well plates (Corning) for simultaneous measurement of cytosolic mature GH mRNA and nuclear GH primary transcripts. After drug treatment, cytosolic and nuclear RNA were isolated by sucrose gradient centrifugation followed by proteinase K and DNase I digestion. These RNA samples were then subjected to ribonuclease protection assays (RPA) previously validated for grass carp mature GH mRNA and GH primary transcripts (31). A DNA fragment covering the junction between exon II and intron II of grass carp GH gene (GenBank no. X60419) was used to produce the 32P-labeled antisense riboprobe by in vitro transcription. Sense-strand cRNAs were also prepared to serve as RPA standards. In these assays, mature GH mRNA (in the cytosol) and GH primary transcripts (in the nucleus) consistently produced protected fragments of 124 and 207 bp, respectively.
Measurement of GH promoter activity
Decreasing lengths of the grass carp GH promoter (–446 to –155) were PCR isolated and subcloned into pBL.CAT to generate pGH.CAT constructs for transfection studies in GH4C1 cells. GH4C1 cells, a rat pituitary cell line, were maintained in Ham’s F-10 medium (GIBCO) with 10% fetal bovine serum at a seeding density of 0.25 x 106 cells/3 ml per dish. After overnight incubation, transfection was conducted in 300 μl OPTI-MEM (GIBCO) for 6 h with 0.25 μg/dish pGH.CAT constructs, 0.5 μg/dish pEGFP-N1 (Clontech, Palo Alto, CA), and 7 μl lipofectamine (GIBCO). The green fluorescent protein (GFP) expression vector pEGFP-N1 was included to serve as an internal control for potential variations in transfection efficiency between dishes. After transfection, GH4C1 cells were cultured in Ham’s F-10 medium with 10% fetal bovine serum for 7 h before the addition of test substances. The duration of drug treatment was routinely fixed at 15 h. After that, cell lysate was prepared for chloramphenicol acetyltransferase (CAT) activity measurement using a two-phase fluor diffusion assay (33). GFP expression levels in these samples were quantified by fluorescence measurement using a Cytofluor 4000 plate reader (Perspective Biosystems, Framingham, MA).
Immunohistochemical staining of grass carp pituitary sections
Carp pituitaries were fixed in Bouin’s fixative, embedded in paraffin wax, and sectioned into 5-μm-thick sections according to standard procedures. Immunohistochemical staining was conducted using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Antisera for GH (1:50,000) and prolactin (PRL, 1:100,000) are gifts from Dr. R. E. Peter (University of Alberta, Alberta, Canada) and have been previously validated for immunostaining in the grass carp (29). The antiserum for human neurofilament (1:500; UpState, Milford, MA) was used to probe the neuronal structures/nerve fibers in the carp pituitary. PACAP antiserum (1:2000; Peninsula Laboratories, Belmont, CA) for immunostaining of PACAP nerve fibers was raised against PACAP38 and had no cross-reactivity with PACAP27. For immunostaining of somatotrophs, cytospin preparations of pituitary cells were prepared using an Autosmear CF-10 centrifuge (Sakura Technical, Nagano, Japan). GH immunoreactivity was visualized according to the immunostaining procedures described previously (34).
Measurement of intracellular Ca2+ level
Enriched somatotrophs were prepared from grass carp pituitary cells by Percoll gradient centrifugation (34) and preloaded with Indo-1/AM (10 μM) in HEPES-buffered saline medium (31) with 0.1% BSA at 28 C for 30 min. After dye loading, the cells were resuspended at 1.5 x 106 cells/ml in BSA-free HEPES-buffered saline medium, and [Ca]i measurement was performed in a thermostatted (28 C) quartz cuvette using a F-4500 fluorescence spectrophotometer (Hitachi, Indianapolis, IN). Wavelengths for excitation and emission were fixed at 329 nm (5-nm slit width) and 405 nm (10-nm slit width), respectively. In this study, [Ca2+]i concentrations were calibrated using the cell lysis method as described previously (35).
Measurement of cAMP production
Enriched somatotrophs or mixed populations of carp pituitary cells were resuspended in carp MEM and seeded at a density of 2 x 106 cells/2 ml in poly-D-lysine-coated 35-mm petri dishes (Corning). After overnight incubation, carp MEM was replaced with 0.9 ml HHBSA medium (36) and incubated at 28 C for 15 min before adding 0.1 ml x10 stock solutions of test substances. The duration of drug treatment was routinely fixed at 15 min. After that, culture medium was harvested for the measurement of cAMP release and cellular cAMP was extracted by adding 1 ml ice-cold absolute ethanol. These cAMP samples were then freeze-dried and stored at –20 C until their cAMP content was quantified by a BioTrak [125I]cAMP RIA (Amersham Pharmacia Biotech, Piscataway, NJ). In these experiments, cAMP production was defined as the sum of cAMP release and cellular cAMP content.
Data transformation and statistics
For perifusion studies, GH data (in ng/ml) from individual columns were expressed as a percentage of the average GH content of the first six fractions collected at the beginning of the experiment before drug treatment (as percent basal). This transformation was used for pooling of data from separate columns without distorting the profile of GH release. GH response was quantified by calculating the net change in GH release after drug treatment (i.e. a net change in area under the curve). GH transcripts were measured in terms of arbitrary density unit and normalized against the amount of -actin mRNA or 18S RNA in the same sample. The half-life (t1/2) of GH mRNA was deduced based on the one-phase exponential decay model using GraphPad Prism 3.02 (GraphPad, San Diego, CA). CAT activity was normalized against GFP expression in the same sample. Given that the levels of internal controls for GH mRNA and GH promoter activity did not exhibit significant changes in these studies, the normalized data were simply transformed as a percentage of the mean value in the control group without drug treatment (as percent control). In the case of cAMP assays, cAMP levels were measured in terms of pmol cAMP/ml or pmol cAMP/106 cells, and the data were also transformed as a percentage of the control for statistical analysis. For [Ca2+]i measurement, fluorescence data (in arbitrary fluorescence units) were transformed into Ca2+ levels using the single-wavelength calibration equation: [Ca2+]i = Kdx (F – Fmin)/(Fmax – F), where Kd is the dissociation constant for Indo-1, Fmin is basal fluorescence without Ca2+, Fmax is the maximal fluorescence under saturating Ca2+, and F is the fluorescence level at any unknown Ca2+ concentration. Fmin and Fmax were determined empirically for individual dye loading according to the method described by Thomas and Delaville (35). Data presented were analyzed using Student’s t test or ANOVA followed by Fisher’s least significance difference (LSD) test. Differences were considered significant at P < 0.05.
Results
Immunohistochemical staining of PACAP in grass carp pituitary sections
The grass carp pituitary could be divided into the anterior pituitary, including the rostral pars distalis (RPD) and proximal pars distalis (PPD), and the posterior pituitary, generally referred to as the neurointermediate lobe (Fig. 1A). PRL immunoreactivity was located exclusively in the RPD (Fig. 1B), whereas GH immunoreactivity was detected only in the PPD (Fig. 1C). Using an antiserum raised against PACAP38, PACAP immunoreactivity could be noted in the pars distalis of the carp pituitary, including both RPD and PPD (Fig. 1D). These immunostaining signals were not observed with the use of a similar dilution of normal rabbit serum (data not shown) and could be blocked by preabsorbing PACAP antiserum (1:2,000) with PACAP38 (0.1 mM) for 24 h at 4 C (Fig. 1E). Apparently, PACAP immunoreactivity was widely expressed in the anterior pituitary. When examined under a higher magnification with phase-contrast microscopy (Fig. 1, F and G), PACAP immunoreactivity was located only in the nerve fibers/nerve tracts but not in pituitary cells. These nerve fibers/nerve tracts were also stained positively with an antiserum for the neuronal marker neurofilament (Fig. 1, F and G, insets). In parallel studies, no immunostaining signals could be detected in the grass carp pituitary sections using an antiserum raised against PACAP27 (data not shown).
Effects of PACAP and VIP on GH release in grass carp pituitary cells
Using a column perifusion approach, the direct actions of PACAP on GH secretion were tested at the pituitary cell level. Increasing concentrations (0.1–1000 nM) of PACAP38 (Fig. 2A) and PACAP27 (Fig. 2B) were effective in inducing GH release from grass carp pituitary cells in a dose-dependent manner. The kinetics of these GH responses was rapid, and the peak of GH secretion could be noted within 10 min after the initiation of drug treatment. After the 10-min pulses of PACAP stimulation, GH release gradually returned to basal levels. Similar doses of human VIP, however, did not alter basal levels of GH secretion (Fig. 2C). A similar lack of GH responses was also noted when substituting cod VIP for human VIP in these experiments (data not shown).
Effects of PACAP on [Ca2+]i and cAMP production in grass carp somatotrophs
Using density gradient centrifugation, enriched somatotrophs were prepared from mixed populations of grass carp pituitary cells (Fig. 3A). After loading with the Ca2+-sensitive dye Indo-1/AM, [Ca2+]i levels in this cell preparation was increased by treatment with PACAP38 (1 μM). This stimulatory effect, however, was blocked by removing [Ca2+]e using the Ca2+ chelator EGTA (4 mM) (Fig. 3B) or by inactivating L-type VSCC using the dihydropyridine inhibitor nifedipine (10 μM). In these studies, the vehicle for PACAP38 did not modify basal [Ca2+]i levels. Using a 15-min short-term incubation protocol, increasing levels of PACAP38 (0.01–1000 nM) also triggered a dose-dependent increase in cAMP release, cellular cAMP content, and total cAMP production in carp somatotrophs (Fig. 3C).
cAMP- and Ca2+-dependent mechanisms in PACAP-induced GH release
To test whether activation of cAMP production and/or Ca2+ entry can induce GH secretion at the pituitary cell level, a pharmacological approach was used. In this case, increasing levels of the AC activator forskolin (0.1–10 μM) (Fig. 4A) and phosphodiesterase inhibitor IBMX (0.1–100 μM) (Fig. 4B) consistently elevated cAMP release, cAMP content, and cAMP production in pituitary cells cultured under static incubation conditions. In parallel experiments using column perifusion, forskolin (0.1–10 μM) (Fig. 4C) and IBMX (0.01–100 μM) (Fig. 4D) were both effective in triggering a dose-dependent increase in GH release. Similarly, elevating the functional levels of cAMP using a membrane-permeable cAMP analog 8Br-cAMP (30 μM to 3 mM) also induced GH secretion (Fig. 5A), and this GH-releasing effect could be mimicked by the Ca2+ ionophore A23187 (0.3–30 μM) (Fig. 5B) and L-type VSCC activator Bay K8644 (1 nM to 10 μM) (Fig. 5C). To examine the possible involvement of cAMP- and Ca2+-dependent mechanisms in PACAP-induced GH secretion, the GH-releasing action of PACAP38 was tested with the simultaneous treatment of the inhibitors for the AC/cAMP/PKA and Ca2+/ CaM/CaM kinase II pathways. In these studies, the stimulatory effects of forskolin (3 μM) and 8Br-cAMP (3 mM) on GH release were suppressed in a dose-dependent manner by the AC inhibitor MDL 12330A (0.03–30 μM) (Fig. 6A) and PKA inhibitor H89 (1–30 μM) (Fig. 6B), respectively. These results indicate that these two inhibitors were both effective in blocking the AC/cAMP/PKA pathway in fish pituitary cells. In parallel experiments, MDL 12330A (30 μM) and H89 (30 μM) were also effective in inhibiting both basal and PACAP38-stimulated GH release (Fig. 6, C and D). Furthermore, the GH-releasing action of PACAP38 could be blocked by perifusion with a Ca2+-free medium (Fig. 7A) or by the L-type VSCC inhibitor nifedipine (10 μM) (Fig. 7B). Comparable results were also noted by replacing PACAP38 with forskolin (3 μM) as the stimulant for GH secretion (Fig. 7, C and D). Consistent with these findings, the GH-releasing effect of PACAP38 was similarly suppressed by cotreatment with the CaM antagonist calmidazolium (1 μM) (Fig. 8A) or by the CaM kinase II inhibitors KN62 (5 μM) (Fig. 8B) and KN93 (5 μM) (Fig. 8C). Again, these CaM/CaM kinase inhibitors were effective in attenuating the GH responses triggered by forskolin treatment (3 μM) (Table 1).
Effects of PACAP on GH production and GH gene expression
Using a static incubation approach, the direct actions of PACAP38 on GH synthesis were tested at the pituitary cell level. In this case, a 48-h incubation with PACAP38 (1 μM) and forskolin (3 μM) significantly increased GH release (Fig. 9A), cellular GH content (Fig. 9B), and total GH production in grass carp pituitary cells (Fig. 9C). Parallel treatments with human and cod VIP (1 μM) were not effective in these regards (data not shown). As revealed by Northern blot, the GH responses observed at the protein level induced by PACAP (1 μM) and forskolin treatment (3 μM) also occurred with a concurrent rise in steady-state GH mRNA levels. Apparently, these GH mRNA responses did not involve modifications in the form or size of GH transcripts. To increase the throughput of our studies, a slot-blot assay was used to monitor GH mRNA levels in grass carp pituitary cells. In this case, the stimulatory effects of PACAP38 (1 μM) and forskolin (10 μM) on GH mRNA expression were found to be time dependent (Fig. 10, A and B). In parallel experiments, GH mRNA levels were also accentuated in a dose-dependent manner by increasing concentrations of PACAP38 (1 nM to 10 μM) (Fig. 10C) and forskolin (0.1–30 μM) (Fig. 10D). Again, human VIP (1 μM) was not effective in altering basal levels of GH mRNA expression (Fig. 10C, inset).
Given that steady-state mRNA represents a dynamic balance between the synthesis and degradation of target transcripts, the role of GH mRNA stability and GH gene transcription in PACAP-induced GH gene expression was also examined. In pituitary cells pretreated with the transcription inhibitor actinomycin D (8 μM), the clearance curves for GH mRNA were shifted to the right with an increase in t1/2 from 10.2 to 21.8 h and from 9.8 to 17.6 h by treatment with PACAP38 (1 μM) (Fig. 11A) and forskolin (10 μM) (Fig. 11B), respectively. Besides, the levels of cytosolic mature GH mRNA and nuclear GH primary transcripts were found to be up-regulated in carp pituitary cells by a 48-h incubation with PACAP38 (0.3 and 3 μM) and forskolin (10 μM) (Fig. 11C). In GH4C1 cells transfected with CAT constructs with decreasing lengths of grass carp GH promoter (–446 to –155), similar drug treatments for 15 h were also effective in stimulating CAT activity expression (Fig. 11D). PACAP38 (1 μM) and forskolin (10 μM), however, did not alter the promoter activities in the control groups transfected with pBL.CAT and TK.CAT, respectively (data not shown). In this study, basal levels of CAT activity were also elevated by 5' truncation of the GH promoter from –221 to –115, suggesting that inhibitory cis-acting element(s) may be present in this region.
cAMP- and Ca2+-dependent mechanisms in PACAP-induced GH mRNA expression
To study the functional role of cAMP- and Ca2+-dependent cascades in PACAP-induced GH gene expression, the stimulatory action of PACAP38 on GH mRNA levels was tested in the presence of the inhibitors for AC/cAMP/PKA and Ca2+/CaM/CaM kinase II cascades. In pituitary cells cultured under static incubation conditions, basal levels of GH transcripts were consistently elevated by 48-h treatment with PACAP38 (1 μM) (Fig. 12A) and forskolin (10 μM) (Fig. 12B). These stimulatory effects, however, were blocked by simultaneous treatment with the AC inhibitor MDL 12330A (30 μM) and PKA inhibitor H89 (30 μM), and these two inhibitors were also effective in reducing basal levels of GH mRNA expression. Similar to PACAP38, the Ca2+ ionophore A23187 (0.1–100 nM) (Fig. 13A) and L-type VSCC activator Bay K8644 (1–1000 nM) (Fig. 13B) could elevate GH mRNA levels in a dose-dependent manner. However, both basal and PACAP38-stimulated (1 μM) GH mRNA expression were inhibited by incubation with a Ca2+-free medium or by the VSCC blocker nifedipine (10 μM) (Fig. 13C). Similar results were also noted by replacing PACAP38 with forskolin (10 μM) as the trigger for GH mRNA expression (Fig. 13C, inset). In parallel experiments, basal levels of CaM mRNA were up-regulated in carp pituitary cells in a dose-related fashion by increasing concentrations of PACAP38 (1–1000 μM), and this stimulatory effect occurred concurrently with a rise in GH transcripts (Fig. 14A). Furthermore, the CaM kinase II inhibitors KN62 (5–5000 nM) (Fig. 14B) and KN93 (5 μM) (Fig. 14C) were both effective in reducing basal and PACAP38-induced (1 μM) GH mRNA expression. Again, similar results were obtained using forskolin (10 μM) as the stimulant in these experiments (Fig. 14C, inset).
Discussion
In mammals, PACAP serves as a hypophysiotopic factor and plays an active role in the regulation/modulation of pituitary functions. This idea is supported by the findings that 1) PACAP neurons are located in the paraventricular and supraoptic nuclei of the hypothalamus (37), 2) PACAP nerve terminals are present in the external zone of the median eminence (38), 3) PACAP immunoreactivity can be detected in the hypophysial portal blood at a level higher than that of systemic circulation (39), and 4) PACAP, under certain conditions, can induce GH, PRL, LH, and ACTH secretion at the pituitary cell level (40). Although the GH-releasing effect of PACAP is still controversial, PACAP receptors have been identified in rat somatotrophs (41, 42), somatotroph cell lines (43), and GH-secreting pituitary adenomas (44, 45). In the cases of a stimulatory action, the GH-releasing effect of PACAP is either weak (40) or modest (46), susceptible to receptor desensitization (47), and mediated through VPAC2 receptors (48). In this study, nerve fibers/nerve tracts with PACAP38 immunoreactivity were located in the grass carp pituitary overlapping with the distribution of somatotrophs in the pars distalis. Using a perifusion approach, PACAP38 and PACAP27 but not VIP were effective in stimulating GH release from grass carp pituitary cells. These results are in agreement with our recent studies in the goldfish (30) and common carp (49), in which PACAP-induced GH release is mediated through pituitary receptors resembling the mammalian PAC1 subtypes. Given that 1) the pituitary of bony fish is under the direct innervation of the hypothalamus (50) and 2) the perikarya of PACAP neurons can be identified in the hypothalamus of fish species, e.g. European eel (51), it is tempting to speculate that PACAP is produced in the hypothalamus and delivered to the anterior pituitary to serve as a GH-releasing factor in fish models. Because the antiserum for PACAP27 was unable to pick up any immunostaining signals in the carp pituitary, it raises the possibility that PACAP38 but not PACAP27 is the major form of PACAP acting at the pituitary level. In the rat, mainly based on the studies using HPLC and RIA, the protein level of PACAP38 in the hypothalamus was found to be at least 10-fold higher than that of PACAP27 (52), indicating that PACAP38 is the dominant form of PACAP expressed in the hypothalamo-pituitary axis.
Using grass carp pituitary cells as a cell model, we have demonstrated that PACAP38 not only induced GH secretion but also increased GH content, GH production, and GH mRNA expression by acting directly at the pituitary level. Because the stimulatory action of PACAP on GH gene expression could be noted in the nanomolar dose range and was not mimicked by VIP, it would be logical to assume that PACAP by acting on pituitary PAC1 receptors can serve as a potent stimulator for GH synthesis in the carp species. Although PACAP induction of GH mRNA expression has been reported in rat (53) and porcine pituitary cells (27), these stimulatory effects were not dose dependent and no corresponding changes in GH content could be noted in these previous studies. Furthermore, in rat somatotroph cell lines, e.g. GH3 cells, PACAP is known to reduce GH mRNA levels as well as GH content (28). The reasons for these discrepancies are still unknown and may be related to the differences in research methodologies and/or variations in the physiological status of cell models. Using enriched somatotrophs prepared from mixed populations of grass carp pituitary cells, PACAP38 was shown to activate cAMP production and Ca2+ entry via L-type VSCC. In carp pituitary cells, the stimulatory actions of PACAP on GH release and GH mRNA expression were mimicked by increasing cAMP synthesis/functional levels, induction of [Ca2+]e entry, and activation of L-type VSCC. PACAP38-stimulated GH release and GH mRNA expression, however, were blocked by inhibiting AC and PKA, removal of [Ca2+]e, and inactivation of VSCC. Using forskolin instead of PACAP38 as the stimulant for GH secretion and GH gene expression, similar inhibitions could be observed after Ca2+-free treatment or VSCC blockade, suggesting that [Ca2+]e entry through VSCC is operating downstream of the AC/cAMP/PKA pathway. This idea is in agreement with the previous reports that 1) VSCC phosphorylation is required for channel opening in GH-secreting cell lines (56) and 2) the -subunit (57) and 2-subunit (58) of VSCC can be PKA phosphorylated in a cAMP-dependent manner. Because PACAP is also known to activate tetrodotoxin-sensitive Na+ channels in GH3 cells (59), we do not exclude the possibility that the actions of PACAP on VSCC may be indirect via PKA phosphorylation of voltage-sensitive Na+ channels (54, 55).
To elucidate the downstream signaling events after Ca2+ entry, the role of CaM and CaM kinase II in PACAP induction of GH synthesis and secretion were also examined. In carp pituitary cells, PACAP-induced GH gene expression also occurred with a rise in CaM mRNA levels, suggesting that CaM-dependent mechanisms might have been activated. This idea is consistent with the findings that the GH-releasing action of PACAP38 could be abolished by CaM antagonism and CaM kinase II inhibition. Because inactivating CaM kinase II was also effective in blocking the stimulatory effects of PACAP38 and forskolin on GH release and GH mRNA levels, it is conceivable that Ca2+ entry induced by PACAP through cAMP-dependent mechanisms can activate CaM/CaM kinase II cascades to trigger GH exocytosis and GH gene expression. Functional coupling of PACAP with CaM-dependent signaling cascades has been recently reported in mammals. In guinea pig, PACAP reduces somatostatin immunoreactivity in cardiac ganglia neurons, and this inhibitory action can be blocked by CaM inhibitors (60). In mouse cerebellar granule cells, PACAP-induced expression of the circadian clock gene is sensitive to the blockade by CaM antagonists and CaM kinase inhibitors (61). Because the total amount of CaM in living cells is much lower than that of its target proteins (62), the operation of CaM-dependent signaling pathways is determined largely by their effectiveness in the competition for the limiting pool of CaM (63). The present finding of PACAP-induced CaM mRNA expression in carp pituitary cells may suggest that PACAP can not only stimulate GH synthesis and secretion by functional coupling of the Ca2+/CaM/CaM kinase II pathway with the cAMP-dependent cascade but also enhance the functionality of CaM-dependent signaling mechanisms by up-regulation of CaM gene expression.
At present, no information is available regarding the molecular mechanisms for PACAP regulation of GH synthesis. To shed light on this area, we examined the role of GH transcript stability and GH gene transcription in PACAP-induced GH gene expression. In carp pituitary cells pretreated with actinomycin D, the t1/2 of GH mRNA was increased by treatment with PACAP38 and forskolin, indicating that PACAP can improve the transcript stability of GH via activation of the cAMP-dependent pathway. In the rat, glucocorticoid and thyroid hormone can induce GH synthesis by prolonging the t1/2 of GH mRNA, and these stimulatory actions are caused by increasing the length of the poly(A+) tail (64). Similar mechanisms, however, may not be applicable to PACAP-induced GH mRNA expression because the size of GH transcript (1.16 kb) was not modified by PACAP38 or forskolin as revealed by the results of Northern blot. In parallel studies, the levels of cytosolic mature GH mRNA and nuclear GH primary transcripts were both elevated in grass carp pituitary cells by treatment with PACAP38 and forskolin. Given that primary transcripts undergo intron splicing rapidly and translocate into the cytoplasm as mature mRNA, the levels of primary transcripts in general are taken as a faithful index for target gene transcription (65). Therefore, the parallel rises in GH primary transcripts and mature GH mRNA observed in the present study may indicate that GH gene transcription has been activated after PACAP38 stimulation. This idea is also supported by the results of our transfection studies in GH4C1 cells, in which the promoter activity of grass carp GH gene was up-regulated by PACAP38 and forskolin, and the cis-acting element(s) responsive to these drug treatments could be mapped to the promoter region downstream of position –115. These results, taken together, suggest that PACAP by acting through cAMP-dependent mechanisms can increase GH mRNA expression both at the transcriptional level (by activating GH gene transcription in the nucleus) and at the posttranscriptional level (by enhancing GH transcript stability in the cytoplasm). In mammals, the transcription factor cAMP response element binding protein (CREB) by phosphorylation at serine 133 (e.g. by PKA/CaM kinases) serves as a key mediator for Ca2+/cAMP-dependent gene expression (66), and CREB phosphorylation can be observed during PACAP stimulation of melatonin synthesis (67) and mPer1 gene expression (68, 69). Besides, the cis-acting element for CREB, namely CRE, can be located in the 5' promoter of the human GH gene and mediates cAMP inducibility of GH gene transcription (70). Similar findings have also been reported in fish, e.g. Chinook salmon (33), suggesting that the role of CRE in mediating cAMP induction of GH gene expression is conserved during vertebrate evolution. Because putative CRE sites have been reported in the 5' promoter of grass carp GH gene (71), it raises the possibility that PACAP-induced GH gene expression is mediated by CREB transactivation via CRE sites in the GH promoter.
In summary, we have demonstrated that nerve fibers with PACAP immunoreactivity are present in the anterior pituitary of grass carp and PACAP by acting directly at the pituitary cell level can stimulate GH secretion and GH gene expression through receptors resembling mammalian PAC1 receptors. These stimulatory effects probably are mediated through Ca2+ entry and cAMP production in grass carp somatotrophs and involve the sequential activation of the AC/cAMP/PKA and Ca2+/CaM/CaM kinase II signaling pathways. Using grass carp pituitary cells as a model (Fig. 15), we propose that PACAP released from nerve terminals of hypothalamic neurons stimulates PAC1 receptors on somatotrophs. Subsequent activation of the AC/cAMP/PKA pathway triggers Ca2+ entry through L-type VSCC, and the rise in [Ca2+]i then activates CaM/CaM kinase II cascades to induce GH exocytosis. Activation of the Ca2+-dependent signaling events secondarily coupled to the cAMP-dependent pathway also elevates GH production by increasing GH mRNA levels. This increase in GH mRNA levels probably are caused by 1) enhancement of GH mRNA stability in the cytoplasm and 2) activation of GH gene transcription in the nucleus. In carp somatotrophs, the concurrent activation of GH synthesis may be essential to replenish the intracellular GH stores after PACAP-induced GH exocytosis. In mammals, GHRH is known to stimulate GH release through Ca2+ entry via L-type VSCC after activation of the AC/cAMP/PKA pathway (26). The postreceptor signaling mechanisms coupled to GHRH receptors appear to be highly comparable with that of PACAP in the carp model. Because PACAP has been proposed to be the ancestral GHRH (13), it is tempting to speculate that the functional replacement of PACAP by GHRH may occur at the level of membrane receptors (i.e. from PAC1 receptors to GHRH receptors) and the postreceptor signaling events leading to GH exocytosis are largely conserved during the course of vertebrate evolution.
Acknowledgments
This series of papers on the role of PACAP as a GH-releasing factor in fish is dedicated to Drs. R. E. Peter and J. P. Chang (University of Alberta, Canada) for their genuine interest in the training of young scientists. Special thanks are given to Dr. R. E. Peter (University of Alberta, Canada) for the supply of protein standard and antiserum for GH RIA. We are also indebted to Dr. W. K. K. Ho (Chinese University of Hong Kong) for his support in setting up the assay systems for GH mRNA and GH primary transcripts.
Footnotes
The present study was sponsored by grants from the Research Grants Council (Hong Kong) and Committee on Research and Conference Grants (University of Hong Kong) (to A.O.L.W.). Financial support from the Department of Zoology (University of Hong Kong) (to C.Y.L., H.Z., and L.H.) in the form of postgraduate studentship is also acknowledged.
First Published Online August 25, 2005
1 W.L. and C.Y.L. contributed equally to this work.
Abbreviations: AC, Adenylate cyclase; 8Br-cAMP; 8-bromo-cAMP; [Ca2+]e, extracellular Ca2+; [Ca2+]i, intracellular Ca2+; CaM, calmodulin; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element binding protein; DIG, digoxigenin; GFP, green fluorescent protein; IBMX, 3-isobutyl-1-methylxanthine; IP3, inositol trisphosphate; LSD, least significant difference; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PLC, phospholipase C; PRL, prolactin; PPD, proximal pars distalis; RPD, rostral pars distalis; VIP, vasoactive intestinal polypeptide; VSCC, voltage-sensitive Ca2+ channels.
Accepted for publication August 17, 2005.
References
Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H 2000 Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 52:269–324
Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH 1989 Isolation of a novel 38-residue hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164:567–574
Arimura A 1998 Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Jpn J Physiol 48:301–331
Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA 1998 International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 50:265–270
Journot L, Spengler D, Pantaloni C, Dumuis A, Sebben M, Bockaert J 1994 The PACAP receptor: generation by alternative splicing of functional diversity among G protein-coupled receptors in nerve cells. Semin Cell Biol 5:263–272
Laburthe M, Couvineau A 2002 Molecular pharmacology and structure of VPAC receptors for VIP and PACAP. Regul Pept 108:165–173
Chatterjee TK, Sharma RV, Fisher RA 1996 Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels. J Biol Chem 271:32226–32232
Hezareh M, Journot L, Bepoldin L, Schlegel W, Rawlings SR 1996 PACAP/VIP receptor subtypes, signal transducers, and effectors in pituitary cells. Ann NY Acad Sci 805:315–327; discussion 327–328
Rawlings SR, Hezareh M 1996 Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland. Endocr Rev 17:4–29
Sherwood NM, Krueckl SL, McRory JE 2000 The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr Rev 21:619–670
Parker DB, Coe IR, Dixon GH, Sherwood NM 1993 Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagon superfamily. Eur J Biochem 215:439–448
Adams BA, Lescheid DW, Vickers ED, Crim LW, Sherwood NM 2002 Pituitary adenylate cyclase-activating polypeptide and growth hormone-releasing hormone-like peptide in sturgeon, whitefish, grayling, flounder and halibut: cDNA sequence, exon skipping and evolution. Regul Pept 109:27–37
Montero M, Yon L, Kikuyama S, Dufour S, Vaudry H 2000 Molecular evolution of the growth hormone-releasing hormone/pituitary adenylate cyclase-activating polypeptide gene family. Functional implication in the regulation of growth hormone secretion. J Mol Endocrinol 25:157–168
Wong AOL, Li WS, Lee EK, Leung MY, Tse LY, Chow BK, Lin HR, Chang JP 2000 Pituitary adenylate cyclase activating polypeptide as a novel hypophysiotropic factor in fish. Biochem Cell Biol 78:329–343
Yon L, Alexandre D, Montero M, Chartrel N, Jeandel L, Vallarino M, Conlon JM, Kikuyama S, Fournier A, Gracia-Navarro F, Roubos E, Chow B, Arimura A, Anouar Y, Vaudry H 2001 Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians. Microsc Res Tech 54:137–157
Jarry H, Leonhardt S, Schmidt WE, Creutzfeldt W, Wuttke W 1992 Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats. Life Sci 51:823–830
Radcliff RP, Lookingland KJ, Chapin LT, Tucker HA 2001 Pituitary adenylate cyclase-activating polypeptide induces secretion of growth hormone in cattle. Domest Anim Endocrinol 21:187–196
Chiodera P, Volpi R, Capretti L, Caffarri G, Magotti MG, Coiro V 1996 Effects of intravenously infused pituitary adenylate cyclase-activating polypeptide on adenohypophyseal hormone secretion in normal men. Neuroendocrinology 64:242–246
Sawangjaroen K, Curlewis JD 1994 Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on prolactin, luteinizing hormone and growth hormone secretion in the ewe. J Neuroendocrinol 6:549–555
Velkeniers B, Zheng L, Kazemzadeh M, Robberecht P, Vanhaelst L, Hooghe-Peters EL 1994 Effect of pituitary adenylate cyclase-activating polypeptide 38 on growth hormone and prolactin expression. J Endocrinol 143:1–11
Goth MI, Lyons CE, Canny BJ, Thorner MO 1992 Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors. Endocrinology 130:939–944
Culler MD, Paschall CS 1991 Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone. Endocrinology 129:2260–2262
Martinez-Fuentes AJ, Castano JP, Gracia-Navarro F, Malagon MM 1998 Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 activate common and distinct intracellular signaling pathways to stimulate growth hormone secretion from porcine somatotropes. Endocrinology 139:5116–5124
Sawangjaroen K, Anderson ST, Curlewis JD 1997 Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro. J Neuroendocrinol 9:279–286
Murakami Y, Koshimura K, Yamauchi K, Nishiki M, Tanaka J, Kato Y 2001 Roles and mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth hormone and prolactin secretion. Endocr J 48:123–132
Gracia-Navarro F, Castano JP, Malagon MM, Sanchez-Hormigo A, Luque RM, Hickey GJ, Peinado JR, Delgado E, Martinez-Fuentes AJ 2002 Research progress in the stimulatory inputs regulating growth hormone (GH) secretion. Comp Biochem Physiol B Biochem Mol Biol 132:141–150
Martinez-Fuentes AJ, Malagon MM, Castano JP, Garrido-Gracia JC, Gracia-Navarro F 1998 Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 differentially stimulate growth hormone release and mRNA accumulation in porcine somatotropes. Life Sci 62:2379–2390
Yonehara T, Kanasaki H, Yamamoto H, Fukunaga K, Miyazaki K, Miyamoto E 2001 Involvement of mitogen-activated protein kinase in cyclic adenosine 3',5'-monophosphate-induced hormone gene expression in rat pituitary GH(3) cells. Endocrinology 142:2811–2819
Wong AOL, Ng S, Lee EK, Leung RC, Ho WK 1998 Somatostatin inhibits (D-Arg6, Pro9-NEt) salmon gonadotropin-releasing hormone- and dopamine D1-stimulated growth hormone release from perifused pituitary cells of Chinese grass carp, Ctenopharyngodon idellus. Gen Comp Endocrinol 110:29–45
Wong AOL, Leung MY, Shea WL, Tse LY, Chang JP, Chow BK 1998 Hypophysiotropic action of pituitary adenylate cyclase-activating polypeptide (PACAP) in the goldfish: immunohistochemical demonstration of PACAP in the pituitary, PACAP stimulation of growth hormone release from pituitary cells, and molecular cloning of pituitary type I PACAP receptor. Endocrinology 139:3465–3479
Zhou H, Ko WK, Ho WK, Stojilkovic SS, Wong AOL 2004 Novel aspects of growth hormone (GH) autoregulation: GH-induced GH gene expression in grass carp pituitary cells through autocrine/paracrine mechanisms. Endocrinology 145:4615–4628
Huo L, Lee EK, Leung PC, Wong AOL 2004 Goldfish calmodulin: molecular cloning, tissue distribution, and regulation of transcript expression in goldfish pituitary cells. Endocrinology 145:5056–5067
Wong AOL, Le Drean Y, Liu D, Hu ZZ, Du SJ, Hew CL 1996 Induction of Chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1. Endocrinology 137:1775–1784
Van Goor F, Goldberg IG, Wong AOL, Jobin RM, Chang JP 1994 Morphological identification of live gonadotropin, growth-hormone, and prolactin cells in goldfish (Carasssius auratus) pituitary cell cultures. Cell Tissue Res 276:253–261
Thomas AP, Delaville F 1991 The use of fluorescent indicators for measurements of cytosolic free calcium concentration in cell populations and single cells. In: McCormack JG, Cobbold PH, eds. Cellular calcium: a practical approach. London: IRL Press; 1–54
Yunker WK, Lee EK, Wong AOL, Chang JP 2000 Norepinephrine regulation of growth hormone release from goldfish pituitary cells. II. Intracellular sites of action. J Neuroendocrinol 12:323–333
Takahashi K, Totsune K, Murakami O, Satoh F, Sone M, Ohneda M, Sasano H, Mouri T 1994 Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in human hypothalamus: co-localization with arginine vasopressin. Regul Pept 50:267–275
Koves K, Gorcs TJ, Kausz M, Arimura A 1994 Present status of knowledge about the distribution and colocalization of PACAP in the forebrain. Acta Biol Hung 45:297–321
Dow RC, Bennie J, Fink G 1994 Pituitary adenylate cyclase-activating peptide-38 (PACAP)-38 is released into hypophysial portal blood in the normal male and female rat. J Endocrinol 142:R1–R4
Hart GR, Gowing H, Burrin JM 1992 Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats. J Endocrinol 134:33–41
Bresson-Bepoldin L, Jacquot MC, Schlegel W, Rawlings SR 1998 Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells. J Mol Endocrinol 21:109–120
Vertongen P, Velkeniers B, Hooghe-Peters E, Robberecht P 1995 Differential alternative splicing of PACAP receptor in pituitary cell subpopulations. Mol Cell Endocrinol 113:131–135
Rawlings SR, Piuz I, Schlegel W, Bockaert J, Journot L 1995 Differential expression of pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptor subtypes in clonal pituitary somatotrophs and gonadotrophs. Endocrinology 136:2088–2098
Robberecht P, Vertongen P, Velkeniers B, de Neef P, Vergani P, Raftopoulos C, Brotchi J, Hooghe-Peters EL, Christophe J 1993 Receptors for pituitary adenylate cyclase activating peptides in human pituitary adenomas. J Clin Endocrinol Metab 77:1235–1239
Oka H, Jin L, Reubi JC, Qian X, Scheithauer BW, Fujii K, Kameya T, Lloyd RV 1998 Pituitary adenylate-cyclase-activating polypeptide (PACAP) binding sites and PACAP/vasoactive intestinal polypeptide receptor expression in human pituitary adenomas. Am J Pathol 153:1787–1796
Propato-Mussafiri R, Kanse SM, Ghatei MA, Bloom SR 1992 Pituitary adenylate cyclase-activating polypeptide releases 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3. J Endocrinol 132:107–113
Wei L, Chan WW, Butler B, Cheng K 1993 Pituitary adenylate cyclase activating polypeptide-induced desensitization on growth hormone release from rat primary pituitary cells. Biochem Biophys Res Commun 197:1396–1401
Murakami Y, Koshimura K, Yamauchi K, Nishiki M, Tanaka J, Furuya H, Miyake T, Kato Y 1995 Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates growth hormone release from GH3 cells through type II PACAP receptor. Regul Pept 56:35–40
Xiao D, Chu MM, Lee EK, Lin HR, Wong AOL 2002 Regulation of growth hormone release in common carp pituitary cells by pituitary adenylate cyclase-activating polypeptide: signal transduction involves cAMP- and calcium-dependent mechanisms. Neuroendocrinology 76:325–338
Peter RE, Yu KL, Marchant TA, Rosenblum PM 1990 Direct neural regulation of the teleost adenohypophysis. J Exp Zool 4(Suppl):84–98
Montero M, Yon L, Rousseau K, Arimura A, Fournier A, Dufour S, Vaudry H 1998 Distribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla. Endocrinology 139:4300–4310
Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy DH, Kitada C 1991 Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 129:2787–2799
Shuto Y, Somogyvari-Vigh A, Vigh S, Wakabayashi I, Arimura A 1996 Effect of pituitary adenylate cyclase-activating polypeptide on GH gene expression in rat pituitary cells. Ann NY Acad Sci 805:684–691
Alarcon P, Garcia-Sancho J 2000 Differential calcium responses to the pituitary adenylate cyclase-activating polypeptide (PACAP) in the five main cell types of rat anterior pituitary. Pflugers Arch 440:685–691
Martinez-Fuentes AJ, Castano JP, Malagon MM, Vazquez-Martinez R, Gracia-Navarro F 1998 Pituitary adenylate cyclase-activating polypeptides 38 and 27 increase cytosolic free Ca2+ concentration in porcine somatotropes through common and distinct mechanisms. Cell Calcium 23:369–378
Chad J, Kalman D, Armstrong D 1987 The role of cyclic AMP-dependent phosphorylation in the maintenance and modulation of voltage-activated calcium channels. Soc Gen Physiol Ser 42:167–186
Mikala G, Klockner U, Varadi M, Eisfeld J, Schwartz A, Varadi G 1998 cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels. Mol Cell Biochem 185:95–109
Bunemann M, Gerhardstein BL, Gao T, Hosey MM 1999 Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the 2 subunit. J Biol Chem 274:33851–33854
Koshimura K, Murakami Y, Mitsushima M, Hori T, Kato Y 1997 Activation of Na+ channels in GH3 cells and human pituitary adenoma cells by PACAP. Peptides 18:877–883
Braas KM, Rossignol TM, Girard BM, May V, Parsons RL 2004 Pituitary adenylate cyclase activating polypeptide (PACAP) decreases neuronal somatostatin immunoreactivity in cultured guinea-pig parasympathetic cardiac ganglia. Neuroscience 126:335–346
Akiyama M, Minami Y, Nakajima T, Moriya T, Shibata S 2001 Calcium and pituitary adenylate cyclase-activating polypeptide induced expression of circadian clock gene mPer1 in the mouse cerebellar granule cell culture. J Neurochem 78:499–508
Persechini A, Stemmer PM 2002 Calmodulin is a limiting factor in the cell. Trends Cardiovasc Med 12:32–37
Tran QK, Black DJ, Persechini A 2003 Intracellular coupling via limiting calmodulin. J Biol Chem 278:24247–24250
Nielsen DA, Shapiro DJ 1990 Insights into hormonal control of messenger RNA stability. Mol Endocrinol 4:953–957
Beyersmann D 2000 Regulation of mammalian gene expression. Exs 89:11–28
Impey S, Goodman RH 2001 CREB signaling: timing is everything. Sci STKE 82:PE1–PE4
Maronde E, Schomerus C, Stehle JH, Korf HW 1997 Control of CREB phosphorylation and its role for induction of melatonin synthesis in rat pinealocytes. Biol Cell 89:505–511
Minami Y, Furuno K, Akiyama M, Moriya T, Shibata S 2002 Pituitary adenylate cyclase-activating polypeptide produces a phase shift associated with induction of mPer expression in the mouse suprachiasmatic nucleus. Neuroscience 113:37–45
von Gall C, Duffield GE, Hastings MH, Kopp MD, Dehghani F, Korf HW, Stehle JH 1998 CREB in the mouse SCN: a molecular interface coding the phase-adjusting stimuli light, glutamate, PACAP, and melatonin for clockwork access. J Neurosci 18:10389–10397
Shepard AR, Zhang W, Eberhardt NL 1994 Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells. J Biol Chem 269:1804–1814
Ho WK, Wong MW, Chan AP 1991 Cloning and sequencing of the grass carp (Ctenopharyngodon idellus) growth hormone gene. Biochim Biophys Acta 1090:245–248(Anderson O. L. Wong, Wensheng Li1, Ching)