Prolactin-Regulated Tyrosine Hydroxylase Activity and Messenger Ribonucleic Acid Expression in Mediobasal Hypothalamic Cultures: The Differe
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内分泌学杂志 2005年第1期
The Centre for Neuroendocrinology (Y.F.M., D.R.G., S.J.B), Department of Anatomy and Structural Biology, The University of Otago, Dunedin 9001, New Zealand; and Institut National de la Santé et de la Recherche Médicale (V.G.), U-584, Molecular Endocrinology, Faculté de Medécine Necker, 75730 Paris, France
Address all correspondence and requests for reprints to: Stephen Bunn, The Centre for Neuroendocrinology, Department of Anatomy and Structural Biology, The University of Otago, P.O. Box 913, Dunedin 9001, New Zealand. E-mail: stephen.bunn@stonebow.otago.ac.nz.
Abstract
Prolactin secretion from the anterior pituitary is tightly regulated by feedback onto the hypothalamic neuroendocrine dopaminergic (NEDA) neurons. Prolactin stimulates these neurons to synthesize and secrete dopamine, which acts via the pituitary portal vasculature to inhibit prolactin secretion from the pituitary lactotrophs. Despite the physiological importance of this feedback, relatively little is known about the signaling mechanisms responsible for prolactin activation of NEDA neurons. This issue has been examined here using a cell culture preparation of the fetal rat mediobasal hypothalamus. Prolactin stimulated a time- and concentration-dependent increase in catecholamine synthesis, which was maximal after 60–120 min (1 μg/ml prolactin) and inhibited by the prolactin antagonist 1–9-G129R-hPRL. This prolactin response was accompanied by a rise in the site-specific (ser-19, -31, and -40) phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Consistent with this observation, the prolactin-induced increase in catecholamine synthesis was abolished by inhibitors of protein kinase A and protein kinase C (PKC). Prolactin incubation also resulted in a PKC-dependent activation of the MAPK pathway, although this was not required for the stimulation of catecholamine synthesis. In addition to increasing TH phosphorylation and catecholamine synthesis, prolactin also increased TH mRNA expression. In contrast to catecholamine synthesis, this latter response was not suppressed by inhibition of protein kinase A or PKC. These results indicate that although prolactin controls catecholamine synthesis in NEDA neurons by regulating both TH activity and TH mRNA expression, it employs distinct, nonoverlapping, signaling pathways to achieve these ends.
Introduction
PROLACTIN IS A HORMONE of major physiological importance with many actions in addition to its well-described role in lactation (1, 2). The secretion of prolactin from the anterior pituitary lactotrophs is subject to a number of regulatory factors, the most important being an inhibitory input from dopamine carried in the hypophysial portal system (1). Dopamine found within these vessels is synthesized and secreted by the neuroendocrine dopaminergic (NEDA) neurons located in the arcuate and periventricular nuclei of the mediobasal hypothalamus. The NEDA neurons are present as three distinct groups, referred to as the tuberoinfundibular, tuberohypophyseal, and periventricular-hypophyseal dopaminergic neurons. The relative contribution of each of these NEDA populations to the control of prolactin secretion is not fully resolved, although the tuberoinfundibular dopaminergic (TIDA) neurons, which deliver dopamine directly into the median eminence, appear to be particularly important (1). Circulating prolactin stimulates the activity of all three populations of NEDA neurons, thus increasing dopamine release and ultimately suppressing its own secretion from the anterior pituitary (3). Whereas this short-loop feedback pathway is well established, the cellular mechanisms by which prolactin stimulates NEDA neuron activity remains to be resolved.
Prolactin receptors have been localized within the hypothalamus including the arcuate nucleus and median eminence and have been identified colocalized on TIDA neurons (4, 5). Isolated dopaminergic neurons from the fetal rat hypothalamus have also been shown to express prolactin receptors (6). When maintained in culture, these neurons respond to prolactin receptor activation by increasing their rate of dopamine synthesis (6). Thus, it would appear likely that TIDA, and perhaps other NEDA neurons, are directly responsive to prolactin. As noted above, the cellular mechanisms underlying such a response are not fully resolved. Whereas data from signal transducer and activator of transcription-5b-deficient mice suggest that this transcription factor is required for prolactin-induced activation of these neurons (7), other studies indicate that prolactin may also exert nontranscription regulation of dopamine synthesis and release. In vivo studies suggest that the TIDA neuron response to prolactin is relatively rapid (8) with the initial increase in dopamine synthesis being independent of transcription and translation, followed by a delayed (12 h) further increase, which was sensitive to cycloheximide inhibition (9). In vitro studies using rat hypothalamic slices have also reported a relatively rapid (2 h) prolactin-induced increase in dopamine synthesis (10), whereas studies using hypothalamic fetal cells in culture reported the need for long-term prolactin exposure (1–10 d) to significantly increase dopamine levels (6).
The rate of dopamine synthesis in TIDA and other dopaminergic neurons is controlled by the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis (11, 12). TH is subject to many regulatory inputs both chronically at the level of gene transcription and translation and acutely at the level of the enzyme activity. TH activity is tightly regulated by end-product inhibition, in that dopamine suppresses its activity by competing with the binding of the tetrahydrobiopterin BH4 cofactor (12). Phosphorylation of TH, within its N-terminal regulatory domain, reduces the affinity of dopamine binding, thus relieving the inhibition and elevating TH activity. Physiologically relevant regulation of TH through its phosphorylation has been well described in a number of tissues and appears to involve three specific serine residues, Ser-19, -31, and -40 (13). The protein kinases responsible for the phosphorylation of each of these residues and their precise roles in the activation of the enzyme are still relatively unclear. At present, however, it is probable that Ser-19 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), MAPK-activated protein kinase (MAPKAPK) 2, and p38-regulated/activated kinase (14, 15, 16). Ser-31 appears to be exclusively phosphorylated by the MAPKSs ERK1/2 (17). In contrast, Ser-40 may be phosphorylated by a number of protein kinases including protein kinase A (PKA), protein kinase C (PKC), protein kinase G, CaMKII, MAPKAPK1/2, and the mitogen- and stress-activated protein kinase 1 (11, 16).
Despite its physiological importance, our understanding of the intracellular pathways by which prolactin receptor activation couples to either TH expression or TH phosphorylation is still incomplete. The aim of this current study was therefore to conduct a detailed investigation into the kinase pathways activated in TIDA neurons by prolactin and determine their influence on the level of dopamine synthesis. To achieve this aim, we employed cell cultures prepared from the mediobasal hypothalamus of the embryonic rat pup and determined the effect of protein kinase inhibitors on the ability of prolactin to increase both TH activity (as reflected in the level of catecholamine synthesis) and TH mRNA expression.
Materials and Methods
Hypothalamic cell cultures
Cultures of the rat mediobasal hypothalamus were prepared using the method developed by Arbogast and Voogt (6) modified as described previously (18). All animal procedures were approved by the University of Otago Animal Ethics Committee. Briefly, mediobasal hypothalami from 28–56 embryonic d 18–21 fetal rats were excised to a depth of approximately 1 mm, limited anteriorly by optic chiasm, posteriorly by the border of the mamillary bodies, and laterally by the hypothalamic fissures. Tissue blocks were pooled, rinsed with buffer (137 mM NaCl, 5.4 mM KCl, 59 mM sucrose, 0.2 mM Na2HPO4, 0.2 mM KH2PO4, 5.5 mM glucose, 100,000 U/liter penicillin, 100 mg/liter streptomycin, and 250 μg/liter Fungizone adjusted to pH 7.4), and roughly chopped. Tissue fragments were then incubated with 10 ml trypsin (2.5 mg/ml in the same buffer) at 37 C for 5 min at which time 1 ml DNase solution (1 mg/ml) was added and the incubation continued for a further 5 min. The digestion was terminated by the addition of 12 ml of soybean trypsin inhibitor solution (0.3 mg/ml) followed by centrifugation at 800 x g for 5 min. The resultant pellet was resuspended, washed with buffer, and again centrifuged at 800 x g for 5 min before being dispersed at a density of approximately 106 cells/ml in DMEM (Life Technologies, Inc., Rockville, MD) containing 2.5% fetal bovine serum and 5% horse serum. Cells were plated out at 1 ml/well (106 cells/well) onto poly-D-lysine-coated 24-well culture plates or for immunocytochemical investigations poly-D-lysine-coated glass coverslips in 24-well culture plates. Plated cells were then transferred to 5% CO2 humidified incubator at 37 C. After the first 24 h in culture, the medium was removed and replaced with a serum- and phenol red-free DMEM containing high glucose and KCl (each at 25 mM) and the following reagents (each obtained from Sigma, St. Louis, MO): linolenic acid (1 μg/ml), linoleic acid (1.5 μg/ml), putrescine (4.4 μg/ml), and ITS+ (insulin-transferrin-sodium selenite supplement) (1:100 dilution). The medium was replaced every second day and the cells routinely used for experimentation after 14–21 d in culture.
Immunocytochemistry
Cells cultured on glass coverslips were removed from the incubator, washed twice (5 min each) with 1 ml of 10 mM PBS, fixed with 2% formaldehyde in PBS for 20 min at room temperature, and then permeabilized with 90% ethanol/10% acetic acid (5 min at –20 C). Cells were then washed three times for 5 min each in 1 ml PBS followed by 1 h in PBS containing 5% goat serum and then 48 h at 4 C with a combination of both primary antibodies, polyclonal anti-TH (AB151 diluted 1:200, from Chemicon, Temecula, CA) and monoclonal antiprolactin receptor (rat) (MA1–610 diluted 1:100, from Affinity Bioreagents, Golden, CO) made up in PBS containing 5% goat serum. The cells were then washed (6 x 5 min in PBS) and incubated with a combination of secondary antibodies antimouse Alexa488 and antirabbit Alexa568 both diluted 1:1000 in PBS (Molecular Probes, Eugene, OR) for 30 min at room temperature. Coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined under fluorescence microscopy using an AX70 microscope (Olympus, Tokyo, Japan) fitted with a Spot-RT color digital camera (Diagnostic Instruments Inc., Sterling Heights, MI).
Catecholamine synthesis
Catecholamine synthesis was measured using the method originally developed for adrenal chromaffin cells (19) modified as described previously (18). Cells were removed from the incubator and washed briefly (twice for 5 min) with 400 μl carbogen-gassed HEPES-buffered saline (HBS) of the following composition: 150 mM NaCl, 15 mM HEPES, 5.5 mM glucose, 3.8 mM K2HPO4, 1 mM MgSO4, 1.5 mM CaCl2, and 0.5 mM sodium ascorbate, adjusted to pH 7.4 at 37 C. The last wash was replaced with 200 μl HBS containing 20 μM L-[carboxyl-14C] tyrosine (0.22 μCi per well, purchased from Amersham Biosciences NZ Ltd. Auckland, New Zealand) in the presence or absence of a stimulating agent, routinely 1 μg/ml prolactin (ovine prolactin obtained from Sigma). Each well was immediately fitted with a tube sealed with a rubber stopper from which was suspended a small plastic cap containing 200 μl of 1 M NaOH to absorb the 14CO2 produced by the cells during the decarboxylation step in catecholamine synthesis. The incubation was terminated by the injection of 400 μl of ice-cold 10% trichloroacetic acid through the rubber stopper. The plates, with their sealed wells in place, were then kept at 4 C for 2 h before an aliquot of the NaOH solution was collected and the 14C radioactivity measured by liquid scintillation spectrometry. When examining the effect of prolactin antagonist or protein kinase inhibitors, these agents were included during both a 15-min preincubation period and the subsequent stimulation. PD-98059, KN-92, KN-93, and H89 were purchased from Sigma; H85 from Seikagaku (Tokyo, Japan); and bisindomaleimide I and V, actinomycin D, and cycloheximide from Calbiochem (San Diego, CA). Recombinant prolactin receptor antagonist 1–9-G129R-hPRL was prepared and purified as described previously (20). Catecholamine synthesis was expressed relative to basal levels (cells incubated with buffer alone for the same time period) or, where appropriate, a control value obtained from cells incubated in the absence of an antagonist and statistical comparisons performed using a Mann-Whitney U test (unless otherwise stated).
TH mRNA expression
Cells were removed from the incubator and briefly washed with 500 μl serum-free DMEM and then returned to the incubator in a further 500 μl serum-free DMEM with or without prolactin (1 μg/ml) in the presence or absence of an appropriate protein kinase inhibitor. After 4 h incubation, each well was extracted into 200 μl TRIzol reagent and total RNA isolated as per manufacturer’s instructions. The resultant RNA was reverse transcribed using GeneAmp Gold RNA PCR kit (PE Applied Biosystems, Foster City, CA) and quantitative real-time RT-PCR for TH and ?-actin performed using the Taqman system (PE Applied Biosystems). Primer and probe details were exactly as described previously (18). The reaction mix was also prepared as previously described and an ABI PRISM 7700 sequence detection system (Centre for Gene Research, University of Otago) used to detect fluorescence during each PCR under conditions exactly as described previously (18). Data were captured and analyzed using sequence detector software (SDS, version 3.0, PE Applied Biosystems). TH mRNA levels in each sample were normalized with reference to its ?-actin mRNA levels and then expressed as relative to TH mRNA in control cells (i.e. those incubated with prolactin in the absence of kinase inhibitors) determined in the same experiment. Statistical analysis was performed using a Mann-Whitney U test.
Reverse-transcribed RNA extracted from cells was also probed for the long and short forms of the prolactin receptor. The forward primer for the prolactin receptor short and long form was 5'-ATACTG GAG TAG ATG GAG CCA GGA GAG TTC-3' corresponding to nucleotides 624–653 of the prolactin receptor cDNA sequence. The reverse primer for the short form was 5'-TCC TAT TTG AGT CTG CAG CTT CAG TAG TCA-3' corresponding to nucleotides 924–953 of the cDNA sequence, and the reverse primer for the long form was 5'-CTT CCG TGA CCA GAG TCA CTG TCG GGA TCT-3' corresponding to nucleotides 1014–1043 of the cDNA sequence (21). The cDNA was amplified through 30 PCR cycles, the products resolved on 1.5% agarose gels, and visualized with ethidium bromide staining.
Activation of ERK1/2
The phosphorylation of ERK1/2 was measured by immunoblotting using activation state-specific antibodies. Cells were washed twice for 5 min with 400 μl HBS and then preincubated for 15 min with PD98059 (50 μM) or bisindolylmaleimide I (3 μM) before being stimulated for 15 min with or without prolactin (1 μg/ml) in the continued presence or absence of the appropriate inhibitor. The cells were then lysed in ice-cold 10 mM Tris buffer (three wells collected in a total of 200 μl) containing 5 mM EDTA, 50 mM NaF, 50 mM NaCl, 1% Triton-X100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 μl /ml of protease inhibitor cocktail (Sigma). The lysate was sonicated, centrifuged at 12,000 x g for 10 min and the resultant supernatant fractionated by SDS-PAGE and transferred onto nitrocellulose membranes. These membranes were probed overnight at 4 C using a phospho-p44/42 MAPK (Thr202/Tyr204)-specific monoclonal antibody (at 1:1000 dilution, Cell Signaling Technology, Beverly, MA). The membranes were then washed with 10 mM Tris-buffered saline containing 0.05% Tween 20 and reincubated with horseradish peroxidase-coupled anti-IgG antibody (1:5000 dilution for 60 min at room temperature), washed again, and the image developed using enhanced chemiluminescence. Relative levels of phosphorylation of ERK1/2 were determined using densitometric image analysis (National Institutes of Health Image) and expressed as a percentage of that obtained from cells incubated with buffer alone and examined on the same immunoblot. Statistical analysis was performed using a Mann-Whitney U test.
TH phosphorylation
Cells were washed and stimulated with or without prolactin (1 μg/ml for 120 min), extracted in lysis buffer, and processed for immunoblotting as described above. Blots were probed overnight at 4 C using antibodies raised against TH (AB151 diluted 1:200, Chemicon) or specific TH phosphorylation sites (ser-19, ser-31, or ser-40; 1:10,000, kindly provided by Prof. Peter Dunkley, University of Newcastle, New South Wales, Australia) and then washed and labeled with secondary antibodies as described above. The density of immunoreactive bands was visualized and quantitated on the Typhoon system (Molecular Dynamics, Sunnyvale, CA) and expressed as a percentage of those detected in cells incubated with buffer alone and examined on the same immunoblot. Details regarding the generation and characterization of these antibodies were described by Cammarota et al. (22).
Results
Cultures prepared from the fetal rat mediobasal hypothalamus contained a population of TH immunoreactive cells. Cell counts indicated that these TH-positive cells constituted approximately 7 ± 1% of the total cell population (determined from six separate cell culture preparations). Some, although not all, of these TH-positive cells also expressed prolactin receptors, as shown by immunocytochemistry (Fig. 1A). It should be noted that prolactin receptor expression was not limited to TH-positive cells, in that other unidentified cell types were also immunoreactive for this antibody (Fig. 1A). RT-PCR performed on extracts of these cultures indicated that the long, rather than short, form of prolactin receptor was the predominant form expressed in these cultures (Fig. 1B).
FIG. 1. The expression of prolactin receptors on TH immunoreactive neurons in mediobasal hypothalamic cultures. A, Cell cultures were prepared as described in Materials and Methods and then dual labeled with antibodies directed against TH (left panel) and prolactin receptors (right panel). Note that prolactin receptor immunoreactivity was present on, but not limited to, cells that were also immunoreactive for TH (a non-TH-immunoreactive cell indicated by arrow, immediately adjacent to a TH-positive cell). B, Mediobasal hypothalamic cell cultures were prepared and maintained as described in Materials and Methods. After 14 d in culture, total mRNA was extracted and mRNA for ?-actin (lane 1), short-form prolactin receptor (lane 2), long-form prolactin receptor (lane 3), TH (lane 4), and short and long forms of the prolactin in choroid plexus (lane 5 and 6, respectively) detected by RT-PCR as described in Materials and Methods.
Incubation with prolactin stimulated a time- and concentration-dependent increase in catecholamine synthesis, presumably reflecting an increase in TH activity (Fig. 2, A and B). This response was slow and fairly modest but highly consistent with a statistically significant increase in catecholamine production (120% of the basal response) seen after a 60-min incubation with prolactin (1 μg/ml). Extending the incubation period to 120 min did not further increase catecholamine synthesis (Fig. 2A). A prolactin-stimulated increase in catecholamine synthesis was evident with concentrations of prolactin as low as 10 ng/ml and was concentration dependent, although no apparent maximum was reached with concentrations up to 1 μg/ml (Fig. 2B). The response to this highest concentration of prolactin was inhibited by the prolactin antagonist, 1–9-G129R-hPRL (10 μg/ml), although it should be noted that catecholamine production in these cells was still significantly higher than that in cells incubated with the antagonist alone (Fig. 2B). The inclusion of serum (2.5% fetal calf serum and 5% horse serum) in the culture medium did not alter responsiveness to prolactin, with a 120-min incubation with prolactin (1 μg/ml), resulting in a 119 ± 5% basal response in the presence of serum, compared with 119 ± 3% basal in its absence (n = 3 from separate cell culture preparations).
FIG. 2. Prolactin stimulates a time- and concentration-dependent increase in catecholamine synthesis. Cells were incubated with or without prolactin (1 μg/ml) for 15–120 min (A) or increasing concentrations of prolactin for 120 min (B). The presence of the prolactin antagonist 1–9-G129R-hPRL (10 μg/ml) is indicated by the dark shaded columns and resulted in a significant suppression of prolactin-induced catecholamine synthesis. Catecholamine synthesis was determined as described in Materials and Methods and expressed as a percentage of the response obtained under basal conditions for the same time period. Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01, compared with the response in cells incubated for the same time period with buffer alone; #, P < 0.01, compared with the matched prolactin response in the absence of antagonist; +, P < 0.05, compared with the basal response in the presence of the prolactin antagonist (Mann-Whitney U test, n = 6–12 from three to four separate cell culture preparations).
It is well recognized that TH activity, and thus the rate of catecholamine synthesis, can be regulated by the phosphorylation of specific serine residues located at the N terminal of the enzyme. To determine whether this was the mechanism underlying prolactin’s action on these hypothalamic neurons, the effect of selected protein kinase inhibitors was determined. Incubation with H89 (3 μM), an inhibitor of PKA, resulted in a small but significant decrease in the basal catecholamine synthesis measured over 120 min (Fig. 3A). This inhibitor also markedly reduced the prolactin-induced increase in this response activity, such that it was no longer significantly greater than the basal activity over this time period (Fig. 3A). In contrast, the same concentration of H85, an inactive analog of H89, was without effect on either the basal or prolactin-induced response (Fig. 3A). Bisindolymaleimide I (3 μM), a PKC antagonist, also fully inhibited the prolactin-induced increase in catecholamine synthesis but unlike H89 had no significant effect on the basal response (Fig. 3B). Bisindolymaleimide V, an inactive analog of bisindolymaleimide I, was without effect on either basal or prolactin-stimulated activity (Fig. 3B). KN-93 (3 μM), which is known to inhibit Ca2+-dependent protein kinases including CaMKII, had no effect on basal catecholamine synthesis but caused a partial inhibition (about 50%) of the prolactin-induced response (Fig. 3C). It is important to note, however, that KN-92, an inactive analog of KN-93, also significantly suppressed the prolactin-stimulated response, albeit to a lesser extent than KN-93 (Fig. 3C).
FIG. 3. The effect of selected protein kinase inhibitors on prolactin-stimulated catecholamine synthesis in mediobasal hypothalamic cultures. Cell cultures were preincubated with or without (open columns) selected protein kinase inhibitors (all at 3 μM), H89 (dark columns) or H85 (light columns) (A), bisindolymaleimide I (dark columns) or bisindolymaleimide VI (light columns) (B), or KN-93 (dark columns) or KN-92 (light columns) (C), for 15 min and then incubated for a further 120 min with or without prolactin (1 μg/ml) in the continued presence or absence of the appropriate protein kinase inhibitor. Catecholamine synthesis was measured as described in Materials and Methods and expressed as a percentage of the basal response in the absence of inhibitors. Data are presented as the mean ± SEM (Mann-Whitney U test, n = 9–18 from three to six separate cell preparations). *, P < 0.001, compared with matched basal response; #, P < 0.05, compared with control basal; ##, P < 0.01, compared with control prolactin response.
The possible involvement of MAPKs in the prolactin-induced increase in catecholamine synthesis in these cells was first investigated by determining whether the MAPKs ERK1/2 were activated in response to prolactin. As depicted in Fig. 4A, incubation with prolactin (1 μg/ml for 15 min) resulted in an increased phosphorylation and thus activation of ERK1 (Fig. 4A). Phosphorylation of ERK1 was abolished by both the MAPK/ERK kinase (MEK) inhibitor PD98059 (50 μM) and the PKC inhibitor bisindolymaleimide I (3 μM) (Fig. 4A). Interestingly, inhibition of MEK, but not PKC, resulted in a significant decrease in ERK1 phosphorylation seen under basal conditions. The effect of prolactin and the above inhibitors on ERK2 phosphorylation was essentially identical with that on ERK1 (data not shown). The level of TH expression detected on these Western blots was not significantly altered after stimulation with prolactin at 115.7 ± 16% basal levels (P > 0.05 using a Wilcoxon sign-ranks test). In agreement with its action on ERK phosphorylation, incubation with PD98059 (50 μM) also caused a marked reduction in the basal rate of catecholamine synthesis (Fig. 4B). In contrast, whereas the overall rate of catecholamine synthesis in the presence of prolactin was reduced in the presence of PD98059, this apparent inhibition was entirely due to the decreased basal activity, with the prolactin response being 122% of basal in the absence of the inhibitor, compared with 123% of basal in its presence (Fig. 4B). Thus, whereas PKC appears to be involved in both prolactin-induced ERK1/2 activation and prolactin-induced TH activity, the ERK1/2 pathway is not itself required for the stimulation of catecholamine synthesis. To examine this proposal further, the effect of PD98059 on the ability of phorbol-12-myristate-13-acetate (PMA), a PKC activator, to stimulate catecholamine synthesis was determined. As described previously (18), PMA (100 nM for 60 min) significantly increased the level of catecholamine synthesis to about 115% of basal levels (Fig. 4C). The inclusion of PD98059 caused a concentration-dependent decrease in the net response to PMA, but, as with prolactin, this resulted from a parallel concentration-dependent decrease in basal activity (Fig. 4C).
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FIG. 4. Prolactin activates the ERK1/2 pathway in mediobasal hypothalamic cell cultures. A, Cells were preincubated with or without (open columns) PD98059 (50 μM, filled columns) or bisindolymaleimide I (3 μM, light columns) for 15 min and then for a further 15 min with or without prolactin (1 μg/ml) in the continued presence or absence of the appropriate inhibitor. Cells were lysed and phosphorylated ERK1/2 levels determined as described in Materials and Methods. Data are presented as a percentage of phospho-ERK1 levels measured under control basal conditions, determined on the same immunoblot. *, P < 0.001 and #, P < 0.05, compared with control basal levels (Mann-Whitney U test, n = 3–6 from independent experiments performed on separate cell culture preparation). Inserts present representative phospho-ERK immunoblots positioned above the appropriate columns. B, Cells were preincubated with or without (open columns) PD98059 (50 μM, filled columns) for 15 min followed by a further 120 min with or without prolactin (1 μg/ml) in the continuing presence or absence of the inhibitor. Catecholamine synthesis was measured as described in Materials and Methods and expressed as a percentage of the basal response in the absence of inhibitor. Data are presented as the mean ± SEM. *, P < 0.001, compared with the control basal response; #, P < 0.001, compared with the basal response in the presence of PD98059 (Mann-Whitney U test, n = 9 from three separate cell preparations). C, Cells were preincubated with increasing concentrations of PD98059 for 15 min and then for a further 60 min with (shaded columns) or without (open columns) PMA (100 nM) in the continued presence or absence of the inhibitor. Catecholamine synthesis was measured as described in Materials and Methods and expressed as a percentage of the basal response in the absence of inhibitor. Data are presented as the mean ± SEM. *, P < 0.01, compared with the matched basal response; #, P < 0.05, compared with the appropriate response in the absence of PD98059 (ANOVA, n = 3).
The involvement of multiple protein kinases in this prolactin-stimulated response was supported by the finding that TH protein extracted from these cells was phosphorylated on multiple serine residues. As presented in Fig. 5, the use of phosphorylation site-specific antibodies revealed that prolactin (1 μg/ml for 60 min) stimulated a significant increase in phosphorylation of ser-19, -31, and -40 residues when compared with cells incubated under basal condition. Whereas the increase in ser-40 phosphorylation appears to be larger than the other two sites, this apparent difference was not statistically significant. The level of total TH expression detected on these Western blots was not significantly altered after stimulation with prolactin at 110.7 ± 9.5% basal levels (P > 0.05 using Wilcoxon sign-ranks test on the original densitometric data).
FIG. 5. Prolactin increases the phosphorylation of specific serine residues on TH from mediobasal hypothalamic cultures. Cells were incubated with (shaded columns) or without (open columns) prolactin (1 μg/ml) for 60 min. After incubation cells were lysed and the phosphorylation of ser-19, -31, and -40 residues of TH determined using phosphorylation site-specific antibodies as described in Materials and Methods. Data are expressed as a percentage of the site-specific phosphorylation measured in cells incubated with buffer alone and represent the mean ± SEM (*, P < 0.01, compared with the relevant basal response; Mann-Whitney U test, n = 4 independent experiments performed on separate cell culture preparations). Inserts present representative phospho-TH immunoblot positions above the appropriate columns.
In addition to increasing TH activity (as reflected by a rise in catecholamine synthesis), incubation with prolactin significantly increased the level of TH mRNA expression after 240 min incubation (Fig. 6A). In contrast to the prolactin-induced increase in catecholamine synthesis, this rise in TH mRNA was not inhibited by H89, bisindolymaleimide I, KN-93, or PD98059 (Fig. 6A). Interestingly, inhibition of transcription or translation with actinomycin D (1 μM) or cyclohexamide (1 μM) decreased basal catecholamine synthesis but failed to suppress the stimulated response to prolactin (Fig. 6B).
FIG. 6. Prolactin increases TH mRNA levels in mediobasal hypothalamic cultures. A, Cells were incubated for 4 h with or without prolactin (1 μg/ml) in the presence (shaded columns) or absence (open columns) of the indicated protein kinase inhibitor, H89 (10 μM), KN-93 (3 μM), bisindolymaleimide I (BisM, 3 μM), or PD98059 (3 μM). Cells were then extracted and TH mRNA levels measured using real-time RT-PCR as described in Materials and Methods. Data are expressed as a percentage of the level detected in cells stimulated with prolactin in the absence of kinase inhibitors and are presented as means ± SEM. *, P < 0.05, compared with cells incubated under basal condition in the presence of appropriate kinase inhibitor (Mann-Whitney U test, n = 3–6 from separate cell culture preparations). B, Cells were preincubated for 15 min with or without actinomycin D (1 μM, dark columns) or cycloheximide (1 μM, light columns) and then for a further 120 min with or without prolactin (1 μg/ml) in the continued presence of the appropriate inhibitor. Catecholamine synthesis was determined as described in Materials and Methods and is expressed as a percentage of that detected in cells incubated under basal condition in the absence of inhibitor. *, P < 0.01, compared with activity in cells incubated under matched basal conditions; #, P < 0.05, compared with cells incubated with buffer alone (Mann-Whitney U test, n = 4 from separate cell culture preparations).
Discussion
The primary aim of this project was to investigate the intracellular mechanisms by which prolactin activity regulates dopamine synthesis in NEDA neurons. As outlined in the introductory text, the key regulatory step in this pathway is the activity of TH. The experimental approach used here to examine TH activity in cultured mediobasal hypothalamic neurons was based on the liberation of 14CO2 from cells loaded with 14C-tyrosine. TH converts the 14C-tyrosine to 14C-DOPA with is then decarboxylated by aromatic acid decarboxylase to dopamine, releasing 14CO2. Thus, whereas the assay is dependent on the activity of both enzymes, the latter is not rate limiting and has been shown to be relatively insensitive to acute regulation (23). Thus, the rate of 14CO2 accumulation provides a sensitive and reliable index of in situ TH activity (19). Whereas the ability to measure cellular dopamine synthesis in the intact living cell has clear advantages, there are also limitations in terms of identifying the locus of an action. TH activity may, for example, be sensitive to multiple factors in addition to its phosphorylation status, including the rate of tyrosine uptake and cofactor availability, which may themselves be influenced by receptor stimulation or protein kinase activity.
Preliminary immunocyotochemical studies confirmed earlier reports that prolactin receptors are expressed on TH immunoreactive cells in these cultures (6). Whereas not fully quantitated, our observations suggest that prolactin receptors are present on most of the TH-expressing cells in these cultures. Prolactin receptor immunoreactivity was also evident on a population of non-TH expressing cells. The identity of these cells was not investigated here, but an earlier study suggested that they were neuronal rather than glial in origin (6). Whereas most TH-immunoreactive cells express prolactin receptors and are thus are presumably capable of responding directly to prolactin, other non-TH immunoreactive cells could also respond to prolactin and thereby potentially mediate a secondary, indirect, action on the TH-positive neurons. RT-PCR examination suggested that regardless of specific cellular location, the vast majority of prolactin receptor expression in these hypothalamic cultures was of the long, rather than short form of the receptor, an observation in agreement with in vivo studies (24, 25). It should be noted, however, that the short-form primers were able to detect the short-form prolactin receptor in choroid plexus (Fig. 1B) and a number of other rat brain regions (21).
The concentration of prolactin required to stimulate a significant increase in catecholamine synthesis in these cultures is in excellent agreement with those reported previously for more chronic prolactin actions on similar cultures (6, 10, 26). The majority of experiments reported here have used a prolactin concentration of 1 μg/ml, which gave a reliable and relatively robust response. This concentration is a little higher than the high end of the physiological range, such as during the preovulatory surge but less than concentrations reported in the portal blood (27). The specificity of prolactin action in these experiments is supported by the inhibition observed using the prolactin antagonist 1–9-G129R-hPRL. This prolactin analog acts as a pure competitive antagonist at the prolactin receptor, and similar concentrations of this or related analogs (20, 28, 29) have been shown to inhibit a variety of prolactin-induced responses in other cells and tissues. The partial inhibition of prolactin response observed in the current studies reflects the fact that the antagonist has a 10-fold lower affinity for the prolactin receptor than wild-type human prolactin (22).
It should be noted that the culture preparations used in the current study have been maintained in a serum-free environment for at least 14 d before stimulation with prolactin. In the earlier studies mentioned above (6), cells were found to be unresponsive to prolactin unless maintained in the presence of serum. The reason for this difference, or indeed the explanation underlying the need for serum in the earlier studies, is unknown, but the ability to maintain prolactin-responsive cells in a defined culture environment is clearly an experimental advantage. Thus, in the absence of postnatal exposure to prolactin, circulating steroids, or other serum-derived factors, TH-containing cells from the mediobasal hypothalamus of the rat fetus appear capable of expressing functional prolactin receptors.
The ability of prolactin to stimulate a relatively rapid increase in catecholamine synthesis in these cultures strongly suggests that it is promoting the phosphorylation and thus activation of TH. As outlined in the introductory text, TH in other cell types can be phosphorylated on three specific serine residues, ser-19, -31, and -40, through a variety of protein kinases (11, 12). In many cases this phosphorylation leads to a rise in catalytic activity of TH. Previous in vivo studies strongly suggested that protein kinases play a key role in regulating TH activity in the arcuate nucleus and median eminence (10, 30, 31), although the pathway by which prolactin mediates these actions is not well documented. The data presented in Fig. 3a suggests that PKA activity may be required for prolactin stimulation of TH. The increase in catecholamine synthesis induced by prolactin was abolished by H89 and unaffected by its inactive analog H85. Previous studies demonstrated that increased cAMP levels stimulate the rate of catecholamine synthesis in both hypothalamic slices and cultures (30, 32).
Given that ser-40 phosphorylation of TH leads to its activation (11, 13) and that prolactin increases the phosphorylation of this residue (Fig. 5), it is tempting to suggest that prolactin achieves this activation via a PKA-mediated pathway. Caution should be taken, however, before reaching this conclusion. There is little evidence that prolactin receptors are coupled to adenylyl cyclase, although it is possible that this enzyme could be activated secondarily to a prolactin-induced increase in intracellular Ca2+ (33, 34). More importantly, however, we and others have shown that H89 inhibits TH activation by other agents that are unlikely to act via PKA. The PMA-induced increase in catecholamine synthesis in these hypothalamic cultures, for example, is abolished by H89 (18). Similarly, TH activity in adrenal medullary chromaffin cells is inhibited by H89, even when this appears to involve little or no stimulation of PKA (35). Thus, whereas it can be reasonably concluded that PKA activity, or at least an H89-sensitive event, is required for the activation of TH in NEDA neurons, its precise role in the prolactin-driven response remains somewhat equivocal. As outlined in the introductory text, TH ser-40 is the substrate for a number of protein kinases in addition to PKA including CaMKII, PKC, and MAPKAPKs downstream of ERK1/2. It is possible that one or more of these kinases is responsible for prolactin stimulation of catecholamine biosynthesis.
In addition to increasing TH ser-40 phosphorylation, prolactin incubation stimulated the phosphorylation of ser-19 (Fig. 5). Whereas this residue is believed to be a substrate for CaMKII, the data in Fig. 2B provide only weak evidence to support involvement of CaMKII in this prolactin response. Preincubation with KN-93, an inhibitor of CaMKII, resulted in only a partial reduction in the prolactin response. A significant inhibition was also seen, however, with its inactive analog KN-92. In contrast, we previously demonstrated that activation of catecholamine synthesis in these cultures by angiotensin II or K+-depolarization was completely inhibited by KN-93 and unaffected by the inactive analog (18). The ability of KN-93 and KN-92 to partially inhibit the prolactin response may result from suppression of Ca2+ influx (36), a recognized action of these compounds. Whereas not characterized in NEDA neurons, prolactin has been reported to increase Ca2+ influx in some transfected cells (33). If this is the case here, then it suggests that Ca2+ influx may play a role in the prolactin stimulation of catecholamine synthesis, although the significance of that role cannot be assessed without knowing the extent of KN-93/92 action on the Ca2+ influx.
The data provide good evidence for an involvement of PKC in prolactin-induced activation of catecholamine synthesis in these hypothalamic cultures. The response to prolactin was abolished by bisindolymaleimide I but unaffected by its inactive analog (Fig. 3B). PKC has been shown to be activated in response to prolactin in a number of tissues including the hypothalamus (2, 10, 37). A previous study measuring prolactin-stimulated TH activity in rat hypothalamic slices employed selected protein kinase inhibitors to conclude that PKC but not CaMKII or PKA was involved in prolactin’s action (10). The importance of PKC in regulating TH activity in these cells is further supported by data from ourselves and others showing that responses to angiotensin II and neurotensin were also inhibited by PKC antagonists (18, 38). In contrast to the actions of the PKA antagonist H89 (discussed above), the PKC antagonist appeared to be selective, in that it only reduced responses to PMA and not those to dibutyl cAMP or K+ depolarization (18). The possible mechanism of PKC action is discussed below in the context of the MAPK pathway.
Stimulation of prolactin receptors on many cell types leads to activation of the MAPK pathway (2). As can be seen in Fig. 4A, in agreement with this general finding, prolactin caused an increase in ERK1/2 phosphorylation in these hypothalamic cultures. It should be noted that, whereas these experiments do not preclude a prolactin-induced increase in ERK expression, such a response is unlikely after this relatively short, 15-min incubation period. ERK1/2 activation is well documented to involve both PKC-dependent and PKC-independent pathways (39). The prolactin stimulation of ERK1/2 phosphorylation appeared to involve the former pathway in that it was abolished by the PKC inhibitor bisindolymaleimide I. Interestingly, the observation that the MEK inhibitor PD98059 reduced basal TH activity suggests an additional PKC-independent phosphorylation of ERK1/2, which is evident under basal conditions. Despite apparently complete inhibition of prolactin-mediated ERK1/2 phosphorylation, PD98059 did not inhibit prolactin activation of catecholamine synthesis, although it did cause a concentration-dependent decrease in the level of catecholamine synthesis occurring under basal conditions. The role of the ERKs in TH regulation is particularly interesting because they are the only kinases currently identified to phosphorylate ser-31 (13). Whereas data presented here clearly show that prolactin increases ser-31 phosphorylation (Fig. 5) and that this is probably mediated by a PKC-dependent activation of ERK1/2, this response does not appear to be necessary for the prolactin-induced increase in catecholamine synthesis. As noted in the introductory text, an increase in the TH ser-31 phosphorylation has been reported in other cells in response to many different stimuli, but the precise role of this phosphorylation in regulating TH activity is not fully resolved. In contrast to its apparent lack of involvement in the prolactin-stimulated response, MEK activity does appear to be important for maintaining basal levels of catecholamine synthesis. Although it is clear that MEK-inhibition decreases the level of basal ERK1/2 phosphorylation, it remains to be determined whether this is associated with a decrease in TH ser-31 phosphorylation.
Thus, the role of PKC in the prolactin-induced increase in catecholamine synthesis in these NEDA neuronal cultures is complex. PKC activity appears to be essential for prolactin stimulation both of catecholamine synthesis and MAPK activation in these cells, but this latter pathway is not required for prolactin activation of catecholamine synthesis. PKC may therefore be stimulating catecholamine synthesis through an alternative as-yet-unidentified mechanism. One interesting possibility is that PKC is mediating the phosphorylation of the ser-40 residue and thus having a direct effect on TH activity. Alternatively, PKC may be having its stimulatory action by acting on another component of the catecholamine synthesis machinery.
In addition to increasing the rate of catecholamine synthesis, prolactin induced TH mRNA expression in these cultures. As would be expected, this transcriptional response appeared to be slower in onset than the increase in activity, requiring 4 h incubation to reach significance. This observation is in agreement with previous in vivo evidence showing that prolactin induces both an acute and chronic activation of TH (9). Whereas earlier reports indicated that the genomic response may require up to 12 h to be induced, more recent evidence suggests that as little as 3 h exposure to high prolactin is sufficient (40). It should be noted, however, that the data obtained using cycloheximide and actinomycin D indicate that the increased TH mRNA expression in response to prolactin does not make a significant contribution to the rate of catecholamine synthesis during the first 2 h. In a previous study, we reported that angiotensin II also stimulates both TH expression and TH activity in these cultures (18). The former response required PKC and the MAPK activity, in that it was inhibited by their respective antagonists. In contrast, prolactin-induced TH mRNA expression was unaffected by either of these inhibitors. Thus, as discussed earlier, prolactin appears to stimulate the ERK1/2 MAPK pathway in these cells in a PKC-dependent manner, but the observed increase in TH mRNA is not dependent on this pathway. The reasons for these differences are unclear but suggest that, whereas both prolactin and angiotensin II use somewhat similar pathways to acutely activate TH, the pathways diverge to regulate its expression. Such a conclusion is consistent with our previous report that the prolactin activation of TIDA neurons in vivo appears to require the signal transducer and activator of transcription-5b signaling pathway (7). Because both angiotensin II and prolactin are likely to regulate the activity of NEDA neurons in vivo, such convergent and divergent signaling pathways may have important consequences for the integrated regulation of NEDA neuron activity and thus prolactin secretion.
Acknowledgments
We acknowledge the valuable technical and intellectual assistance from Professor Peter Dunkley and Dr. Larisa Bobrovskaya in preparing this manuscript.
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Address all correspondence and requests for reprints to: Stephen Bunn, The Centre for Neuroendocrinology, Department of Anatomy and Structural Biology, The University of Otago, P.O. Box 913, Dunedin 9001, New Zealand. E-mail: stephen.bunn@stonebow.otago.ac.nz.
Abstract
Prolactin secretion from the anterior pituitary is tightly regulated by feedback onto the hypothalamic neuroendocrine dopaminergic (NEDA) neurons. Prolactin stimulates these neurons to synthesize and secrete dopamine, which acts via the pituitary portal vasculature to inhibit prolactin secretion from the pituitary lactotrophs. Despite the physiological importance of this feedback, relatively little is known about the signaling mechanisms responsible for prolactin activation of NEDA neurons. This issue has been examined here using a cell culture preparation of the fetal rat mediobasal hypothalamus. Prolactin stimulated a time- and concentration-dependent increase in catecholamine synthesis, which was maximal after 60–120 min (1 μg/ml prolactin) and inhibited by the prolactin antagonist 1–9-G129R-hPRL. This prolactin response was accompanied by a rise in the site-specific (ser-19, -31, and -40) phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Consistent with this observation, the prolactin-induced increase in catecholamine synthesis was abolished by inhibitors of protein kinase A and protein kinase C (PKC). Prolactin incubation also resulted in a PKC-dependent activation of the MAPK pathway, although this was not required for the stimulation of catecholamine synthesis. In addition to increasing TH phosphorylation and catecholamine synthesis, prolactin also increased TH mRNA expression. In contrast to catecholamine synthesis, this latter response was not suppressed by inhibition of protein kinase A or PKC. These results indicate that although prolactin controls catecholamine synthesis in NEDA neurons by regulating both TH activity and TH mRNA expression, it employs distinct, nonoverlapping, signaling pathways to achieve these ends.
Introduction
PROLACTIN IS A HORMONE of major physiological importance with many actions in addition to its well-described role in lactation (1, 2). The secretion of prolactin from the anterior pituitary lactotrophs is subject to a number of regulatory factors, the most important being an inhibitory input from dopamine carried in the hypophysial portal system (1). Dopamine found within these vessels is synthesized and secreted by the neuroendocrine dopaminergic (NEDA) neurons located in the arcuate and periventricular nuclei of the mediobasal hypothalamus. The NEDA neurons are present as three distinct groups, referred to as the tuberoinfundibular, tuberohypophyseal, and periventricular-hypophyseal dopaminergic neurons. The relative contribution of each of these NEDA populations to the control of prolactin secretion is not fully resolved, although the tuberoinfundibular dopaminergic (TIDA) neurons, which deliver dopamine directly into the median eminence, appear to be particularly important (1). Circulating prolactin stimulates the activity of all three populations of NEDA neurons, thus increasing dopamine release and ultimately suppressing its own secretion from the anterior pituitary (3). Whereas this short-loop feedback pathway is well established, the cellular mechanisms by which prolactin stimulates NEDA neuron activity remains to be resolved.
Prolactin receptors have been localized within the hypothalamus including the arcuate nucleus and median eminence and have been identified colocalized on TIDA neurons (4, 5). Isolated dopaminergic neurons from the fetal rat hypothalamus have also been shown to express prolactin receptors (6). When maintained in culture, these neurons respond to prolactin receptor activation by increasing their rate of dopamine synthesis (6). Thus, it would appear likely that TIDA, and perhaps other NEDA neurons, are directly responsive to prolactin. As noted above, the cellular mechanisms underlying such a response are not fully resolved. Whereas data from signal transducer and activator of transcription-5b-deficient mice suggest that this transcription factor is required for prolactin-induced activation of these neurons (7), other studies indicate that prolactin may also exert nontranscription regulation of dopamine synthesis and release. In vivo studies suggest that the TIDA neuron response to prolactin is relatively rapid (8) with the initial increase in dopamine synthesis being independent of transcription and translation, followed by a delayed (12 h) further increase, which was sensitive to cycloheximide inhibition (9). In vitro studies using rat hypothalamic slices have also reported a relatively rapid (2 h) prolactin-induced increase in dopamine synthesis (10), whereas studies using hypothalamic fetal cells in culture reported the need for long-term prolactin exposure (1–10 d) to significantly increase dopamine levels (6).
The rate of dopamine synthesis in TIDA and other dopaminergic neurons is controlled by the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis (11, 12). TH is subject to many regulatory inputs both chronically at the level of gene transcription and translation and acutely at the level of the enzyme activity. TH activity is tightly regulated by end-product inhibition, in that dopamine suppresses its activity by competing with the binding of the tetrahydrobiopterin BH4 cofactor (12). Phosphorylation of TH, within its N-terminal regulatory domain, reduces the affinity of dopamine binding, thus relieving the inhibition and elevating TH activity. Physiologically relevant regulation of TH through its phosphorylation has been well described in a number of tissues and appears to involve three specific serine residues, Ser-19, -31, and -40 (13). The protein kinases responsible for the phosphorylation of each of these residues and their precise roles in the activation of the enzyme are still relatively unclear. At present, however, it is probable that Ser-19 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), MAPK-activated protein kinase (MAPKAPK) 2, and p38-regulated/activated kinase (14, 15, 16). Ser-31 appears to be exclusively phosphorylated by the MAPKSs ERK1/2 (17). In contrast, Ser-40 may be phosphorylated by a number of protein kinases including protein kinase A (PKA), protein kinase C (PKC), protein kinase G, CaMKII, MAPKAPK1/2, and the mitogen- and stress-activated protein kinase 1 (11, 16).
Despite its physiological importance, our understanding of the intracellular pathways by which prolactin receptor activation couples to either TH expression or TH phosphorylation is still incomplete. The aim of this current study was therefore to conduct a detailed investigation into the kinase pathways activated in TIDA neurons by prolactin and determine their influence on the level of dopamine synthesis. To achieve this aim, we employed cell cultures prepared from the mediobasal hypothalamus of the embryonic rat pup and determined the effect of protein kinase inhibitors on the ability of prolactin to increase both TH activity (as reflected in the level of catecholamine synthesis) and TH mRNA expression.
Materials and Methods
Hypothalamic cell cultures
Cultures of the rat mediobasal hypothalamus were prepared using the method developed by Arbogast and Voogt (6) modified as described previously (18). All animal procedures were approved by the University of Otago Animal Ethics Committee. Briefly, mediobasal hypothalami from 28–56 embryonic d 18–21 fetal rats were excised to a depth of approximately 1 mm, limited anteriorly by optic chiasm, posteriorly by the border of the mamillary bodies, and laterally by the hypothalamic fissures. Tissue blocks were pooled, rinsed with buffer (137 mM NaCl, 5.4 mM KCl, 59 mM sucrose, 0.2 mM Na2HPO4, 0.2 mM KH2PO4, 5.5 mM glucose, 100,000 U/liter penicillin, 100 mg/liter streptomycin, and 250 μg/liter Fungizone adjusted to pH 7.4), and roughly chopped. Tissue fragments were then incubated with 10 ml trypsin (2.5 mg/ml in the same buffer) at 37 C for 5 min at which time 1 ml DNase solution (1 mg/ml) was added and the incubation continued for a further 5 min. The digestion was terminated by the addition of 12 ml of soybean trypsin inhibitor solution (0.3 mg/ml) followed by centrifugation at 800 x g for 5 min. The resultant pellet was resuspended, washed with buffer, and again centrifuged at 800 x g for 5 min before being dispersed at a density of approximately 106 cells/ml in DMEM (Life Technologies, Inc., Rockville, MD) containing 2.5% fetal bovine serum and 5% horse serum. Cells were plated out at 1 ml/well (106 cells/well) onto poly-D-lysine-coated 24-well culture plates or for immunocytochemical investigations poly-D-lysine-coated glass coverslips in 24-well culture plates. Plated cells were then transferred to 5% CO2 humidified incubator at 37 C. After the first 24 h in culture, the medium was removed and replaced with a serum- and phenol red-free DMEM containing high glucose and KCl (each at 25 mM) and the following reagents (each obtained from Sigma, St. Louis, MO): linolenic acid (1 μg/ml), linoleic acid (1.5 μg/ml), putrescine (4.4 μg/ml), and ITS+ (insulin-transferrin-sodium selenite supplement) (1:100 dilution). The medium was replaced every second day and the cells routinely used for experimentation after 14–21 d in culture.
Immunocytochemistry
Cells cultured on glass coverslips were removed from the incubator, washed twice (5 min each) with 1 ml of 10 mM PBS, fixed with 2% formaldehyde in PBS for 20 min at room temperature, and then permeabilized with 90% ethanol/10% acetic acid (5 min at –20 C). Cells were then washed three times for 5 min each in 1 ml PBS followed by 1 h in PBS containing 5% goat serum and then 48 h at 4 C with a combination of both primary antibodies, polyclonal anti-TH (AB151 diluted 1:200, from Chemicon, Temecula, CA) and monoclonal antiprolactin receptor (rat) (MA1–610 diluted 1:100, from Affinity Bioreagents, Golden, CO) made up in PBS containing 5% goat serum. The cells were then washed (6 x 5 min in PBS) and incubated with a combination of secondary antibodies antimouse Alexa488 and antirabbit Alexa568 both diluted 1:1000 in PBS (Molecular Probes, Eugene, OR) for 30 min at room temperature. Coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined under fluorescence microscopy using an AX70 microscope (Olympus, Tokyo, Japan) fitted with a Spot-RT color digital camera (Diagnostic Instruments Inc., Sterling Heights, MI).
Catecholamine synthesis
Catecholamine synthesis was measured using the method originally developed for adrenal chromaffin cells (19) modified as described previously (18). Cells were removed from the incubator and washed briefly (twice for 5 min) with 400 μl carbogen-gassed HEPES-buffered saline (HBS) of the following composition: 150 mM NaCl, 15 mM HEPES, 5.5 mM glucose, 3.8 mM K2HPO4, 1 mM MgSO4, 1.5 mM CaCl2, and 0.5 mM sodium ascorbate, adjusted to pH 7.4 at 37 C. The last wash was replaced with 200 μl HBS containing 20 μM L-[carboxyl-14C] tyrosine (0.22 μCi per well, purchased from Amersham Biosciences NZ Ltd. Auckland, New Zealand) in the presence or absence of a stimulating agent, routinely 1 μg/ml prolactin (ovine prolactin obtained from Sigma). Each well was immediately fitted with a tube sealed with a rubber stopper from which was suspended a small plastic cap containing 200 μl of 1 M NaOH to absorb the 14CO2 produced by the cells during the decarboxylation step in catecholamine synthesis. The incubation was terminated by the injection of 400 μl of ice-cold 10% trichloroacetic acid through the rubber stopper. The plates, with their sealed wells in place, were then kept at 4 C for 2 h before an aliquot of the NaOH solution was collected and the 14C radioactivity measured by liquid scintillation spectrometry. When examining the effect of prolactin antagonist or protein kinase inhibitors, these agents were included during both a 15-min preincubation period and the subsequent stimulation. PD-98059, KN-92, KN-93, and H89 were purchased from Sigma; H85 from Seikagaku (Tokyo, Japan); and bisindomaleimide I and V, actinomycin D, and cycloheximide from Calbiochem (San Diego, CA). Recombinant prolactin receptor antagonist 1–9-G129R-hPRL was prepared and purified as described previously (20). Catecholamine synthesis was expressed relative to basal levels (cells incubated with buffer alone for the same time period) or, where appropriate, a control value obtained from cells incubated in the absence of an antagonist and statistical comparisons performed using a Mann-Whitney U test (unless otherwise stated).
TH mRNA expression
Cells were removed from the incubator and briefly washed with 500 μl serum-free DMEM and then returned to the incubator in a further 500 μl serum-free DMEM with or without prolactin (1 μg/ml) in the presence or absence of an appropriate protein kinase inhibitor. After 4 h incubation, each well was extracted into 200 μl TRIzol reagent and total RNA isolated as per manufacturer’s instructions. The resultant RNA was reverse transcribed using GeneAmp Gold RNA PCR kit (PE Applied Biosystems, Foster City, CA) and quantitative real-time RT-PCR for TH and ?-actin performed using the Taqman system (PE Applied Biosystems). Primer and probe details were exactly as described previously (18). The reaction mix was also prepared as previously described and an ABI PRISM 7700 sequence detection system (Centre for Gene Research, University of Otago) used to detect fluorescence during each PCR under conditions exactly as described previously (18). Data were captured and analyzed using sequence detector software (SDS, version 3.0, PE Applied Biosystems). TH mRNA levels in each sample were normalized with reference to its ?-actin mRNA levels and then expressed as relative to TH mRNA in control cells (i.e. those incubated with prolactin in the absence of kinase inhibitors) determined in the same experiment. Statistical analysis was performed using a Mann-Whitney U test.
Reverse-transcribed RNA extracted from cells was also probed for the long and short forms of the prolactin receptor. The forward primer for the prolactin receptor short and long form was 5'-ATACTG GAG TAG ATG GAG CCA GGA GAG TTC-3' corresponding to nucleotides 624–653 of the prolactin receptor cDNA sequence. The reverse primer for the short form was 5'-TCC TAT TTG AGT CTG CAG CTT CAG TAG TCA-3' corresponding to nucleotides 924–953 of the cDNA sequence, and the reverse primer for the long form was 5'-CTT CCG TGA CCA GAG TCA CTG TCG GGA TCT-3' corresponding to nucleotides 1014–1043 of the cDNA sequence (21). The cDNA was amplified through 30 PCR cycles, the products resolved on 1.5% agarose gels, and visualized with ethidium bromide staining.
Activation of ERK1/2
The phosphorylation of ERK1/2 was measured by immunoblotting using activation state-specific antibodies. Cells were washed twice for 5 min with 400 μl HBS and then preincubated for 15 min with PD98059 (50 μM) or bisindolylmaleimide I (3 μM) before being stimulated for 15 min with or without prolactin (1 μg/ml) in the continued presence or absence of the appropriate inhibitor. The cells were then lysed in ice-cold 10 mM Tris buffer (three wells collected in a total of 200 μl) containing 5 mM EDTA, 50 mM NaF, 50 mM NaCl, 1% Triton-X100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 μl /ml of protease inhibitor cocktail (Sigma). The lysate was sonicated, centrifuged at 12,000 x g for 10 min and the resultant supernatant fractionated by SDS-PAGE and transferred onto nitrocellulose membranes. These membranes were probed overnight at 4 C using a phospho-p44/42 MAPK (Thr202/Tyr204)-specific monoclonal antibody (at 1:1000 dilution, Cell Signaling Technology, Beverly, MA). The membranes were then washed with 10 mM Tris-buffered saline containing 0.05% Tween 20 and reincubated with horseradish peroxidase-coupled anti-IgG antibody (1:5000 dilution for 60 min at room temperature), washed again, and the image developed using enhanced chemiluminescence. Relative levels of phosphorylation of ERK1/2 were determined using densitometric image analysis (National Institutes of Health Image) and expressed as a percentage of that obtained from cells incubated with buffer alone and examined on the same immunoblot. Statistical analysis was performed using a Mann-Whitney U test.
TH phosphorylation
Cells were washed and stimulated with or without prolactin (1 μg/ml for 120 min), extracted in lysis buffer, and processed for immunoblotting as described above. Blots were probed overnight at 4 C using antibodies raised against TH (AB151 diluted 1:200, Chemicon) or specific TH phosphorylation sites (ser-19, ser-31, or ser-40; 1:10,000, kindly provided by Prof. Peter Dunkley, University of Newcastle, New South Wales, Australia) and then washed and labeled with secondary antibodies as described above. The density of immunoreactive bands was visualized and quantitated on the Typhoon system (Molecular Dynamics, Sunnyvale, CA) and expressed as a percentage of those detected in cells incubated with buffer alone and examined on the same immunoblot. Details regarding the generation and characterization of these antibodies were described by Cammarota et al. (22).
Results
Cultures prepared from the fetal rat mediobasal hypothalamus contained a population of TH immunoreactive cells. Cell counts indicated that these TH-positive cells constituted approximately 7 ± 1% of the total cell population (determined from six separate cell culture preparations). Some, although not all, of these TH-positive cells also expressed prolactin receptors, as shown by immunocytochemistry (Fig. 1A). It should be noted that prolactin receptor expression was not limited to TH-positive cells, in that other unidentified cell types were also immunoreactive for this antibody (Fig. 1A). RT-PCR performed on extracts of these cultures indicated that the long, rather than short, form of prolactin receptor was the predominant form expressed in these cultures (Fig. 1B).
FIG. 1. The expression of prolactin receptors on TH immunoreactive neurons in mediobasal hypothalamic cultures. A, Cell cultures were prepared as described in Materials and Methods and then dual labeled with antibodies directed against TH (left panel) and prolactin receptors (right panel). Note that prolactin receptor immunoreactivity was present on, but not limited to, cells that were also immunoreactive for TH (a non-TH-immunoreactive cell indicated by arrow, immediately adjacent to a TH-positive cell). B, Mediobasal hypothalamic cell cultures were prepared and maintained as described in Materials and Methods. After 14 d in culture, total mRNA was extracted and mRNA for ?-actin (lane 1), short-form prolactin receptor (lane 2), long-form prolactin receptor (lane 3), TH (lane 4), and short and long forms of the prolactin in choroid plexus (lane 5 and 6, respectively) detected by RT-PCR as described in Materials and Methods.
Incubation with prolactin stimulated a time- and concentration-dependent increase in catecholamine synthesis, presumably reflecting an increase in TH activity (Fig. 2, A and B). This response was slow and fairly modest but highly consistent with a statistically significant increase in catecholamine production (120% of the basal response) seen after a 60-min incubation with prolactin (1 μg/ml). Extending the incubation period to 120 min did not further increase catecholamine synthesis (Fig. 2A). A prolactin-stimulated increase in catecholamine synthesis was evident with concentrations of prolactin as low as 10 ng/ml and was concentration dependent, although no apparent maximum was reached with concentrations up to 1 μg/ml (Fig. 2B). The response to this highest concentration of prolactin was inhibited by the prolactin antagonist, 1–9-G129R-hPRL (10 μg/ml), although it should be noted that catecholamine production in these cells was still significantly higher than that in cells incubated with the antagonist alone (Fig. 2B). The inclusion of serum (2.5% fetal calf serum and 5% horse serum) in the culture medium did not alter responsiveness to prolactin, with a 120-min incubation with prolactin (1 μg/ml), resulting in a 119 ± 5% basal response in the presence of serum, compared with 119 ± 3% basal in its absence (n = 3 from separate cell culture preparations).
FIG. 2. Prolactin stimulates a time- and concentration-dependent increase in catecholamine synthesis. Cells were incubated with or without prolactin (1 μg/ml) for 15–120 min (A) or increasing concentrations of prolactin for 120 min (B). The presence of the prolactin antagonist 1–9-G129R-hPRL (10 μg/ml) is indicated by the dark shaded columns and resulted in a significant suppression of prolactin-induced catecholamine synthesis. Catecholamine synthesis was determined as described in Materials and Methods and expressed as a percentage of the response obtained under basal conditions for the same time period. Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01, compared with the response in cells incubated for the same time period with buffer alone; #, P < 0.01, compared with the matched prolactin response in the absence of antagonist; +, P < 0.05, compared with the basal response in the presence of the prolactin antagonist (Mann-Whitney U test, n = 6–12 from three to four separate cell culture preparations).
It is well recognized that TH activity, and thus the rate of catecholamine synthesis, can be regulated by the phosphorylation of specific serine residues located at the N terminal of the enzyme. To determine whether this was the mechanism underlying prolactin’s action on these hypothalamic neurons, the effect of selected protein kinase inhibitors was determined. Incubation with H89 (3 μM), an inhibitor of PKA, resulted in a small but significant decrease in the basal catecholamine synthesis measured over 120 min (Fig. 3A). This inhibitor also markedly reduced the prolactin-induced increase in this response activity, such that it was no longer significantly greater than the basal activity over this time period (Fig. 3A). In contrast, the same concentration of H85, an inactive analog of H89, was without effect on either the basal or prolactin-induced response (Fig. 3A). Bisindolymaleimide I (3 μM), a PKC antagonist, also fully inhibited the prolactin-induced increase in catecholamine synthesis but unlike H89 had no significant effect on the basal response (Fig. 3B). Bisindolymaleimide V, an inactive analog of bisindolymaleimide I, was without effect on either basal or prolactin-stimulated activity (Fig. 3B). KN-93 (3 μM), which is known to inhibit Ca2+-dependent protein kinases including CaMKII, had no effect on basal catecholamine synthesis but caused a partial inhibition (about 50%) of the prolactin-induced response (Fig. 3C). It is important to note, however, that KN-92, an inactive analog of KN-93, also significantly suppressed the prolactin-stimulated response, albeit to a lesser extent than KN-93 (Fig. 3C).
FIG. 3. The effect of selected protein kinase inhibitors on prolactin-stimulated catecholamine synthesis in mediobasal hypothalamic cultures. Cell cultures were preincubated with or without (open columns) selected protein kinase inhibitors (all at 3 μM), H89 (dark columns) or H85 (light columns) (A), bisindolymaleimide I (dark columns) or bisindolymaleimide VI (light columns) (B), or KN-93 (dark columns) or KN-92 (light columns) (C), for 15 min and then incubated for a further 120 min with or without prolactin (1 μg/ml) in the continued presence or absence of the appropriate protein kinase inhibitor. Catecholamine synthesis was measured as described in Materials and Methods and expressed as a percentage of the basal response in the absence of inhibitors. Data are presented as the mean ± SEM (Mann-Whitney U test, n = 9–18 from three to six separate cell preparations). *, P < 0.001, compared with matched basal response; #, P < 0.05, compared with control basal; ##, P < 0.01, compared with control prolactin response.
The possible involvement of MAPKs in the prolactin-induced increase in catecholamine synthesis in these cells was first investigated by determining whether the MAPKs ERK1/2 were activated in response to prolactin. As depicted in Fig. 4A, incubation with prolactin (1 μg/ml for 15 min) resulted in an increased phosphorylation and thus activation of ERK1 (Fig. 4A). Phosphorylation of ERK1 was abolished by both the MAPK/ERK kinase (MEK) inhibitor PD98059 (50 μM) and the PKC inhibitor bisindolymaleimide I (3 μM) (Fig. 4A). Interestingly, inhibition of MEK, but not PKC, resulted in a significant decrease in ERK1 phosphorylation seen under basal conditions. The effect of prolactin and the above inhibitors on ERK2 phosphorylation was essentially identical with that on ERK1 (data not shown). The level of TH expression detected on these Western blots was not significantly altered after stimulation with prolactin at 115.7 ± 16% basal levels (P > 0.05 using a Wilcoxon sign-ranks test). In agreement with its action on ERK phosphorylation, incubation with PD98059 (50 μM) also caused a marked reduction in the basal rate of catecholamine synthesis (Fig. 4B). In contrast, whereas the overall rate of catecholamine synthesis in the presence of prolactin was reduced in the presence of PD98059, this apparent inhibition was entirely due to the decreased basal activity, with the prolactin response being 122% of basal in the absence of the inhibitor, compared with 123% of basal in its presence (Fig. 4B). Thus, whereas PKC appears to be involved in both prolactin-induced ERK1/2 activation and prolactin-induced TH activity, the ERK1/2 pathway is not itself required for the stimulation of catecholamine synthesis. To examine this proposal further, the effect of PD98059 on the ability of phorbol-12-myristate-13-acetate (PMA), a PKC activator, to stimulate catecholamine synthesis was determined. As described previously (18), PMA (100 nM for 60 min) significantly increased the level of catecholamine synthesis to about 115% of basal levels (Fig. 4C). The inclusion of PD98059 caused a concentration-dependent decrease in the net response to PMA, but, as with prolactin, this resulted from a parallel concentration-dependent decrease in basal activity (Fig. 4C).
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FIG. 4. Prolactin activates the ERK1/2 pathway in mediobasal hypothalamic cell cultures. A, Cells were preincubated with or without (open columns) PD98059 (50 μM, filled columns) or bisindolymaleimide I (3 μM, light columns) for 15 min and then for a further 15 min with or without prolactin (1 μg/ml) in the continued presence or absence of the appropriate inhibitor. Cells were lysed and phosphorylated ERK1/2 levels determined as described in Materials and Methods. Data are presented as a percentage of phospho-ERK1 levels measured under control basal conditions, determined on the same immunoblot. *, P < 0.001 and #, P < 0.05, compared with control basal levels (Mann-Whitney U test, n = 3–6 from independent experiments performed on separate cell culture preparation). Inserts present representative phospho-ERK immunoblots positioned above the appropriate columns. B, Cells were preincubated with or without (open columns) PD98059 (50 μM, filled columns) for 15 min followed by a further 120 min with or without prolactin (1 μg/ml) in the continuing presence or absence of the inhibitor. Catecholamine synthesis was measured as described in Materials and Methods and expressed as a percentage of the basal response in the absence of inhibitor. Data are presented as the mean ± SEM. *, P < 0.001, compared with the control basal response; #, P < 0.001, compared with the basal response in the presence of PD98059 (Mann-Whitney U test, n = 9 from three separate cell preparations). C, Cells were preincubated with increasing concentrations of PD98059 for 15 min and then for a further 60 min with (shaded columns) or without (open columns) PMA (100 nM) in the continued presence or absence of the inhibitor. Catecholamine synthesis was measured as described in Materials and Methods and expressed as a percentage of the basal response in the absence of inhibitor. Data are presented as the mean ± SEM. *, P < 0.01, compared with the matched basal response; #, P < 0.05, compared with the appropriate response in the absence of PD98059 (ANOVA, n = 3).
The involvement of multiple protein kinases in this prolactin-stimulated response was supported by the finding that TH protein extracted from these cells was phosphorylated on multiple serine residues. As presented in Fig. 5, the use of phosphorylation site-specific antibodies revealed that prolactin (1 μg/ml for 60 min) stimulated a significant increase in phosphorylation of ser-19, -31, and -40 residues when compared with cells incubated under basal condition. Whereas the increase in ser-40 phosphorylation appears to be larger than the other two sites, this apparent difference was not statistically significant. The level of total TH expression detected on these Western blots was not significantly altered after stimulation with prolactin at 110.7 ± 9.5% basal levels (P > 0.05 using Wilcoxon sign-ranks test on the original densitometric data).
FIG. 5. Prolactin increases the phosphorylation of specific serine residues on TH from mediobasal hypothalamic cultures. Cells were incubated with (shaded columns) or without (open columns) prolactin (1 μg/ml) for 60 min. After incubation cells were lysed and the phosphorylation of ser-19, -31, and -40 residues of TH determined using phosphorylation site-specific antibodies as described in Materials and Methods. Data are expressed as a percentage of the site-specific phosphorylation measured in cells incubated with buffer alone and represent the mean ± SEM (*, P < 0.01, compared with the relevant basal response; Mann-Whitney U test, n = 4 independent experiments performed on separate cell culture preparations). Inserts present representative phospho-TH immunoblot positions above the appropriate columns.
In addition to increasing TH activity (as reflected by a rise in catecholamine synthesis), incubation with prolactin significantly increased the level of TH mRNA expression after 240 min incubation (Fig. 6A). In contrast to the prolactin-induced increase in catecholamine synthesis, this rise in TH mRNA was not inhibited by H89, bisindolymaleimide I, KN-93, or PD98059 (Fig. 6A). Interestingly, inhibition of transcription or translation with actinomycin D (1 μM) or cyclohexamide (1 μM) decreased basal catecholamine synthesis but failed to suppress the stimulated response to prolactin (Fig. 6B).
FIG. 6. Prolactin increases TH mRNA levels in mediobasal hypothalamic cultures. A, Cells were incubated for 4 h with or without prolactin (1 μg/ml) in the presence (shaded columns) or absence (open columns) of the indicated protein kinase inhibitor, H89 (10 μM), KN-93 (3 μM), bisindolymaleimide I (BisM, 3 μM), or PD98059 (3 μM). Cells were then extracted and TH mRNA levels measured using real-time RT-PCR as described in Materials and Methods. Data are expressed as a percentage of the level detected in cells stimulated with prolactin in the absence of kinase inhibitors and are presented as means ± SEM. *, P < 0.05, compared with cells incubated under basal condition in the presence of appropriate kinase inhibitor (Mann-Whitney U test, n = 3–6 from separate cell culture preparations). B, Cells were preincubated for 15 min with or without actinomycin D (1 μM, dark columns) or cycloheximide (1 μM, light columns) and then for a further 120 min with or without prolactin (1 μg/ml) in the continued presence of the appropriate inhibitor. Catecholamine synthesis was determined as described in Materials and Methods and is expressed as a percentage of that detected in cells incubated under basal condition in the absence of inhibitor. *, P < 0.01, compared with activity in cells incubated under matched basal conditions; #, P < 0.05, compared with cells incubated with buffer alone (Mann-Whitney U test, n = 4 from separate cell culture preparations).
Discussion
The primary aim of this project was to investigate the intracellular mechanisms by which prolactin activity regulates dopamine synthesis in NEDA neurons. As outlined in the introductory text, the key regulatory step in this pathway is the activity of TH. The experimental approach used here to examine TH activity in cultured mediobasal hypothalamic neurons was based on the liberation of 14CO2 from cells loaded with 14C-tyrosine. TH converts the 14C-tyrosine to 14C-DOPA with is then decarboxylated by aromatic acid decarboxylase to dopamine, releasing 14CO2. Thus, whereas the assay is dependent on the activity of both enzymes, the latter is not rate limiting and has been shown to be relatively insensitive to acute regulation (23). Thus, the rate of 14CO2 accumulation provides a sensitive and reliable index of in situ TH activity (19). Whereas the ability to measure cellular dopamine synthesis in the intact living cell has clear advantages, there are also limitations in terms of identifying the locus of an action. TH activity may, for example, be sensitive to multiple factors in addition to its phosphorylation status, including the rate of tyrosine uptake and cofactor availability, which may themselves be influenced by receptor stimulation or protein kinase activity.
Preliminary immunocyotochemical studies confirmed earlier reports that prolactin receptors are expressed on TH immunoreactive cells in these cultures (6). Whereas not fully quantitated, our observations suggest that prolactin receptors are present on most of the TH-expressing cells in these cultures. Prolactin receptor immunoreactivity was also evident on a population of non-TH expressing cells. The identity of these cells was not investigated here, but an earlier study suggested that they were neuronal rather than glial in origin (6). Whereas most TH-immunoreactive cells express prolactin receptors and are thus are presumably capable of responding directly to prolactin, other non-TH immunoreactive cells could also respond to prolactin and thereby potentially mediate a secondary, indirect, action on the TH-positive neurons. RT-PCR examination suggested that regardless of specific cellular location, the vast majority of prolactin receptor expression in these hypothalamic cultures was of the long, rather than short form of the receptor, an observation in agreement with in vivo studies (24, 25). It should be noted, however, that the short-form primers were able to detect the short-form prolactin receptor in choroid plexus (Fig. 1B) and a number of other rat brain regions (21).
The concentration of prolactin required to stimulate a significant increase in catecholamine synthesis in these cultures is in excellent agreement with those reported previously for more chronic prolactin actions on similar cultures (6, 10, 26). The majority of experiments reported here have used a prolactin concentration of 1 μg/ml, which gave a reliable and relatively robust response. This concentration is a little higher than the high end of the physiological range, such as during the preovulatory surge but less than concentrations reported in the portal blood (27). The specificity of prolactin action in these experiments is supported by the inhibition observed using the prolactin antagonist 1–9-G129R-hPRL. This prolactin analog acts as a pure competitive antagonist at the prolactin receptor, and similar concentrations of this or related analogs (20, 28, 29) have been shown to inhibit a variety of prolactin-induced responses in other cells and tissues. The partial inhibition of prolactin response observed in the current studies reflects the fact that the antagonist has a 10-fold lower affinity for the prolactin receptor than wild-type human prolactin (22).
It should be noted that the culture preparations used in the current study have been maintained in a serum-free environment for at least 14 d before stimulation with prolactin. In the earlier studies mentioned above (6), cells were found to be unresponsive to prolactin unless maintained in the presence of serum. The reason for this difference, or indeed the explanation underlying the need for serum in the earlier studies, is unknown, but the ability to maintain prolactin-responsive cells in a defined culture environment is clearly an experimental advantage. Thus, in the absence of postnatal exposure to prolactin, circulating steroids, or other serum-derived factors, TH-containing cells from the mediobasal hypothalamus of the rat fetus appear capable of expressing functional prolactin receptors.
The ability of prolactin to stimulate a relatively rapid increase in catecholamine synthesis in these cultures strongly suggests that it is promoting the phosphorylation and thus activation of TH. As outlined in the introductory text, TH in other cell types can be phosphorylated on three specific serine residues, ser-19, -31, and -40, through a variety of protein kinases (11, 12). In many cases this phosphorylation leads to a rise in catalytic activity of TH. Previous in vivo studies strongly suggested that protein kinases play a key role in regulating TH activity in the arcuate nucleus and median eminence (10, 30, 31), although the pathway by which prolactin mediates these actions is not well documented. The data presented in Fig. 3a suggests that PKA activity may be required for prolactin stimulation of TH. The increase in catecholamine synthesis induced by prolactin was abolished by H89 and unaffected by its inactive analog H85. Previous studies demonstrated that increased cAMP levels stimulate the rate of catecholamine synthesis in both hypothalamic slices and cultures (30, 32).
Given that ser-40 phosphorylation of TH leads to its activation (11, 13) and that prolactin increases the phosphorylation of this residue (Fig. 5), it is tempting to suggest that prolactin achieves this activation via a PKA-mediated pathway. Caution should be taken, however, before reaching this conclusion. There is little evidence that prolactin receptors are coupled to adenylyl cyclase, although it is possible that this enzyme could be activated secondarily to a prolactin-induced increase in intracellular Ca2+ (33, 34). More importantly, however, we and others have shown that H89 inhibits TH activation by other agents that are unlikely to act via PKA. The PMA-induced increase in catecholamine synthesis in these hypothalamic cultures, for example, is abolished by H89 (18). Similarly, TH activity in adrenal medullary chromaffin cells is inhibited by H89, even when this appears to involve little or no stimulation of PKA (35). Thus, whereas it can be reasonably concluded that PKA activity, or at least an H89-sensitive event, is required for the activation of TH in NEDA neurons, its precise role in the prolactin-driven response remains somewhat equivocal. As outlined in the introductory text, TH ser-40 is the substrate for a number of protein kinases in addition to PKA including CaMKII, PKC, and MAPKAPKs downstream of ERK1/2. It is possible that one or more of these kinases is responsible for prolactin stimulation of catecholamine biosynthesis.
In addition to increasing TH ser-40 phosphorylation, prolactin incubation stimulated the phosphorylation of ser-19 (Fig. 5). Whereas this residue is believed to be a substrate for CaMKII, the data in Fig. 2B provide only weak evidence to support involvement of CaMKII in this prolactin response. Preincubation with KN-93, an inhibitor of CaMKII, resulted in only a partial reduction in the prolactin response. A significant inhibition was also seen, however, with its inactive analog KN-92. In contrast, we previously demonstrated that activation of catecholamine synthesis in these cultures by angiotensin II or K+-depolarization was completely inhibited by KN-93 and unaffected by the inactive analog (18). The ability of KN-93 and KN-92 to partially inhibit the prolactin response may result from suppression of Ca2+ influx (36), a recognized action of these compounds. Whereas not characterized in NEDA neurons, prolactin has been reported to increase Ca2+ influx in some transfected cells (33). If this is the case here, then it suggests that Ca2+ influx may play a role in the prolactin stimulation of catecholamine synthesis, although the significance of that role cannot be assessed without knowing the extent of KN-93/92 action on the Ca2+ influx.
The data provide good evidence for an involvement of PKC in prolactin-induced activation of catecholamine synthesis in these hypothalamic cultures. The response to prolactin was abolished by bisindolymaleimide I but unaffected by its inactive analog (Fig. 3B). PKC has been shown to be activated in response to prolactin in a number of tissues including the hypothalamus (2, 10, 37). A previous study measuring prolactin-stimulated TH activity in rat hypothalamic slices employed selected protein kinase inhibitors to conclude that PKC but not CaMKII or PKA was involved in prolactin’s action (10). The importance of PKC in regulating TH activity in these cells is further supported by data from ourselves and others showing that responses to angiotensin II and neurotensin were also inhibited by PKC antagonists (18, 38). In contrast to the actions of the PKA antagonist H89 (discussed above), the PKC antagonist appeared to be selective, in that it only reduced responses to PMA and not those to dibutyl cAMP or K+ depolarization (18). The possible mechanism of PKC action is discussed below in the context of the MAPK pathway.
Stimulation of prolactin receptors on many cell types leads to activation of the MAPK pathway (2). As can be seen in Fig. 4A, in agreement with this general finding, prolactin caused an increase in ERK1/2 phosphorylation in these hypothalamic cultures. It should be noted that, whereas these experiments do not preclude a prolactin-induced increase in ERK expression, such a response is unlikely after this relatively short, 15-min incubation period. ERK1/2 activation is well documented to involve both PKC-dependent and PKC-independent pathways (39). The prolactin stimulation of ERK1/2 phosphorylation appeared to involve the former pathway in that it was abolished by the PKC inhibitor bisindolymaleimide I. Interestingly, the observation that the MEK inhibitor PD98059 reduced basal TH activity suggests an additional PKC-independent phosphorylation of ERK1/2, which is evident under basal conditions. Despite apparently complete inhibition of prolactin-mediated ERK1/2 phosphorylation, PD98059 did not inhibit prolactin activation of catecholamine synthesis, although it did cause a concentration-dependent decrease in the level of catecholamine synthesis occurring under basal conditions. The role of the ERKs in TH regulation is particularly interesting because they are the only kinases currently identified to phosphorylate ser-31 (13). Whereas data presented here clearly show that prolactin increases ser-31 phosphorylation (Fig. 5) and that this is probably mediated by a PKC-dependent activation of ERK1/2, this response does not appear to be necessary for the prolactin-induced increase in catecholamine synthesis. As noted in the introductory text, an increase in the TH ser-31 phosphorylation has been reported in other cells in response to many different stimuli, but the precise role of this phosphorylation in regulating TH activity is not fully resolved. In contrast to its apparent lack of involvement in the prolactin-stimulated response, MEK activity does appear to be important for maintaining basal levels of catecholamine synthesis. Although it is clear that MEK-inhibition decreases the level of basal ERK1/2 phosphorylation, it remains to be determined whether this is associated with a decrease in TH ser-31 phosphorylation.
Thus, the role of PKC in the prolactin-induced increase in catecholamine synthesis in these NEDA neuronal cultures is complex. PKC activity appears to be essential for prolactin stimulation both of catecholamine synthesis and MAPK activation in these cells, but this latter pathway is not required for prolactin activation of catecholamine synthesis. PKC may therefore be stimulating catecholamine synthesis through an alternative as-yet-unidentified mechanism. One interesting possibility is that PKC is mediating the phosphorylation of the ser-40 residue and thus having a direct effect on TH activity. Alternatively, PKC may be having its stimulatory action by acting on another component of the catecholamine synthesis machinery.
In addition to increasing the rate of catecholamine synthesis, prolactin induced TH mRNA expression in these cultures. As would be expected, this transcriptional response appeared to be slower in onset than the increase in activity, requiring 4 h incubation to reach significance. This observation is in agreement with previous in vivo evidence showing that prolactin induces both an acute and chronic activation of TH (9). Whereas earlier reports indicated that the genomic response may require up to 12 h to be induced, more recent evidence suggests that as little as 3 h exposure to high prolactin is sufficient (40). It should be noted, however, that the data obtained using cycloheximide and actinomycin D indicate that the increased TH mRNA expression in response to prolactin does not make a significant contribution to the rate of catecholamine synthesis during the first 2 h. In a previous study, we reported that angiotensin II also stimulates both TH expression and TH activity in these cultures (18). The former response required PKC and the MAPK activity, in that it was inhibited by their respective antagonists. In contrast, prolactin-induced TH mRNA expression was unaffected by either of these inhibitors. Thus, as discussed earlier, prolactin appears to stimulate the ERK1/2 MAPK pathway in these cells in a PKC-dependent manner, but the observed increase in TH mRNA is not dependent on this pathway. The reasons for these differences are unclear but suggest that, whereas both prolactin and angiotensin II use somewhat similar pathways to acutely activate TH, the pathways diverge to regulate its expression. Such a conclusion is consistent with our previous report that the prolactin activation of TIDA neurons in vivo appears to require the signal transducer and activator of transcription-5b signaling pathway (7). Because both angiotensin II and prolactin are likely to regulate the activity of NEDA neurons in vivo, such convergent and divergent signaling pathways may have important consequences for the integrated regulation of NEDA neuron activity and thus prolactin secretion.
Acknowledgments
We acknowledge the valuable technical and intellectual assistance from Professor Peter Dunkley and Dr. Larisa Bobrovskaya in preparing this manuscript.
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