The Role of Calcitonin Gene-Related Peptide in the in Vivo Pituitary-Adrenocortical Response to Acute Hypoxemia in the Late-Gestation Sheep
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《内分泌学杂志》
Department of Physiology, University of Cambridge, Cambridge CB2 3EG, United Kingdom
Abstract
This study tested the hypothesis that calcitonin gene-related peptide (CGRP) has a role in mediating the in vivo fetal adrenal glucocorticoid response to acute stress. The hypothesis was tested by investigating the effects of fetal treatment with a selective CGRP antagonist on plasma ACTH and cortisol responses to acute hypoxemia in the late-gestation sheep fetus. Under anesthesia, six fetuses at 0.8 of gestation were surgically instrumented with vascular catheters. Five days later, fetuses were subjected to 0.5-h hypoxemia during treatment with either iv saline or a CGRP antagonist, in randomized order, on different days. Treatment started 30 min before hypoxemia and ran continuously until the end of the challenge. Arterial blood samples were collected for plasma ACTH and cortisol measurements (RIA) and blood gas monitoring. CGRP antagonism did not alter basal arterial blood gas or endocrine status. During hypoxemia, similar falls in arterial partial pressure of oxygen occurred in all fetuses. During saline infusion, acute hypoxemia induced significant increases in fetal ACTH and cortisol concentrations. During CGRP antagonism, the pituitary-adrenal responses were markedly attenuated. Correlation of paired plasma ACTH and cortisol values from all individual fetuses during normoxia and hypoxemia showed positive linear relationships; however, neither the slope nor the intercept of the peptide-steroid relationship was affected by CGRP antagonism. These data support the hypothesis that CGRP is involved in the in vivo regulation of fetal adrenocortical steroidogenesis during acute hypoxemia. In addition, the data reveal that CGRP may have a role in the control of other components of the hypothalamo-pituitary-adrenal axis during stimulated conditions in fetal life.
Introduction
DURING THREATENING SITUATIONS, the stress system coordinates adaptive responses that adjust homeostatic mechanisms to increase the chance of survival of the individual. The hypothalamo-pituitary-adrenal (HPA) axis constitutes one of the efferent limbs of the stress system (1). In the adult individual, it has long been known that secretion of adrenal glucocorticoids is principally controlled by the release of ACTH from the anterior pituitary, but only comparatively recently has it become appreciated that this is not an exclusive mechanism. Compelling evidence suggests that the splanchnic innervation to the adrenal gland plays an important role in modulating adrenocortical output during stimulated conditions. Edwards and Jones (2) have shown, in conscious hypophysectomized calves, that stimulation of the splanchnic innervation to the gland doubles, whereas section of the splanchnic nerves halves (3), the output of cortisol in response to an exogenous infusion of ACTH.
In the fetus, similar neural mechanisms may operate in the control of adrenocortical sensitivity during stimulated conditions. Three pieces of evidence support this assertion. First, functional innervation of the ovine fetal adrenal gland is present by the final third of gestation (4, 5). Second, Myers et al. (6) showed that splanchnic nerve section in the ovine fetus had no effect on basal cortisol but that denervation significantly attenuated the cortisol response to acute fetal hypotension. Third, our group has previously reported that in fetal mammals during acute hypoxemia, bilateral section of the carotid sinus nerves can completely prevent the increase in plasma cortisol without affecting either the increase in plasma ACTH concentrations or the adrenal vasodilatation and, hence, the delivery of plasma ACTH to the fetal adrenal cortex (7, 8). Taken together, past data, therefore, suggest that a chemoreflex triggered by the carotid chemoreceptors, and mediated via splanchnic efferent pathways, may operate to regulate adrenocortical responsiveness to ACTH during episodes of acute hypoxemic stress in the fetal period.
A component of splanchnic control of adrenal function may be regulated by the release of peptides, which may directly stimulate steroidogenesis and/or increase adrenal blood flow to accelerate the presentation of ACTH to the adrenal cortex (9, 10, 11). A plausible candidate for this peptidergic regulation of adrenocortical function includes calcitonin gene-related peptide (CGRP), because CGRP-stained fibers have been shown within the adrenal cortex, even in fetal animals during late gestation (4). Furthermore, exogenous treatment with CGRP promotes an increase in steroid output from the adrenal glands of adult rats, calves, and frogs (12, 13, 14), an effect that may or may not be mediated via the nitric oxide-dependent dilator action of the peptide within the adrenal gland (12, 14). However, no study has investigated the function of CGRP in the in vivo regulation of adrenocortical steroidogenic output during hypoxemic stress in either adult or fetal animals. Therefore, by investigating the effects of fetal treatment with a selective CGRP antagonist on the plasma ACTH and cortisol responses to acute hypoxemia in the chronically instrumented late-gestation ovine fetus, this study tested the hypothesis that CGRP has a role in mediating the in vivo adrenal glucocorticoid response to acute stress in the mature fetus.
Materials and Methods
Surgical preparation
All procedures were performed under the United Kingdom Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Cambridge. Six Welsh Mountain sheep fetuses were surgically instrumented for long-term recording at 120 ± 1 d gestation (term is 145 d) using strict aseptic conditions as described previously in detail (15). In brief, food, but not water, was withheld from the pregnant ewes for 24 h before surgery. After induction with 20 mg·kg–1 iv sodium thiopentone (Intraval Sodium; Merial Animal Health Ltd., Rhone Mérieux, Dublin, Ireland), general anesthesia (1.5–2.0% halothane in 50% O2:50% N2O) was maintained using positive-pressure ventilation. Midline abdominal and uterine incisions were made, the fetal hindlimbs were exteriorized, and, on one side, femoral arterial (inner diameter, 0.86 mm; outer diameter, 1.52 mm; Critchly Electrical Products, New South Wales, Australia) and venous (inner diameter, 0.56 mm; outer diameter, 0.96 mm) catheters were inserted. The catheter tips were advanced carefully to the descending aorta and inferior vena cava, respectively. Another catheter was anchored onto the fetal hindlimb for recording of the reference amniotic pressure. The uterine incisions were closed in layers, the dead space of the catheters was filled with heparinized saline (80 IU heparin·ml–1 in 0.9% NaCl), and the catheter ends were plugged with sterile brass pins. The catheters were then exteriorized via a keyhole incision in the maternal flank and kept inside a plastic pouch sewn onto the maternal skin.
Postoperative care
During recovery, ewes were housed in individual pens in rooms with a 12-h light, 12-h dark cycle where they had free access to hay and water and were fed concentrates twice daily (100 g sheep nuts no. 6; H & C Beart Ltd., Kings Lynn, UK). Antibiotics were administered daily to the ewe (0.20–0.25 mg·kg–1 im Depocillin; Mycofarm, Cambridge, UK) and fetus intravenously and into the amniotic cavity (150 mg·kg–1 Penbritin; SmithKline Beecham Animal Health, Welwyn Garden City, Hertfordshire, UK). The ewes also received 2 d of postoperative analgesia if in pain (10–20 mg·kg–1 oral Phenylbutazone; Equipalozone paste, Arnolds Veterinary Products Ltd., Shropshire, UK), as assessed by their general demeanor and feeding patterns. Generally, normal feeding patterns were restored within 48 h of recovery. After 72 h of postoperative recovery, ewes were transferred to metabolic crates where they were housed for the remainder of the protocol. The arterial and amniotic catheters were connected to sterile pressure transducers (COBE; Argon Division, Maxxim Medical, Athens, TX). Calibrated mean fetal arterial blood pressure (corrected for amniotic pressure) and fetal heart rate (triggered via a tachometer from the pulsatility in the arterial blood pressure) were recorded continually at 1-sec intervals using a computerized Data Acquisition System (Department of Physiology, Cambridge University). While on the metabolic crates, the patency of the fetal catheters was maintained by a slow continuous infusion of heparinized saline (80 IU heparin·ml–1 at 0.1 ml·h–1 in 0.9% NaCl) containing antibiotic (1 mg·ml–1 benzylpenicillin; Crystapen, Schering-Plough, Animal Health Division, Welwyn Garden City, UK).
Experimental protocol
After at least 5 d of postoperative recovery, all fetuses were subjected to two experimental protocols, carried out on consecutive days in a randomized order (Fig. 1). Each protocol consisted of a 2.5-h period divided into 1-h normoxia, 0.5-h hypoxemia, and 1-h recovery. At the midpoint (30 min) of the first normoxic period, fetal treatment with either a slow iv infusion of vehicle (80 IU heparin·ml–1 in 0.9% NaCl) or the CGRP antagonist dissolved in heparinized saline (42.6 ± 1.8 μg·kg–1 intraarterial bolus followed by 8.5 ± 0.4 μg·kg–1·min–1 iv infusion; Calcitonin Gene Related Peptide fragment 8–37, CGRP8–37; C-2806; Sigma Chemicals, Dorset, UK) was commenced (Fig. 1). The dose of CGRP antagonist used has been calculated retrospectively from the fetal weights (2.4 ± 0.1 kg) obtained at postmortem. Acute hypoxemia in the fetus was induced by maternal inhalational hypoxia. In brief, a large transparent respiratory hood was placed over the heads of the ewes into which air was passed at a rate of approximately 50 liter·min–1 for the 1-h period of normoxia. After this control period, acute fetal hypoxemia was induced for 30 min by changing the concentrations of gases breathed by the ewe to 6% O2 in N2 with small amounts of CO2 (15 liter·min–1 air:35 liter·min–1 N2:1.5–2.5 liter·min–1 CO2). This mixture was designed to reduce fetal arterial partial pressure of oxygen (PaO2) to approximately 10 mm Hg while maintaining arterial partial pressure of CO2 (PaCO2). After the 0.5-h period of hypoxemia, the ewe was returned to breathing air for the 1-h recovery period. At the end of the experimental protocol, the ewes and fetuses were humanely killed using a lethal dose of sodium pentobarbitone (200 mg·kg–1 iv Pentoject; Animal Ltd., York, UK). Postmortems were carried out at 130 ± 1 d gestation, during which time the positions of the implanted catheters were confirmed and the fetuses were weighed.
Blood sampling regimen
During any acute hypoxemia protocol, descending aortic blood samples (0.3 ml) were taken using sterile techniques from the fetus at set time intervals (Fig. 1, arrows) to determine arterial blood gas and acid base status (ABL5 Blood Gas Analyser, Radiometer; Copenhagen, Denmark; measurements corrected to 39.5 C). Values for percentage saturation of hemoglobin with oxygen (Sat Hb) and the blood hemoglobin concentration ([Hb]) were determined using a hemoximeter (OSM2; Radiometer). An additional 2 ml of arterial blood was withdrawn at set intervals for hormone analyses (Fig. 1, arrows). These samples were collected under sterile conditions into chilled EDTA tubes (2 ml K+/EDTA; L.I.P., Ltd., Shipley, West Yorkshire, UK) for ACTH and cortisol analysis. All samples were then centrifuged at 4000 rpm for 4 min at 4 C. The plasma obtained was then dispensed into prelabeled tubes, and the samples were stored at –20 C until analysis.
Hormone analyses
All hormone measurements were performed within 2 months of sample collection. Plasma ACTH and cortisol concentrations were determined by RIA validated for use in ovine plasma.
ACTH assay
Fetal plasma ACTH concentrations were measured using a commercially available double antibody 125I RIA kit (DiaSorin Inc., Stillwater, MN) as previously described (16). The kit contained a set of ACTH standards (porcine; range, 20–500 pg·ml–1), ACTH quality control samples, lyophilized rabbit anti-ACTH antiserum, 125I-labeled synthetic ACTH, and a second antibody-precipitating complex (goat antirabbit -globulin serum and polyethylene glycol in buffer). Reagents were reconstituted in deionized water and were kept on crushed ice. Duplicate 100-μl aliquots of the ACTH standards, quality controls, and unknown plasma samples (taken from previously unthawed K+/EDTA-treated plasma aliquots) were incubated overnight at 5 C with 200 μl of anti-ACTH antiserum and 200 μl of 125I-labeled ACTH. Bound and free ACTH fractions were separated by immunoprecipitation with the second antibody-precipitating complex (500 μl). After centrifugation (3600 rpm; 20 min; 20–25 C), the supernatant was rapidly decanted and discarded and the residue pellets allowed to dry. A scintillation counter (Cobra II Auto-Gamma Packard Bioscience Co., Pangbourne, Berks, UK) was used to count the radioactivity of each pellet for 2 min, together with that of duplicate nonspecific binding samples (anti-ACTH antiserum omitted) and that of total counts tubes (200 μl of 125I-labeled ACTH). A standard curve was produced by the computer using a logistic curve fit, and ACTH concentrations were calculated for each pellet.
The lower limit of detection of the assay (90% bound·free–1) was between 10–25 pg·ml–1. The intraassay coefficients of variation (CV) for three plasma pools (mean concentration: 69, 281, and 600 pg·ml–1) were 12.5, 6.3, and 8.1%, respectively. The interassay CV for two plasma pools (mean concentration: 36 and 136 pg·ml–1) were 6.0 and 6.7%, respectively. The anti-ACTH antiserum showed less than 0.01% cross-reactivity against -MSH, -endorphin, -lipotropin, leucine enkephalin, methionine enkephalin, bombesin, calcitonin, parathyroid hormone, follicle-stimulating hormone, arginine vasopressin, oxytocin, and substance P.
Cortisol assay
Fetal plasma cortisol concentrations were measured by RIA using tritium-labeled cortisol as tracer, as previously described (17). Duplicate 20-μl plasma samples were taken from previously unthawed K+/EDTA-treated sample aliquots and were extracted twice in 300 μl of ethanol. After each extraction, the aliquots were centrifuged and the ethanol layer decanted into a bacteriological test tube. The combined ethanol extracts were evaporated in a drier (Speedvac Concentrator, SVC 200H; Stratech Scientific, Luton, UK) overnight. Residues were reconstituted in 100 μl of PBS (pH 7.0) and were allowed to stand at 5 C for at least 2 h. Samples in which cortisol concentration values were predicted to be in the upper range of the standard curve were diluted using PBS to reduce the concentration to within the most sensitive range (1–10 ng·ml–1) of the standard curve. Values for these samples were subsequently corrected for the dilution factor used in each case. The plasma extracts, quality control samples, and working standards (1, 2.5, 5, 10, 25, 50, and 100 ng·ml–1) were incubated overnight (5 C) with 100 μl of antiserum (Tenovus Institute, Cardiff, UK) and 100 μl of [1,2,6,7-3H]-labeled cortisol (TRK407; Amersham Pharmacia Biotech United Kingdom Ltd., Little Chalfont, Buckinghamshire, UK). Bound and free cortisol fractions were separated using stirred dextran-coated charcoal suspension (300 μl; 0.075 g/100 ml dextran and 0.75 g/100 ml activated charcoal in PBS). After immediate centrifugation (3600 rpm; 7 min; 4 C), the supernatant was rapidly decanted into scintillation vial inserts, and 4 ml of scintillant (OptiPhase HiSafe; Fisons Chemicals, Loughborough, UK) was added to each insert. The inserts were capped and the contents thoroughly mixed. After a 1-h equilibration period, a scintillation counter (1216 RackBeta Liquid Scintillation Counter; LKB Wallac, Sweden) was used to count the radioactivity of each insert for 5 min, including the radioactive content of duplicate nonspecific binding samples (antiserum omitted) and total counts tubes (100 μl of 3H-labeled cortisol). Recoveries averaged 90%. A standard curve was produced by the computer using a spline curve fit, and cortisol concentrations were calculated for each sample.
The lower limit of detection of the assay was between 1.0 and 1.5 ng·ml–1. The intraassay CV was 5.3% for a mean value of 13 ng·ml–1. The interassay CV for two plasma pools (mean concentration, 13 and 28 ng·ml–1) were 13.6 and 11.4%, respectively. The cross-reactivities of the antiserum at 50% binding with other cortisol-related compounds were as follows: 0.5% cortisone, 2.3% corticosterone, 0.3% progesterone, 4.6% deoxycortisol.
Data and statistical analyses
Values for all arterial blood gas and endocrine variables are expressed as mean ± SEM at 0 (N0) and 45 (N45) min of normoxia, 15 (H15) and 30 (H30) min of hypoxemia, and at 30 (R30) and 60 (R60) min of recovery for fetuses exposed to 30 min of hypoxemia during saline infusion or during treatment with CGRP antagonist. The cardiovascular variables are expressed as minute averages at the corresponding time points. All measured variables were first analyzed for normality of distribution and then assessed using two-way ANOVA with repeated measures (Sigma-Stat; SPSS Inc., Chicago, IL) comparing the effect of time (normoxia vs. hypoxemia/recovery), group (saline vs. CGRP antagonist), and interactions between time and group. Where a significant effect of time or group was indicated, the post hoc Tukey test was used to isolate the statistical differences. The relationship between parallel measurements of plasma concentrations of ACTH and cortisol in all individual fetuses was assessed using the Pearson Product Moment correlation. For all comparisons, statistical significance was accepted when P < 0.05.
Results
Fetal arterial blood gas and acid base status
Basal values for arterial blood gas and acid base status were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal arterial blood gas and acid-base status (Table 1). In all fetuses, acute hypoxemia induced significant falls in arterial pH (pHa), acid base excess (ABE), PaO2, and Sat Hb, without any alteration to PaCO2. The magnitude of these changes was similar during saline infusion or during treatment with the CGRP antagonist (Table 1). However, the maximal increment in fetal [Hb] during acute hypoxemia was significantly diminished during treatment with the CGRP antagonist (0.77 ± 0.46 g·dl–1) when compared with saline-infused controls (2.15 ± 0.42 g·dl–1; P < 0.05). During recovery, pHa and ABE remained significantly depressed by the end of the experimental protocol in all fetuses, whereas PaO2, Sat Hb, and [Hb] returned to basal values.
Fetal pituitary-adrenocortical function
Plasma ACTH concentrations
Basal plasma concentrations of ACTH were similar in all fetuses before saline infusion (22 ± 1 pg·ml–1) or CGRP antagonist treatment (27 ± 4 pg·ml–1). Infusion with saline or treatment with the CGRP antagonist had no effect on basal plasma concentrations of ACTH (Fig. 2). Acute hypoxemia resulted in a pronounced increase in plasma ACTH in all fetuses. However, the increment in plasma ACTH during hypoxemia was more than halved during treatment with the CGRP antagonist (472 ± 108 pg·ml–1) when compared with the increment in saline-infused controls (968 ± 187 pg·ml–1; P < 0.05). During recovery, plasma ACTH concentrations returned to basal values in all fetuses.
Plasma cortisol concentrations
Basal plasma concentrations of cortisol were also similar in all fetuses before saline infusion (14 ± 2 ng·ml–1) or CGRP antagonist treatment (14 ± 4 ng·ml–1). Infusion with saline or treatment with the CGRP antagonist had no effect on basal plasma concentrations of cortisol (Fig. 2). Acute hypoxemia resulted in a pronounced increase in plasma cortisol in all fetuses. However, the increment in plasma cortisol during acute hypoxemia was significantly diminished during treatment with the CGRP antagonist (22.6 ± 4.8 ng·ml–1) when compared with the increment in saline-infused controls (35.2 ± 8.2 ng·ml–1; P < 0.05). During recovery, plasma cortisol concentrations returned to basal values in all fetuses.
Correlation analysis of paired plasma ACTH and cortisol values for all individual fetuses during basal and hypoxemic conditions, during either saline infusion (r = 0.69, n = 24, P < 0.001) or during treatment with the CGRP antagonist (r = 0.59, n = 24, P < 0.001), revealed significant positive relationships (Fig. 3A). Treatment with the CGRP antagonist did not affect the slope or the intercept of the peptide-steroid relationship compared with saline infusion (Fig. 3B).
Fetal cardiovascular function
Basal values for arterial blood pressure and heart rate were similar in all fetuses before saline infusion or CGRP antagonist treatment. Infusion with saline or treatment with the CGRP antagonist had no effect on the basal cardiovascular variables (Table 2). During acute hypoxemia, a significant increase in arterial blood pressure (from 48.9 ± 2.0 to 56.8 ± 2.0 mm Hg) and fall in heart rate (from 170 ± 11 to 140 ± 13 beats·min–1) occurred in all fetuses during saline infusion (P < 0.05). The magnitude of these cardiovascular responses during acute hypoxemia were not affected during treatment with the CGRP antagonist, because arterial blood pressure increased from 46.9 ± 1.7 to 56.0 ± 2.6 mm Hg and fetal heart rate fell from 170 ± 4 to 144 ± 14 beats·min–1. During recovery, fetal arterial blood pressure remained significantly elevated by the end of the experimental protocol in all fetuses, whereas fetal heart rate returned to basal values.
Discussion
CGRP is a 37-amino acid peptide discovered in 1983 by alternative processing of RNA transcripts of the gene encoding calcitonin (18). In addition to being one of the most potent vasodilator peptides known (19), CGRP can also acts as a neuropeptide, often found colocalized with substance P (20) throughout the central and peripheral nervous systems (21, 22, 23). In the adult, immunocytochemical studies have demonstrated several nerve plexuses and terminals, which stain positively for CGRP (4, 14) as well as the presence of CGRP receptors at the level of the adrenal cortex (24), consistent with a potential role for CGRP in regulating adrenal function. In vivo studies in hypophysectomized calves with the adrenal clamp technique (12) or in isolated frog or rat adrenal glands perfused in situ (13, 14) have confirmed a functional role for CGRP in stimulating adrenal glucocorticoid output. However, in vitro studies using dispersed adrenocortical cells in the rat have reported that CGRP does not affect either basal or ACTH-stimulated glucocorticoid production (25). The apparent discrepancy has been attributed to the type of preparation (14). Although, in isolated cellular studies, CGRP appears to have an inhibitory effect on adrenocortical secretion, this effect appears to be overridden when the integrity and functional architecture of the adrenal tissue is preserved and when the adrenal gland is exposed to systemic influences. A net stimulatory mechanism may rely on the close and direct relationship between the rate of blood flow through the intact adrenal gland and the rate of glucocorticoid release (14). Hence, in the intact adrenal, CGRP may also act as a powerful vasodilator increasing adrenal blood flow and, thereby, the presentation rate of trophic agents, such as ACTH, resulting in an overall increase in adrenocortical secretion. However, Bloom et al. (12) suggested that CGRP might have blood flow-independent, direct steroidogenic effects in the conscious hypophysectomized calf, because in their study, the increase in glucocorticoid output could not be completely accounted for by an increase in ACTH presentation to the adrenal gland. Data from our in vivo study in the ovine fetus show that during CGRP antagonism, there was a marked attenuation in the fetal plasma cortisol response to acute hypoxemia, implying for the first time in fetal life, that CGRP may also have a stimulatory role in the control of adrenal function. However, in the present study, CGRP antagonism also caused a marked attenuation in the fetal plasma ACTH concentrations during acute hypoxemia, and detailed assessment of the paired plasma ACTH and cortisol concentrations during basal and hypoxemic conditions in all individual fetuses showed no effects of CGRP antagonism on the slope or the intercept of the peptide-steroid relationship. Combined, these results suggest that in the ovine fetus, CGRP antagonism may have diminished plasma concentrations of cortisol during acute hypoxemia purely by virtue of an effect on plasma concentrations of ACTH. Therefore, the data also emphasize a regulatory role for CGRP higher up the HPA axis in the fetus, either in the control of hypothalamic CRH and/or pituitary ACTH release.
Several reports support a functional role for CGRP in the control of pituitary ACTH release in the adult individual. Peptidergic fibers containing CGRP have been found to innervate the anterior pituitary of the monkey, dog, rat, and human (26, 27), where CGRP-binding sites have also been detected (23, 28). These fibers exhibit morphological, distributional, and numerical changes after stress and adrenalectomy (29, 30). In addition, a close anatomical arrangement of peptidergic fibers exists with pituitary glandular cells, with synaptic contacts being made specifically with corticotropes (30). In cultured rat anterior pituitary cells, Iino et al. (31) have shown that CGRP stimulates ACTH secretion and that this effect is prevented by inhibition of protein kinase A. This suggests that CGRP may be directly involved in the synthesis and/or release of pituitary ACTH by increasing intracellular cAMP, which, in turn, may activate the adenylate-cyclase-protein kinase system (32). Similarly, evidence exists to show the presence of CGRP fibers and CGRP-binding sites in adult rat (22, 23) and human (28) hypothalamus. Two recent studies in adult rats support a functional role of CGRP in hypothalamic stimulation. Li et al. (33) have shown that central administration of CGRP into the lateral cerebral ventricles induces c-Fos expression in the preoptic area and paraventricular nucleus of the hypothalamus. Furthermore, Dhillo et al. (34) have reported that central administration of CGRP into the paraventricular nucleus leads to an increase in plasma ACTH and corticosterone concentrations, an effect that is prevented by treatment with a CRH antagonist. Our study is the first to suggest regulation of pituitary ACTH by CGRP during stimulated conditions in fetal life; however, whether this control is secondary to an effect at the hypothalamic level remains unknown.
It is well established that CRH release is regulated by central sympathetic pathways (1) and that central sympathetic outflow can be affected by CGRP (35, 36, 37). Indirect evidence from the present study showing an effect of CGRP antagonism on the concentration of fetal hemoglobin during hypoxemia may support a central effect of the antagonist on sympathetic outflow and, thereby, on central sympathetic regulation of CRH release. During acute hypoxemia, increased sympathetic outflow stimulates contraction of the spleen and other venous reservoirs (38). This decreases venous capacitance and shifts blood into the systemic circulation, promoting an acute increase in red blood cell count and, hence, [Hb] concentration. Increased sympathetic outflow during acute hypoxemia also promotes an increase in total peripheral vascular resistance and, hence, an increase in arterial blood pressure in the ovine fetus (39). In the present study, although CGRP antagonism markedly diminished the increase in [Hb] concentration, it did not affect the magnitude of the pressor response to acute hypoxemia, partially supporting an effect on sympathetic outflow. However, it must be recognized that multiple mechanisms in addition to sympathetic stimulation mediate the fetal arterial blood pressure response to acute hypoxemia (40). Hence, studies comparing the effects of central vs. systemic administration of CGRP antagonism, or those addressing the effects of CGRP antagonism on actual changes in peripheral vascular resistance in the ovine fetus, may provide greater insight on the central role of CGRP on HPA and cardiovascular function in fetal life.
Although measurement of plasma concentrations of ACTH and cortisol after exogenous intravenous treatment of the sheep fetus with CGRP may be an interesting experiment to perform, it may offer little additional insight into the effects of CGRP on pituitary-adrenal function. The current study proposed that in the fetus, sympathetic activity would modify pituitary-adrenocortical function via a mechanism involving CGRP. Exogenous, systemic treatment of the ovine fetus with CGRP will not mimic the synaptic concentrations of the peptide within the sympathetic nervous system. Hence, a negative effect of exogenous, systemic CGRP treatment on plasma glucocorticoid concentration will not necessarily imply that CGRP is not involved in pituitary or adrenocortical regulation.
In conclusion, the present study tested the hypothesis that CGRP has a role in mediating the in vivo adrenal glucocorticoid response to acute hypoxemic stress in the mature fetus. The results from this study show that fetal treatment with a CGRP antagonist significantly diminished both the plasma ACTH and cortisol responses to acute hypoxemia. Furthermore, correlation analysis of paired plasma ACTH and cortisol concentrations in all individual fetuses during basal and hypoxemic conditions revealed significant positive relationships. However, neither the slope nor the intercept of the peptide-steroid relationship was affected by CGRP antagonist treatment. These data partly support the hypothesis tested in this study but reveal that CGRP may have a role in the control of other components of the HPA axis during stimulated conditions in the late-gestation ovine fetus.
Footnotes
This work was supported by the International Journal of Experimental Pathology and the Lister Institute for Preventive Medicine. D.A.G. is a Fellow of the Lister Institute for Preventive Medicine.
Abbreviations: ABE, Acid base excess; CGRP, calcitonin gene-related peptide; CV, coefficient(s) of variation; [Hb], blood hemoglobin concentration; HPA, hypothalamo-pituitary-adrenal; PaCO2, arterial partial pressure of CO2; PaO2, arterial partial pressure of oxygen; pHa, arterial pH; Sat Hb, saturation of hemoglobin with oxygen.
References
Tsigos C, Chrousos GP 2002 Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress. J Psychosom Res 53:865–871
Edwards AV, Jones CT 1987 The effect of splanchnic nerve stimulation on adrenocortical activity in conscious calves. J Physiol 382:385–396
Edwards AV, Jones CT 1987 The effect of splanchnic nerve section on the sensitivity of the adrenal cortex to adrenocorticotrophin in the calf. J Physiol 390:23–31
Engeland WC, Wotus C, Rose JC 1998 Ontogeny of innervation of rat and ovine fetal adrenals. Endocr Res 24:889–898
Comline RS, Silver M 1961 The release of adrenaline and noradrenaline from the adrenal glands of the foetal sheep. J Physiol 156:424–444
Myers DA, Robertshaw D, Nathanielsz PW 1990 Effect of bilateral splanchnic nerve section on adrenal function in the ovine fetus. Endocrinology 127:2328–2335
Giussani DA, McGarrigle HH, Moore PJ, Bennet L, Spencer JA, Hanson MA 1994 Carotid sinus nerve section and the increase in plasma cortisol during acute hypoxia in fetal sheep. J Physiol. 477(Pt 1):75–80
Riquelme RA, Llanos JA, McGarrigle HH, Sanhueza EM, Hanson MA, Giussani DA 1998 Chemoreflex contribution to adrenocortical function during acute hypoxemia in the llama fetus at 0.6 to 0.7 of gestation. Endocrinology 139:2564–2570
Edwards AV, Jones CT 1993 Autonomic control of adrenal function. J Anat. 183 (Pt 2):291–307
Ehrhart-Bornstein M, Hinson JP, Bornstein SR, Scherbaum WA, Vinson GP 1998 Intraadrenal interactions in the regulation of adrenocortical steroidogenesis. Endocr Rev 19:101–143
Vinson GP, Hinson JP, Toth IE 1994 The neuroendocrinology of the adrenal cortex. J Neuroendocrinol 6:235–246
Bloom SR, Edwards AV, Jones CT 1989 Adrenal responses to calcitonin gene-related peptide in conscious hypophysectomized calves. J Physiol 409:29–41
Esneu M, Delarue C, Remy-Jouet I, Manzardo E, Fasolo A, Fournier A, Saint-Pierre S, Conlon JM, Vaudry H 1994 Localization, identification, and action of calcitonin gene-related peptide in the frog adrenal gland. Endocrinology 135:423–430
Hinson JP, Vinson GP 1990 Calcitonin gene-related peptide stimulates adrenocortical function in the isolated perfused rat adrenal gland in situ. Neuropeptides 16:129–133
Giussani DA, Gardner DS, Cox DT, Fletcher AJ 2001 Purinergic contribution to circulatory, metabolic, and adrenergic responses to acute hypoxemia in fetal sheep. Am J Physiol Regul Integr Comp Physiol 280:R678–R685
Gardner DS, Fletcher AJ, Bloomfield MR, Fowden AL, Giussani DA 2002 Effects of prevailing hypoxaemia, acidaemia or hypoglycaemia upon the cardiovascular, endocrine and metabolic responses to acute hypoxaemia in the ovine fetus. J Physiol 540:351–366
Robinson PM, Comline RS, Fowden AL, Silver M 1983 Adrenal cortex of fetal lamb: changes after hypophysectomy and effects of Synacthen on cytoarchitecture and secretory activity. Q J Exp Physiol 68:15–27
Rosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vale WW, Evans RM 1983 Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304:129–135
Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I 1985 Calcitonin gene-related peptide is a potent vasodilator. Nature 313:54–56
Franco-Cereceda A, Henke H, Lundberg JM, Petermann JB, Hokfelt T, Fischer JA 1987 Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin. Peptides 8:399–410
Maggi CA 1995 Tachykinins and calcitonin gene-related peptide (CGRP) as co-transmitters released from peripheral endings of sensory nerves. Prog Neurobiol 45:1–98
Skofitsch G, Jacobowitz DM 1985 Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6:721–745
Wimalawansa SJ, Emson PC, MacIntyre I 1987 Regional distribution of calcitonin gene-related peptide and its specific binding sites in rats with particular reference to the nervous system. Neuroendocrinology 46:131–136
Renshaw D, Cruchley AT, Kapas S, Hinson JP 1998 Receptors for calcitonin gene-related peptide (CGRP) in the rat adrenal cortex. Endocr Res 24:773–776
Mazzocchi G, Musajo F, Neri G, Gottardo G, Nussdorfer GG 1996 Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: comparison with calcitonin gene-related peptide effects. Peptides 17:853–857
Ju G, Liu SJ, Zhang X 1991 Peptidergic innervation of the pars distalis of the adenohypophysis. Neuroendocrinology 53(Suppl 1):41–44
Liu S 2004 Peptidergic innervation in pars distalis of the human anterior pituitary. Brain Res 1008:61–68
Tschopp FA, Henke H, Petermann JB, Tobler PH, Janzer R, Hokfelt T, Lundberg JM, Cuello C, Fischer JA 1985 Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary. Proc Natl Acad Sci USA 82:248–252
Hu D, Chen B, Wang B, Ma D 2002 The changes in the morphology and distribution of substance P and calcitonin-gene related peptide nerves in the anterior pituitary of scalded rats. Zhonghua Shao Shang Za Zhi 18:176–179
Ju G, Ma D, Fan SC 1994 Response to calcitonin-gene-related peptide-like immunoreactive nerve fibres of the anterior pituitary to adrenalectomy in the rat. Neuroendocrinology 505–510
Iino K, Oki Y, Tominaga T, Iwabuchi M, Ozawa M, Watanabe F, Yoshimi T 1998 Stimulatory effect of calcitonin gene-related peptide on adrenocorticotropin release from rat anterior pituitary cells. J Neuroendocrinol 10:325–329
Poyner DR 1992 Calcitonin gene-related peptide: multiple actions, multiple receptors. Pharmacol Ther 56:23–51
Li XF, Bowe JE, Mitchell JC, Brain SD, Lightman SL, O’Byrne KT 2004 Stress-induced suppression of the gonadotropin-releasing hormone pulse generator in the female rat: a novel neural action for calcitonin gene-related peptide. Endocrinology 145:1556–1563
Dhillo WS, Small CJ, Jethwa PH, Russell SH, Gardiner JV, Bewick GA, Seth A, Murphy KG, Ghatei MA, Bloom SR 2003 Paraventricular nucleus administration of calcitonin gene-related peptide inhibits food intake and stimulates the hypothalamo-pituitary-adrenal axis. Endocrinology 144:1420–1425
Bereiter DA, Benetti AP 1991 Microinjections of calcitonin gene-related peptide within the trigeminal subnucleus caudalis of the cat affects adrenal and autonomic function. Brain Res 558:53–62
Fisher LA, Kikkawa DO, Rivier JE, Amara SG, Evans RM, Rosenfeld MG, Vale WW, Brown MR 1983 Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature 305:534–536
Hasegawa T, Yokotani K, Okuma Y, Manabe M, Hirakawa M, Osumi Y 1993 Microinjection of alpha-calcitonin gene-related peptide into the hypothalamus activates sympathetic outflow in rats. Jpn J Pharmacol 61:325–332
Hoka S, Bosnjak ZJ, Arimura H, Kampine JP 1989 Regional venous outflow, blood volume, and sympathetic nerve activity during severe hypoxia. Am J Physiol 256:H162–H170
Giussani DA, Spencer JA, Moore PJ, Bennet L, Hanson MA 1993 Afferent and efferent components of the cardiovascular reflex responses to acute hypoxia in term fetal sheep. J Physiol 461:431–449
Giussani DA, Spencer JA, Hanson MA 1994 Fetal cardiovascular reflex responses to acute hypoxaemia. Fetal Matern Med Rev 6:17–37(A. S. Thakor and D. A. Gi)
Abstract
This study tested the hypothesis that calcitonin gene-related peptide (CGRP) has a role in mediating the in vivo fetal adrenal glucocorticoid response to acute stress. The hypothesis was tested by investigating the effects of fetal treatment with a selective CGRP antagonist on plasma ACTH and cortisol responses to acute hypoxemia in the late-gestation sheep fetus. Under anesthesia, six fetuses at 0.8 of gestation were surgically instrumented with vascular catheters. Five days later, fetuses were subjected to 0.5-h hypoxemia during treatment with either iv saline or a CGRP antagonist, in randomized order, on different days. Treatment started 30 min before hypoxemia and ran continuously until the end of the challenge. Arterial blood samples were collected for plasma ACTH and cortisol measurements (RIA) and blood gas monitoring. CGRP antagonism did not alter basal arterial blood gas or endocrine status. During hypoxemia, similar falls in arterial partial pressure of oxygen occurred in all fetuses. During saline infusion, acute hypoxemia induced significant increases in fetal ACTH and cortisol concentrations. During CGRP antagonism, the pituitary-adrenal responses were markedly attenuated. Correlation of paired plasma ACTH and cortisol values from all individual fetuses during normoxia and hypoxemia showed positive linear relationships; however, neither the slope nor the intercept of the peptide-steroid relationship was affected by CGRP antagonism. These data support the hypothesis that CGRP is involved in the in vivo regulation of fetal adrenocortical steroidogenesis during acute hypoxemia. In addition, the data reveal that CGRP may have a role in the control of other components of the hypothalamo-pituitary-adrenal axis during stimulated conditions in fetal life.
Introduction
DURING THREATENING SITUATIONS, the stress system coordinates adaptive responses that adjust homeostatic mechanisms to increase the chance of survival of the individual. The hypothalamo-pituitary-adrenal (HPA) axis constitutes one of the efferent limbs of the stress system (1). In the adult individual, it has long been known that secretion of adrenal glucocorticoids is principally controlled by the release of ACTH from the anterior pituitary, but only comparatively recently has it become appreciated that this is not an exclusive mechanism. Compelling evidence suggests that the splanchnic innervation to the adrenal gland plays an important role in modulating adrenocortical output during stimulated conditions. Edwards and Jones (2) have shown, in conscious hypophysectomized calves, that stimulation of the splanchnic innervation to the gland doubles, whereas section of the splanchnic nerves halves (3), the output of cortisol in response to an exogenous infusion of ACTH.
In the fetus, similar neural mechanisms may operate in the control of adrenocortical sensitivity during stimulated conditions. Three pieces of evidence support this assertion. First, functional innervation of the ovine fetal adrenal gland is present by the final third of gestation (4, 5). Second, Myers et al. (6) showed that splanchnic nerve section in the ovine fetus had no effect on basal cortisol but that denervation significantly attenuated the cortisol response to acute fetal hypotension. Third, our group has previously reported that in fetal mammals during acute hypoxemia, bilateral section of the carotid sinus nerves can completely prevent the increase in plasma cortisol without affecting either the increase in plasma ACTH concentrations or the adrenal vasodilatation and, hence, the delivery of plasma ACTH to the fetal adrenal cortex (7, 8). Taken together, past data, therefore, suggest that a chemoreflex triggered by the carotid chemoreceptors, and mediated via splanchnic efferent pathways, may operate to regulate adrenocortical responsiveness to ACTH during episodes of acute hypoxemic stress in the fetal period.
A component of splanchnic control of adrenal function may be regulated by the release of peptides, which may directly stimulate steroidogenesis and/or increase adrenal blood flow to accelerate the presentation of ACTH to the adrenal cortex (9, 10, 11). A plausible candidate for this peptidergic regulation of adrenocortical function includes calcitonin gene-related peptide (CGRP), because CGRP-stained fibers have been shown within the adrenal cortex, even in fetal animals during late gestation (4). Furthermore, exogenous treatment with CGRP promotes an increase in steroid output from the adrenal glands of adult rats, calves, and frogs (12, 13, 14), an effect that may or may not be mediated via the nitric oxide-dependent dilator action of the peptide within the adrenal gland (12, 14). However, no study has investigated the function of CGRP in the in vivo regulation of adrenocortical steroidogenic output during hypoxemic stress in either adult or fetal animals. Therefore, by investigating the effects of fetal treatment with a selective CGRP antagonist on the plasma ACTH and cortisol responses to acute hypoxemia in the chronically instrumented late-gestation ovine fetus, this study tested the hypothesis that CGRP has a role in mediating the in vivo adrenal glucocorticoid response to acute stress in the mature fetus.
Materials and Methods
Surgical preparation
All procedures were performed under the United Kingdom Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Cambridge. Six Welsh Mountain sheep fetuses were surgically instrumented for long-term recording at 120 ± 1 d gestation (term is 145 d) using strict aseptic conditions as described previously in detail (15). In brief, food, but not water, was withheld from the pregnant ewes for 24 h before surgery. After induction with 20 mg·kg–1 iv sodium thiopentone (Intraval Sodium; Merial Animal Health Ltd., Rhone Mérieux, Dublin, Ireland), general anesthesia (1.5–2.0% halothane in 50% O2:50% N2O) was maintained using positive-pressure ventilation. Midline abdominal and uterine incisions were made, the fetal hindlimbs were exteriorized, and, on one side, femoral arterial (inner diameter, 0.86 mm; outer diameter, 1.52 mm; Critchly Electrical Products, New South Wales, Australia) and venous (inner diameter, 0.56 mm; outer diameter, 0.96 mm) catheters were inserted. The catheter tips were advanced carefully to the descending aorta and inferior vena cava, respectively. Another catheter was anchored onto the fetal hindlimb for recording of the reference amniotic pressure. The uterine incisions were closed in layers, the dead space of the catheters was filled with heparinized saline (80 IU heparin·ml–1 in 0.9% NaCl), and the catheter ends were plugged with sterile brass pins. The catheters were then exteriorized via a keyhole incision in the maternal flank and kept inside a plastic pouch sewn onto the maternal skin.
Postoperative care
During recovery, ewes were housed in individual pens in rooms with a 12-h light, 12-h dark cycle where they had free access to hay and water and were fed concentrates twice daily (100 g sheep nuts no. 6; H & C Beart Ltd., Kings Lynn, UK). Antibiotics were administered daily to the ewe (0.20–0.25 mg·kg–1 im Depocillin; Mycofarm, Cambridge, UK) and fetus intravenously and into the amniotic cavity (150 mg·kg–1 Penbritin; SmithKline Beecham Animal Health, Welwyn Garden City, Hertfordshire, UK). The ewes also received 2 d of postoperative analgesia if in pain (10–20 mg·kg–1 oral Phenylbutazone; Equipalozone paste, Arnolds Veterinary Products Ltd., Shropshire, UK), as assessed by their general demeanor and feeding patterns. Generally, normal feeding patterns were restored within 48 h of recovery. After 72 h of postoperative recovery, ewes were transferred to metabolic crates where they were housed for the remainder of the protocol. The arterial and amniotic catheters were connected to sterile pressure transducers (COBE; Argon Division, Maxxim Medical, Athens, TX). Calibrated mean fetal arterial blood pressure (corrected for amniotic pressure) and fetal heart rate (triggered via a tachometer from the pulsatility in the arterial blood pressure) were recorded continually at 1-sec intervals using a computerized Data Acquisition System (Department of Physiology, Cambridge University). While on the metabolic crates, the patency of the fetal catheters was maintained by a slow continuous infusion of heparinized saline (80 IU heparin·ml–1 at 0.1 ml·h–1 in 0.9% NaCl) containing antibiotic (1 mg·ml–1 benzylpenicillin; Crystapen, Schering-Plough, Animal Health Division, Welwyn Garden City, UK).
Experimental protocol
After at least 5 d of postoperative recovery, all fetuses were subjected to two experimental protocols, carried out on consecutive days in a randomized order (Fig. 1). Each protocol consisted of a 2.5-h period divided into 1-h normoxia, 0.5-h hypoxemia, and 1-h recovery. At the midpoint (30 min) of the first normoxic period, fetal treatment with either a slow iv infusion of vehicle (80 IU heparin·ml–1 in 0.9% NaCl) or the CGRP antagonist dissolved in heparinized saline (42.6 ± 1.8 μg·kg–1 intraarterial bolus followed by 8.5 ± 0.4 μg·kg–1·min–1 iv infusion; Calcitonin Gene Related Peptide fragment 8–37, CGRP8–37; C-2806; Sigma Chemicals, Dorset, UK) was commenced (Fig. 1). The dose of CGRP antagonist used has been calculated retrospectively from the fetal weights (2.4 ± 0.1 kg) obtained at postmortem. Acute hypoxemia in the fetus was induced by maternal inhalational hypoxia. In brief, a large transparent respiratory hood was placed over the heads of the ewes into which air was passed at a rate of approximately 50 liter·min–1 for the 1-h period of normoxia. After this control period, acute fetal hypoxemia was induced for 30 min by changing the concentrations of gases breathed by the ewe to 6% O2 in N2 with small amounts of CO2 (15 liter·min–1 air:35 liter·min–1 N2:1.5–2.5 liter·min–1 CO2). This mixture was designed to reduce fetal arterial partial pressure of oxygen (PaO2) to approximately 10 mm Hg while maintaining arterial partial pressure of CO2 (PaCO2). After the 0.5-h period of hypoxemia, the ewe was returned to breathing air for the 1-h recovery period. At the end of the experimental protocol, the ewes and fetuses were humanely killed using a lethal dose of sodium pentobarbitone (200 mg·kg–1 iv Pentoject; Animal Ltd., York, UK). Postmortems were carried out at 130 ± 1 d gestation, during which time the positions of the implanted catheters were confirmed and the fetuses were weighed.
Blood sampling regimen
During any acute hypoxemia protocol, descending aortic blood samples (0.3 ml) were taken using sterile techniques from the fetus at set time intervals (Fig. 1, arrows) to determine arterial blood gas and acid base status (ABL5 Blood Gas Analyser, Radiometer; Copenhagen, Denmark; measurements corrected to 39.5 C). Values for percentage saturation of hemoglobin with oxygen (Sat Hb) and the blood hemoglobin concentration ([Hb]) were determined using a hemoximeter (OSM2; Radiometer). An additional 2 ml of arterial blood was withdrawn at set intervals for hormone analyses (Fig. 1, arrows). These samples were collected under sterile conditions into chilled EDTA tubes (2 ml K+/EDTA; L.I.P., Ltd., Shipley, West Yorkshire, UK) for ACTH and cortisol analysis. All samples were then centrifuged at 4000 rpm for 4 min at 4 C. The plasma obtained was then dispensed into prelabeled tubes, and the samples were stored at –20 C until analysis.
Hormone analyses
All hormone measurements were performed within 2 months of sample collection. Plasma ACTH and cortisol concentrations were determined by RIA validated for use in ovine plasma.
ACTH assay
Fetal plasma ACTH concentrations were measured using a commercially available double antibody 125I RIA kit (DiaSorin Inc., Stillwater, MN) as previously described (16). The kit contained a set of ACTH standards (porcine; range, 20–500 pg·ml–1), ACTH quality control samples, lyophilized rabbit anti-ACTH antiserum, 125I-labeled synthetic ACTH, and a second antibody-precipitating complex (goat antirabbit -globulin serum and polyethylene glycol in buffer). Reagents were reconstituted in deionized water and were kept on crushed ice. Duplicate 100-μl aliquots of the ACTH standards, quality controls, and unknown plasma samples (taken from previously unthawed K+/EDTA-treated plasma aliquots) were incubated overnight at 5 C with 200 μl of anti-ACTH antiserum and 200 μl of 125I-labeled ACTH. Bound and free ACTH fractions were separated by immunoprecipitation with the second antibody-precipitating complex (500 μl). After centrifugation (3600 rpm; 20 min; 20–25 C), the supernatant was rapidly decanted and discarded and the residue pellets allowed to dry. A scintillation counter (Cobra II Auto-Gamma Packard Bioscience Co., Pangbourne, Berks, UK) was used to count the radioactivity of each pellet for 2 min, together with that of duplicate nonspecific binding samples (anti-ACTH antiserum omitted) and that of total counts tubes (200 μl of 125I-labeled ACTH). A standard curve was produced by the computer using a logistic curve fit, and ACTH concentrations were calculated for each pellet.
The lower limit of detection of the assay (90% bound·free–1) was between 10–25 pg·ml–1. The intraassay coefficients of variation (CV) for three plasma pools (mean concentration: 69, 281, and 600 pg·ml–1) were 12.5, 6.3, and 8.1%, respectively. The interassay CV for two plasma pools (mean concentration: 36 and 136 pg·ml–1) were 6.0 and 6.7%, respectively. The anti-ACTH antiserum showed less than 0.01% cross-reactivity against -MSH, -endorphin, -lipotropin, leucine enkephalin, methionine enkephalin, bombesin, calcitonin, parathyroid hormone, follicle-stimulating hormone, arginine vasopressin, oxytocin, and substance P.
Cortisol assay
Fetal plasma cortisol concentrations were measured by RIA using tritium-labeled cortisol as tracer, as previously described (17). Duplicate 20-μl plasma samples were taken from previously unthawed K+/EDTA-treated sample aliquots and were extracted twice in 300 μl of ethanol. After each extraction, the aliquots were centrifuged and the ethanol layer decanted into a bacteriological test tube. The combined ethanol extracts were evaporated in a drier (Speedvac Concentrator, SVC 200H; Stratech Scientific, Luton, UK) overnight. Residues were reconstituted in 100 μl of PBS (pH 7.0) and were allowed to stand at 5 C for at least 2 h. Samples in which cortisol concentration values were predicted to be in the upper range of the standard curve were diluted using PBS to reduce the concentration to within the most sensitive range (1–10 ng·ml–1) of the standard curve. Values for these samples were subsequently corrected for the dilution factor used in each case. The plasma extracts, quality control samples, and working standards (1, 2.5, 5, 10, 25, 50, and 100 ng·ml–1) were incubated overnight (5 C) with 100 μl of antiserum (Tenovus Institute, Cardiff, UK) and 100 μl of [1,2,6,7-3H]-labeled cortisol (TRK407; Amersham Pharmacia Biotech United Kingdom Ltd., Little Chalfont, Buckinghamshire, UK). Bound and free cortisol fractions were separated using stirred dextran-coated charcoal suspension (300 μl; 0.075 g/100 ml dextran and 0.75 g/100 ml activated charcoal in PBS). After immediate centrifugation (3600 rpm; 7 min; 4 C), the supernatant was rapidly decanted into scintillation vial inserts, and 4 ml of scintillant (OptiPhase HiSafe; Fisons Chemicals, Loughborough, UK) was added to each insert. The inserts were capped and the contents thoroughly mixed. After a 1-h equilibration period, a scintillation counter (1216 RackBeta Liquid Scintillation Counter; LKB Wallac, Sweden) was used to count the radioactivity of each insert for 5 min, including the radioactive content of duplicate nonspecific binding samples (antiserum omitted) and total counts tubes (100 μl of 3H-labeled cortisol). Recoveries averaged 90%. A standard curve was produced by the computer using a spline curve fit, and cortisol concentrations were calculated for each sample.
The lower limit of detection of the assay was between 1.0 and 1.5 ng·ml–1. The intraassay CV was 5.3% for a mean value of 13 ng·ml–1. The interassay CV for two plasma pools (mean concentration, 13 and 28 ng·ml–1) were 13.6 and 11.4%, respectively. The cross-reactivities of the antiserum at 50% binding with other cortisol-related compounds were as follows: 0.5% cortisone, 2.3% corticosterone, 0.3% progesterone, 4.6% deoxycortisol.
Data and statistical analyses
Values for all arterial blood gas and endocrine variables are expressed as mean ± SEM at 0 (N0) and 45 (N45) min of normoxia, 15 (H15) and 30 (H30) min of hypoxemia, and at 30 (R30) and 60 (R60) min of recovery for fetuses exposed to 30 min of hypoxemia during saline infusion or during treatment with CGRP antagonist. The cardiovascular variables are expressed as minute averages at the corresponding time points. All measured variables were first analyzed for normality of distribution and then assessed using two-way ANOVA with repeated measures (Sigma-Stat; SPSS Inc., Chicago, IL) comparing the effect of time (normoxia vs. hypoxemia/recovery), group (saline vs. CGRP antagonist), and interactions between time and group. Where a significant effect of time or group was indicated, the post hoc Tukey test was used to isolate the statistical differences. The relationship between parallel measurements of plasma concentrations of ACTH and cortisol in all individual fetuses was assessed using the Pearson Product Moment correlation. For all comparisons, statistical significance was accepted when P < 0.05.
Results
Fetal arterial blood gas and acid base status
Basal values for arterial blood gas and acid base status were similar in all fetuses. Infusion with saline or treatment with the CGRP antagonist had no effect on basal arterial blood gas and acid-base status (Table 1). In all fetuses, acute hypoxemia induced significant falls in arterial pH (pHa), acid base excess (ABE), PaO2, and Sat Hb, without any alteration to PaCO2. The magnitude of these changes was similar during saline infusion or during treatment with the CGRP antagonist (Table 1). However, the maximal increment in fetal [Hb] during acute hypoxemia was significantly diminished during treatment with the CGRP antagonist (0.77 ± 0.46 g·dl–1) when compared with saline-infused controls (2.15 ± 0.42 g·dl–1; P < 0.05). During recovery, pHa and ABE remained significantly depressed by the end of the experimental protocol in all fetuses, whereas PaO2, Sat Hb, and [Hb] returned to basal values.
Fetal pituitary-adrenocortical function
Plasma ACTH concentrations
Basal plasma concentrations of ACTH were similar in all fetuses before saline infusion (22 ± 1 pg·ml–1) or CGRP antagonist treatment (27 ± 4 pg·ml–1). Infusion with saline or treatment with the CGRP antagonist had no effect on basal plasma concentrations of ACTH (Fig. 2). Acute hypoxemia resulted in a pronounced increase in plasma ACTH in all fetuses. However, the increment in plasma ACTH during hypoxemia was more than halved during treatment with the CGRP antagonist (472 ± 108 pg·ml–1) when compared with the increment in saline-infused controls (968 ± 187 pg·ml–1; P < 0.05). During recovery, plasma ACTH concentrations returned to basal values in all fetuses.
Plasma cortisol concentrations
Basal plasma concentrations of cortisol were also similar in all fetuses before saline infusion (14 ± 2 ng·ml–1) or CGRP antagonist treatment (14 ± 4 ng·ml–1). Infusion with saline or treatment with the CGRP antagonist had no effect on basal plasma concentrations of cortisol (Fig. 2). Acute hypoxemia resulted in a pronounced increase in plasma cortisol in all fetuses. However, the increment in plasma cortisol during acute hypoxemia was significantly diminished during treatment with the CGRP antagonist (22.6 ± 4.8 ng·ml–1) when compared with the increment in saline-infused controls (35.2 ± 8.2 ng·ml–1; P < 0.05). During recovery, plasma cortisol concentrations returned to basal values in all fetuses.
Correlation analysis of paired plasma ACTH and cortisol values for all individual fetuses during basal and hypoxemic conditions, during either saline infusion (r = 0.69, n = 24, P < 0.001) or during treatment with the CGRP antagonist (r = 0.59, n = 24, P < 0.001), revealed significant positive relationships (Fig. 3A). Treatment with the CGRP antagonist did not affect the slope or the intercept of the peptide-steroid relationship compared with saline infusion (Fig. 3B).
Fetal cardiovascular function
Basal values for arterial blood pressure and heart rate were similar in all fetuses before saline infusion or CGRP antagonist treatment. Infusion with saline or treatment with the CGRP antagonist had no effect on the basal cardiovascular variables (Table 2). During acute hypoxemia, a significant increase in arterial blood pressure (from 48.9 ± 2.0 to 56.8 ± 2.0 mm Hg) and fall in heart rate (from 170 ± 11 to 140 ± 13 beats·min–1) occurred in all fetuses during saline infusion (P < 0.05). The magnitude of these cardiovascular responses during acute hypoxemia were not affected during treatment with the CGRP antagonist, because arterial blood pressure increased from 46.9 ± 1.7 to 56.0 ± 2.6 mm Hg and fetal heart rate fell from 170 ± 4 to 144 ± 14 beats·min–1. During recovery, fetal arterial blood pressure remained significantly elevated by the end of the experimental protocol in all fetuses, whereas fetal heart rate returned to basal values.
Discussion
CGRP is a 37-amino acid peptide discovered in 1983 by alternative processing of RNA transcripts of the gene encoding calcitonin (18). In addition to being one of the most potent vasodilator peptides known (19), CGRP can also acts as a neuropeptide, often found colocalized with substance P (20) throughout the central and peripheral nervous systems (21, 22, 23). In the adult, immunocytochemical studies have demonstrated several nerve plexuses and terminals, which stain positively for CGRP (4, 14) as well as the presence of CGRP receptors at the level of the adrenal cortex (24), consistent with a potential role for CGRP in regulating adrenal function. In vivo studies in hypophysectomized calves with the adrenal clamp technique (12) or in isolated frog or rat adrenal glands perfused in situ (13, 14) have confirmed a functional role for CGRP in stimulating adrenal glucocorticoid output. However, in vitro studies using dispersed adrenocortical cells in the rat have reported that CGRP does not affect either basal or ACTH-stimulated glucocorticoid production (25). The apparent discrepancy has been attributed to the type of preparation (14). Although, in isolated cellular studies, CGRP appears to have an inhibitory effect on adrenocortical secretion, this effect appears to be overridden when the integrity and functional architecture of the adrenal tissue is preserved and when the adrenal gland is exposed to systemic influences. A net stimulatory mechanism may rely on the close and direct relationship between the rate of blood flow through the intact adrenal gland and the rate of glucocorticoid release (14). Hence, in the intact adrenal, CGRP may also act as a powerful vasodilator increasing adrenal blood flow and, thereby, the presentation rate of trophic agents, such as ACTH, resulting in an overall increase in adrenocortical secretion. However, Bloom et al. (12) suggested that CGRP might have blood flow-independent, direct steroidogenic effects in the conscious hypophysectomized calf, because in their study, the increase in glucocorticoid output could not be completely accounted for by an increase in ACTH presentation to the adrenal gland. Data from our in vivo study in the ovine fetus show that during CGRP antagonism, there was a marked attenuation in the fetal plasma cortisol response to acute hypoxemia, implying for the first time in fetal life, that CGRP may also have a stimulatory role in the control of adrenal function. However, in the present study, CGRP antagonism also caused a marked attenuation in the fetal plasma ACTH concentrations during acute hypoxemia, and detailed assessment of the paired plasma ACTH and cortisol concentrations during basal and hypoxemic conditions in all individual fetuses showed no effects of CGRP antagonism on the slope or the intercept of the peptide-steroid relationship. Combined, these results suggest that in the ovine fetus, CGRP antagonism may have diminished plasma concentrations of cortisol during acute hypoxemia purely by virtue of an effect on plasma concentrations of ACTH. Therefore, the data also emphasize a regulatory role for CGRP higher up the HPA axis in the fetus, either in the control of hypothalamic CRH and/or pituitary ACTH release.
Several reports support a functional role for CGRP in the control of pituitary ACTH release in the adult individual. Peptidergic fibers containing CGRP have been found to innervate the anterior pituitary of the monkey, dog, rat, and human (26, 27), where CGRP-binding sites have also been detected (23, 28). These fibers exhibit morphological, distributional, and numerical changes after stress and adrenalectomy (29, 30). In addition, a close anatomical arrangement of peptidergic fibers exists with pituitary glandular cells, with synaptic contacts being made specifically with corticotropes (30). In cultured rat anterior pituitary cells, Iino et al. (31) have shown that CGRP stimulates ACTH secretion and that this effect is prevented by inhibition of protein kinase A. This suggests that CGRP may be directly involved in the synthesis and/or release of pituitary ACTH by increasing intracellular cAMP, which, in turn, may activate the adenylate-cyclase-protein kinase system (32). Similarly, evidence exists to show the presence of CGRP fibers and CGRP-binding sites in adult rat (22, 23) and human (28) hypothalamus. Two recent studies in adult rats support a functional role of CGRP in hypothalamic stimulation. Li et al. (33) have shown that central administration of CGRP into the lateral cerebral ventricles induces c-Fos expression in the preoptic area and paraventricular nucleus of the hypothalamus. Furthermore, Dhillo et al. (34) have reported that central administration of CGRP into the paraventricular nucleus leads to an increase in plasma ACTH and corticosterone concentrations, an effect that is prevented by treatment with a CRH antagonist. Our study is the first to suggest regulation of pituitary ACTH by CGRP during stimulated conditions in fetal life; however, whether this control is secondary to an effect at the hypothalamic level remains unknown.
It is well established that CRH release is regulated by central sympathetic pathways (1) and that central sympathetic outflow can be affected by CGRP (35, 36, 37). Indirect evidence from the present study showing an effect of CGRP antagonism on the concentration of fetal hemoglobin during hypoxemia may support a central effect of the antagonist on sympathetic outflow and, thereby, on central sympathetic regulation of CRH release. During acute hypoxemia, increased sympathetic outflow stimulates contraction of the spleen and other venous reservoirs (38). This decreases venous capacitance and shifts blood into the systemic circulation, promoting an acute increase in red blood cell count and, hence, [Hb] concentration. Increased sympathetic outflow during acute hypoxemia also promotes an increase in total peripheral vascular resistance and, hence, an increase in arterial blood pressure in the ovine fetus (39). In the present study, although CGRP antagonism markedly diminished the increase in [Hb] concentration, it did not affect the magnitude of the pressor response to acute hypoxemia, partially supporting an effect on sympathetic outflow. However, it must be recognized that multiple mechanisms in addition to sympathetic stimulation mediate the fetal arterial blood pressure response to acute hypoxemia (40). Hence, studies comparing the effects of central vs. systemic administration of CGRP antagonism, or those addressing the effects of CGRP antagonism on actual changes in peripheral vascular resistance in the ovine fetus, may provide greater insight on the central role of CGRP on HPA and cardiovascular function in fetal life.
Although measurement of plasma concentrations of ACTH and cortisol after exogenous intravenous treatment of the sheep fetus with CGRP may be an interesting experiment to perform, it may offer little additional insight into the effects of CGRP on pituitary-adrenal function. The current study proposed that in the fetus, sympathetic activity would modify pituitary-adrenocortical function via a mechanism involving CGRP. Exogenous, systemic treatment of the ovine fetus with CGRP will not mimic the synaptic concentrations of the peptide within the sympathetic nervous system. Hence, a negative effect of exogenous, systemic CGRP treatment on plasma glucocorticoid concentration will not necessarily imply that CGRP is not involved in pituitary or adrenocortical regulation.
In conclusion, the present study tested the hypothesis that CGRP has a role in mediating the in vivo adrenal glucocorticoid response to acute hypoxemic stress in the mature fetus. The results from this study show that fetal treatment with a CGRP antagonist significantly diminished both the plasma ACTH and cortisol responses to acute hypoxemia. Furthermore, correlation analysis of paired plasma ACTH and cortisol concentrations in all individual fetuses during basal and hypoxemic conditions revealed significant positive relationships. However, neither the slope nor the intercept of the peptide-steroid relationship was affected by CGRP antagonist treatment. These data partly support the hypothesis tested in this study but reveal that CGRP may have a role in the control of other components of the HPA axis during stimulated conditions in the late-gestation ovine fetus.
Footnotes
This work was supported by the International Journal of Experimental Pathology and the Lister Institute for Preventive Medicine. D.A.G. is a Fellow of the Lister Institute for Preventive Medicine.
Abbreviations: ABE, Acid base excess; CGRP, calcitonin gene-related peptide; CV, coefficient(s) of variation; [Hb], blood hemoglobin concentration; HPA, hypothalamo-pituitary-adrenal; PaCO2, arterial partial pressure of CO2; PaO2, arterial partial pressure of oxygen; pHa, arterial pH; Sat Hb, saturation of hemoglobin with oxygen.
References
Tsigos C, Chrousos GP 2002 Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress. J Psychosom Res 53:865–871
Edwards AV, Jones CT 1987 The effect of splanchnic nerve stimulation on adrenocortical activity in conscious calves. J Physiol 382:385–396
Edwards AV, Jones CT 1987 The effect of splanchnic nerve section on the sensitivity of the adrenal cortex to adrenocorticotrophin in the calf. J Physiol 390:23–31
Engeland WC, Wotus C, Rose JC 1998 Ontogeny of innervation of rat and ovine fetal adrenals. Endocr Res 24:889–898
Comline RS, Silver M 1961 The release of adrenaline and noradrenaline from the adrenal glands of the foetal sheep. J Physiol 156:424–444
Myers DA, Robertshaw D, Nathanielsz PW 1990 Effect of bilateral splanchnic nerve section on adrenal function in the ovine fetus. Endocrinology 127:2328–2335
Giussani DA, McGarrigle HH, Moore PJ, Bennet L, Spencer JA, Hanson MA 1994 Carotid sinus nerve section and the increase in plasma cortisol during acute hypoxia in fetal sheep. J Physiol. 477(Pt 1):75–80
Riquelme RA, Llanos JA, McGarrigle HH, Sanhueza EM, Hanson MA, Giussani DA 1998 Chemoreflex contribution to adrenocortical function during acute hypoxemia in the llama fetus at 0.6 to 0.7 of gestation. Endocrinology 139:2564–2570
Edwards AV, Jones CT 1993 Autonomic control of adrenal function. J Anat. 183 (Pt 2):291–307
Ehrhart-Bornstein M, Hinson JP, Bornstein SR, Scherbaum WA, Vinson GP 1998 Intraadrenal interactions in the regulation of adrenocortical steroidogenesis. Endocr Rev 19:101–143
Vinson GP, Hinson JP, Toth IE 1994 The neuroendocrinology of the adrenal cortex. J Neuroendocrinol 6:235–246
Bloom SR, Edwards AV, Jones CT 1989 Adrenal responses to calcitonin gene-related peptide in conscious hypophysectomized calves. J Physiol 409:29–41
Esneu M, Delarue C, Remy-Jouet I, Manzardo E, Fasolo A, Fournier A, Saint-Pierre S, Conlon JM, Vaudry H 1994 Localization, identification, and action of calcitonin gene-related peptide in the frog adrenal gland. Endocrinology 135:423–430
Hinson JP, Vinson GP 1990 Calcitonin gene-related peptide stimulates adrenocortical function in the isolated perfused rat adrenal gland in situ. Neuropeptides 16:129–133
Giussani DA, Gardner DS, Cox DT, Fletcher AJ 2001 Purinergic contribution to circulatory, metabolic, and adrenergic responses to acute hypoxemia in fetal sheep. Am J Physiol Regul Integr Comp Physiol 280:R678–R685
Gardner DS, Fletcher AJ, Bloomfield MR, Fowden AL, Giussani DA 2002 Effects of prevailing hypoxaemia, acidaemia or hypoglycaemia upon the cardiovascular, endocrine and metabolic responses to acute hypoxaemia in the ovine fetus. J Physiol 540:351–366
Robinson PM, Comline RS, Fowden AL, Silver M 1983 Adrenal cortex of fetal lamb: changes after hypophysectomy and effects of Synacthen on cytoarchitecture and secretory activity. Q J Exp Physiol 68:15–27
Rosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vale WW, Evans RM 1983 Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304:129–135
Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I 1985 Calcitonin gene-related peptide is a potent vasodilator. Nature 313:54–56
Franco-Cereceda A, Henke H, Lundberg JM, Petermann JB, Hokfelt T, Fischer JA 1987 Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin. Peptides 8:399–410
Maggi CA 1995 Tachykinins and calcitonin gene-related peptide (CGRP) as co-transmitters released from peripheral endings of sensory nerves. Prog Neurobiol 45:1–98
Skofitsch G, Jacobowitz DM 1985 Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6:721–745
Wimalawansa SJ, Emson PC, MacIntyre I 1987 Regional distribution of calcitonin gene-related peptide and its specific binding sites in rats with particular reference to the nervous system. Neuroendocrinology 46:131–136
Renshaw D, Cruchley AT, Kapas S, Hinson JP 1998 Receptors for calcitonin gene-related peptide (CGRP) in the rat adrenal cortex. Endocr Res 24:773–776
Mazzocchi G, Musajo F, Neri G, Gottardo G, Nussdorfer GG 1996 Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: comparison with calcitonin gene-related peptide effects. Peptides 17:853–857
Ju G, Liu SJ, Zhang X 1991 Peptidergic innervation of the pars distalis of the adenohypophysis. Neuroendocrinology 53(Suppl 1):41–44
Liu S 2004 Peptidergic innervation in pars distalis of the human anterior pituitary. Brain Res 1008:61–68
Tschopp FA, Henke H, Petermann JB, Tobler PH, Janzer R, Hokfelt T, Lundberg JM, Cuello C, Fischer JA 1985 Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary. Proc Natl Acad Sci USA 82:248–252
Hu D, Chen B, Wang B, Ma D 2002 The changes in the morphology and distribution of substance P and calcitonin-gene related peptide nerves in the anterior pituitary of scalded rats. Zhonghua Shao Shang Za Zhi 18:176–179
Ju G, Ma D, Fan SC 1994 Response to calcitonin-gene-related peptide-like immunoreactive nerve fibres of the anterior pituitary to adrenalectomy in the rat. Neuroendocrinology 505–510
Iino K, Oki Y, Tominaga T, Iwabuchi M, Ozawa M, Watanabe F, Yoshimi T 1998 Stimulatory effect of calcitonin gene-related peptide on adrenocorticotropin release from rat anterior pituitary cells. J Neuroendocrinol 10:325–329
Poyner DR 1992 Calcitonin gene-related peptide: multiple actions, multiple receptors. Pharmacol Ther 56:23–51
Li XF, Bowe JE, Mitchell JC, Brain SD, Lightman SL, O’Byrne KT 2004 Stress-induced suppression of the gonadotropin-releasing hormone pulse generator in the female rat: a novel neural action for calcitonin gene-related peptide. Endocrinology 145:1556–1563
Dhillo WS, Small CJ, Jethwa PH, Russell SH, Gardiner JV, Bewick GA, Seth A, Murphy KG, Ghatei MA, Bloom SR 2003 Paraventricular nucleus administration of calcitonin gene-related peptide inhibits food intake and stimulates the hypothalamo-pituitary-adrenal axis. Endocrinology 144:1420–1425
Bereiter DA, Benetti AP 1991 Microinjections of calcitonin gene-related peptide within the trigeminal subnucleus caudalis of the cat affects adrenal and autonomic function. Brain Res 558:53–62
Fisher LA, Kikkawa DO, Rivier JE, Amara SG, Evans RM, Rosenfeld MG, Vale WW, Brown MR 1983 Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature 305:534–536
Hasegawa T, Yokotani K, Okuma Y, Manabe M, Hirakawa M, Osumi Y 1993 Microinjection of alpha-calcitonin gene-related peptide into the hypothalamus activates sympathetic outflow in rats. Jpn J Pharmacol 61:325–332
Hoka S, Bosnjak ZJ, Arimura H, Kampine JP 1989 Regional venous outflow, blood volume, and sympathetic nerve activity during severe hypoxia. Am J Physiol 256:H162–H170
Giussani DA, Spencer JA, Moore PJ, Bennet L, Hanson MA 1993 Afferent and efferent components of the cardiovascular reflex responses to acute hypoxia in term fetal sheep. J Physiol 461:431–449
Giussani DA, Spencer JA, Hanson MA 1994 Fetal cardiovascular reflex responses to acute hypoxaemia. Fetal Matern Med Rev 6:17–37(A. S. Thakor and D. A. Gi)