001H-7227/H1/1291-0295SIW.00/0 Kndocrinology Copyright i 191)1 by The Endocrine Society
Vol. 129, No. 1
Printed in U.S.A.
Regulation of Basal Adrenocorticotropin and Cortisol Secretion by Arginine Vasopressin in the Fetal Sheep during Late Gestation* EDE MARIE APOSTOLAKIS, LAWRENCE D. LONGO, AND STEVEN M. YELLON Division of Perinatal Biology, Departments of Physiology, Pediatrics, and Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, California 92350
cortisol remained unchanged after the AVP challenge. To further define the role of endogenous AVP in basal ACTH and cortisol secretion, the AVP antagonist was administered (five studies in two fetuses) at 30-min intervals for a total of three injections per fetus. This extended AVP antagonist regimen also failed to alter fetal circulating concentrations of ACTH or cortisol (P > 0.05). Cortisol in the maternal circulation was not affected by any of the fetal AVP or AVP antagonist treatments. Lambs were born at 146 ± 2 days gestation (n = 5), within the range for the normal duration of pregnancy. These data do not support the hypotheses that AVP is involved in the regulation of basal ACTH secretion in the fetal sheep during the 10 days preceding parturition. Rather, the ability of AVP antagonist to block the AVP-induced rise in plasma ACTH and cortisol in the fetus suggests that basal and stimulated ACTH secretion are under separate regulatory mechanisms. (Endocrinology 129: 295-300, 1991)
ABSTRACT. This study tested the hypothesis that arginine vasopressin (AVP) is involved in the regulation of basal ACTH secretion in the ovine fetus near term. In five fetuses challenged with AVP (1 /ug/ml, iv bolus) plasma ACTH concentrations increased to an 8-fold peak within 10 min of the preceding baseline (55 ± 6 to 403 ± 241 pg/ml). Cortisol in fetal circulation subsequently increased 2-fold (11 ± 1 to 28 ± 5 ng/ml) within 15 min of the AVP injection. The AVP-induced rise in plasma ACTH and cortisol concentrations was blocked when the fetus was pretreated with the AVP V] receptor antagonist d(0h..>)5Tyr(Me)AVP. In a total of seven studies, antagonist (10 Aig/kg estimated BW, iv bolus) was administered to three fetuses, aged 137-147 days gestation, followed 40 min later by the exogenous AVP challenge, as described above. After AVP antagonist treatment, basal ACTH and cortisol concentrations were not significantly different from the preinjection baseline levels (P > 0.05, by analysis of variance). Moreover, plasma ACTH and
I
N THE fetal sheep near term, activation of the pituitary-adrenal axis is part of the endocrine cascade that initiates parturition (1, 2). Cortisol in fetal circulation gradually increases over the last third of gestation, abruptly rising during the final 72 h to peak on the day of birth (~147 days in sheep). During this time period, fetal ACTH secretion also increases, but whether ACTH drives the prepartum cortisol rise is controversial (3-5). The importance of ACTH secretion for stimulation of adrenal cortisol in the ovine fetus has led to investigations of the neuroendocrine mechanism controlling its pituitary release. In adult sheep, arginine vasopressin (AVP) is the predominant regulator of ACTH secretion and biosynthesis (6-9). In fetal sheep, both AVP and
CRF given iv induce fetal ACTH release (10, 11), although the response appears to be greater to AVP than to CRF (10). Both releasing factors are present in the hypothalamus of young fetuses by 90 days gestation, and the AVP content is greater than that of CRF (12). In addition to its presence in hypophyseal-portal vessels (13), AVP from fibers in contact with pituitary corticotropes (14) may influence basal ACTH secretion. Moreover, Maclsaac and colleagues (15) found that AVPstimulated ACTH release was blocked by pretreatment with an AVP receptor antagonist in 106- to 118-day-old fetuses. Whether antagonist treatment affects basal ACTH secretion in fetuses later in gestation, when plasma cortisol, ACTH, and AVP concentrations are increasing in the fetal circulation (16, 17), has not been examined. The goal of the present study was to determine whether AVP is involved in the regulation of basal plasma ACTH release and, subsequently, basal cortisol secretion during the last weeks of gestation in the ovine fetus. The specific AVP Vi receptor antagonist d(CH2)5Tyr(Me)AVP was administered to the fetus during late gestation in an
Received December 4, 1990. Address all correspondence and requests for reprints to: Steven M. Yellon, Ph.D., Division of Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California 92350. * This work was supported by grants from the USPHS (HD-03817) and the Center for Nursing Research (NRSA-06042), and the Pediatrics Department Research Fund, Loma Linda University School of Medicine. Preliminary data from the study were presented at the 1990 Annual Meeting of the Society for Gynecologic Investigation, St. Louis, MO (Abstract 369).
295
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
AVP AND BASAL ACTH IN THE OVINE FETUS
296
attempt to block secretion of basal ACTH and reduce plasma cortisol concentrations in the fetal circulation.
Materials and Methods General Studies were performed in five time-dated pregnant singleton ewes of mixed Western breed (Nebeker Farms, Santa Monica, CA), housed in a controlled access light-locked room with constant humidity, temperature, and photoperiod (10 h of light/ day; lights on at 0700 h, PST). A dim red light remained on at night to facilitate collection of nighttime blood samples. Alfalfa pellets (Nebeker Farms) and water were provided in excess ad libitum. Pregnant ewes were acclimated to the animal care facility for 7 days. At 126 days gestation using sterile surgical procedures, catheters were placed in the brachial artery and cephalic vein of each fetal forelimb, as previously described (18). Catheter tips were advanced to obtain blood from the aortic arch and superior vena cava, respectively. At the same time, maternal catheters were inserted in the left external jugular vein and left pedal artery. All catheters were exteriorized through a left flank stab wound and stored in a nylon pouch sutured to the skin of the ewe. Antibiotics were administered to the ewe for 3 days (2.5 ml Combiotic, im; Pfizer, New York, NY) and to the fetus daily (40 mg Gentamycin, iv; ElkinsSinn, Cherry Hill, NJ). Catheters were flushed each day with heparinized saline (100 U/ml). To assess fetal well-being, arterial blood samples (0.3 ml) were drawn into heparinized syringes every morning (1000 h) for determination of pH, PO2, PCO2, hemoglobin, and percent oxyhemoglobin saturation (ABL300 and OSM2, respectively, Radiometer, Copenhagen, Denmark). Fetal and maternal blood samples (2 and 3 ml, respectively) were drawn daily (~1000 and 2200 h) into chilled EDTA-treated syringes. Blood samples were centrifuged at 4000 rpm for 15 min at 4 C. Plasma was harvested and stored at -70 C in polypropylene microcentrifuge tubes (Fisher Scientific Corp., Pittsburgh, PA) for later RIAs (see below). Experimental protocols The following experiments were conducted in the morning, beginning at 1045 h. A VP challenge. To induce a robust physiological rise in plasma ACTH and cortisol, we administered AVP (1 Mg/ml normal saline, iv bolus; Bachem, Torrance, CA), immediately followed by a 3-ml flush of lactated Ringer's solution (RL) to fetuses at 133 days gestation (n = 5). Beginning 15 min before the AVP injection, fetal (2 ml) and maternal (3 ml) arterial blood samples were taken every 5 min for 45 min, and less frequently thereafter. After each blood sample, RL was infused to replace the volume of withdrawn blood. In addition, fetal and maternal erythrocytes were reconstituted with RL (~3 times the volume) and returned every 20 min to the fetus and ewe, respectively. Arterial blood gases, hemoglobin, and percent oxyhemoglobin saturation were measured every 15-60 min to assess fetal wellbeing. We repeated the same protocol for a total of 75 min at 140 days gestation in two fetuses to test the ability of AVP to stimulate ACTH and cortisol later in gestation.
Endo • 1991 Vol 129 • No 1
AVP antagonist and challenge. To assess the contribution of endogenous AVP in the regulation of basal ACTH release and subsequent cortisol secretion, we administered the AVP Vj receptor antagonist d(CH2)5Tyr(Me)AVP (generously provided by M. Manning, Ph.D., Medical College of Toledo, Toledo, OH), to fetuses at various gestational ages. Antagonism of the AVP V, receptor by d(CH2)5Tyr(Me)AVP blocks AVPstimulated ACTH release in several species (19-22). In a total of seven experiments in three fetuses (137, 142, and 147 days gestation), the AVP antagonist was administered (10 fig/kg estimated BW/ml normal saline, iv bolus), followed immediately by 3 ml RL flush. Based on previous data from our laboratory, fetal weight was estimated to be about 3 kg (23). As before, erythrocytes were reconstituted with RL and returned. As part of this protocol, the efficacy of the receptor antagonism was tested. Each fetus was challenged with AVP (1 Mg/ml; as described above) 40 min after the antagonist injection. Beginning 15 min before the antagonist treatment, fetal (2 ml) and maternal (3 ml) blood samples were taken every 5 min for 115 min, and less frequently thereafter. To assess fetal well-being, arterial blood gases, hemoglobin, and percent oxyhemoglobin saturation were measured every 15-60 min. In another experiment, the duration of the AVP receptor antagonist treatment was extended to further define the role of AVP in the regulation of basal ACTH and cortisol secretion. Five studies were performed in two fetuses beginning at 137 days gestation (n = 2) and repeated at 142 and 147 days gestation (n = 2 and 1, respectively). The AVP antagonist was administered (10 Mg/kg estimated BW) at 30-min intervals (total of three injections). Fetal and maternal blood samples were taken every 5 min for 115 min, beginning 15 min before the first antagonist injection. Again to assess fetal well-being, arterial blood gases, hemoglobin, and percent oxyhemoglobin saturation were measured every 15-60 min. ACTH and cortisol RIAs ACTH was measured with a commercial kit (IncStar Corp., Stillwater, MN), previously validated for use in the fetal sheep (24). Parallelism, accuracy, and repeatability were verified in our laboratory using plasma pools from pregnant and fetal sheep. The intra- and interassay coefficients of variation were 6% and 11%, respectively (n = 12 assays). Assay sensitivity was 1 pg/ml. The cortisol RIA used an antiserum provided by J. R. G. Chain's, Ph.D. (University of Western Ontario, London, Ontario, Canada). Validation and cross-reactivity for this assay as applied to the fetal sheep have been previously described (25). The intra- and interassay coefficients of variation were 7% and 13%, respectively (n = 40 assays). Assay sensitivity was 0.2 ng/ ml. Statistics Statistical analyses were performed on log-transformed data to normalize heterogeneity of variance. To assess differences between fetal age groups, two-way analysis of variance (ANOVA) with repeated measures was used. No statistical differences in ACTH or cortisol concentrations between ages were found; thus, data were pooled and analyzed by one-way ANOVA with repeated measures, with respect to hormone concentration
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
297
AVP AND BASAL ACTH IN THE OVINE FETUS over time. Individual comparisons were then made with Duncan's multiple range test. P < 0.05 was considered significant. At each gestational age, fetuses served as their own control, i.e. the average baseline hormone value (time —15 min to 0 min, four samples) was used as pretreatment control to assess the effect of agonist and/or antagonist treatment in each sheep (see Fig. 1). To account for gestational increases in baseline hormone concentrations in the fetus, data in some figures are plotted as the percent change from the pretreatment baseline. Values are reported as the mean ± SE and/or percent change from baseline. Correlation and linear regression were calculated with fetal cortisol as the dependent variable and maternal cortisol as the independent variable. One-way ANOVA with
repeated measures was used to analyze fetal blood gas data for changes during experiments.
C AVP II
400 m 200
100
Fetuses were born at 146 ± 2 days gestation (n = 5) and weighed 4 ± 0.5 kg; both values are typical for this breed of sheep. Throughout gestation as well as during the AVP treatment, the percent oxyhemoglobin saturation was stable (pH 7.33 ± 0.02; PCO2 46 ± 0.6 Torr; POo 24 ± 0.4 Torr; hemoglobin, 10.5 ± 0.2 dl/g; oxyhemoglobin saturation, 60 ± 1%) and remained within the normal range (26). Maternal blood gases, hemoglobin, and percent oxyhemoglobin saturation also were stable (pH 7.43 ± 0.01; PCO2 37 ± 1 Torr; PO2 109 ± 3 Torr; hemoglobin, 9 ± 0.3 dl/g; oxyhemoglobin saturation, 98 ± 0.7%) and remained within the normal range throughout the experiment (26). Effects of AVP challenge AVP injection in the fetus increased plasma ACTH concentrations within 5 min to peak at 10 min (Fig. 1A, 133 days gestation; n = 5). This was followed by increased plasma cortisol (Fig. IB); a significant rise occurred within 10 min, and the peak concentration was attained by 15 min. In contrast to the rapid decline from the peak in plasma ACTH, cortisol maintained a plateau lasting up to 45 min after AVP injection and then declined. Relative to baseline, plasma ACTH concentrations increased almost 800% within 10 min of AVP injection (Fig. 1C). Circulating cortisol subsequently peaked about 175% over baseline within 15 min of fetal AVP treatment (Fig. ID). The rises in fetal plasma concentrations of ACTH and cortisol after the AVP challenge were significantly correlated (r = 0.82; P < 0.001). Later in gestation, fetal ACTH and cortisol responses to AVP challenge (two fetuses at 140 days gestation) were comparable to those at 133 days. However, one difference was noteworthy. Instead of a rapid decline from the peak that was observed for ACTH at 133 days, both plasma ACTH and cortisol in fetal circulation at 140 days gestation remained at a plateau for upwards of 30 min and at least 45 min in the fetal circulation, respectively, after the
li
•Jt*
AVP 4-
AVP 4-
100
Li
10
0
Results
600 Z
300
1 2 0 TIME RELATIVE TO AGONIST (hours)
1
2
FIG. 1. Plasma ACTH and cortisol are increased by exogenous AVP (1 Mg/ml normal saline, iv bolus injected at time zero) in chronically catheterized fetuses at 133 days gestation (n = 5). Fetal concentrations (mean ± SE) of plasma ACTH (A) and cortisol (B) are from blood samples collected every 5 min for 45 min, and less frequently thereafter. To normalize for the gestational increase in baseline hormone concentrations, data are plotted as the percent change from the pretreatment baseline. Fetal plasma ACTH (C) and cortisol (D) are plotted as the percent change from the baseline average of four samples (20 min) in each sheep immediately preceding the AVP challenge. The percent change was calculated by subtracting the baseline hormone concentration from the sample value after AVP challenge and dividing by the baseline. Thus, in individual sheep the baseline values were used as a control for the experimental effect at each gestational age.
AVP challenge (data not shown). In contrast to the significant changes in fetal cortisol, maternal cortisol did not change during AVP challenge of the fetus. The mean concentration of maternal cortisol after the fetal AVP challenge (5-60 min) was 23 ± 2 ng/ ml; values ranged between 18-29 ng/ml, in no temporal relation to fetal AVP treatment. In addition, fetal plasma cortisol concentrations were not correlated with maternal circulating cortisol (r = 0.21, by Scatchard analysis; P > 0.05, ANOVA). Effects of AVP antagonist and challenge AVP antagonist failed to alter basal concentrations of plasma ACTH or cortisol (data at 137,142, and 147 days gestation are combined in Fig. 2). The mean basal concentrations of fetal ACTH and cortisol (-15 to 0 min) were 61 ± 5 pg/ml and 45 ± 8 ng/ml, respectively. The percent changes in plasma ACTH and cortisol after antagonist treatment were not significantly different from baseline values. Plasma ACTH and cortisol remained unchanged after the AVP challenge (recall that AVP was administered 40 min after antagonist). The mean concentrations of ACTH and cortisol after AVP challenge preceded by antagonist (45-75 min) were 58 ± 5 pg/ml and 53 ± 6 ng/ml, respectively. Thus, antagonist pretreatment blocked the rise in plasma ACTH and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
298
AVP AND BASAL ACTH IN THE OVINE FETUS
ANT
ACTH
AVP
600 -
300
1 l l ANT
Endo«1991 Vol 129 • No 1 i
i
ANT
ANT
i
A ~
E a.
w 400 m
I
2E
O
i
200
i
CORTISOL
AVP
ANT
1
(ng)
ANT
i
cc
ANT
ANT
40
iW
1 p D -
lTTTi
20 I
o o
1
+
d CO
100
-
rtirrflTtTTt
\ Ixjl1
•i
l
i i
A
0
1
2
TIME RELATIVE TO ANTAGONIST (hours)
O
1
2
TIME RELATIVE TO ANTAGONIST (hours)
FIG. 2. Absence of plasma ACTH and cortisol responses (•) to the AVP challenge in fetuses pretreated with the AVP antagonist d(Ch2)6Tyr(Me)AVP (ANT) at 137-147 days gestation (seven treatments in three fetuses). The antagonist (10 ^g/kg estimated BW, iv bolus) was injected at 0 min, and 40 min later AVP was given (1 /ig/ml normal saline, iv bolus). D, Fetal plasma ACTH and cortisol responses to AVP challenge (1 Mg/kg estimated BW, iv bolus) at 133 days gestation, as described in Fig. 1. Data are plotted as the percent change (mean ± SE; seven studies in three fetuses at 137-147 days gestation). See Fig. 1 and Materials and Methods for details.
cortisol that would normally be induced by the AVP challenge. Episodes of increased fetal plasma ACTH were present in individual fetuses, but were not temporally related to antagonist treatment; plasma cortisol was as likely to be increased, decreased, or not changed compared to baseline. There was no relation between fetal and maternal cortisol concentrations (data not shown). Fetuses administered AVP antagonist at 30-min intervals (for a total of three injections) demonstrated no change in plasma concentrations of ACTH or cortisol compared with baseline (Fig. 3). Endogenous episodes of ACTH and cortisol secretion were evident in individual fetuses at all gestational ages during the extended period of antagonist treatment. Thus, basal ACTH and cortisol values were not significantly different after antagonist treatment from baseline hormone concentrations (P > 0.05, by ANOVA). Over the gestational period studied (137-147 days gestation), significant increases in baseline plasma ACTH and cortisol concentrations were observed regardless of treatment (Fig. 4). After antagonist treatment (30 and 120 min), fetal ACTH and cortisol levels were not different from baseline (P > 0.05), but were significantly increased with gestational age at 147 days compared with values at 133 days (P < 0.05). Increased fetal plasma
FIG. 3. Basal plasma ACTH (A) and cortisol (B) concentrations (mean ± SE) are unchanged after administration of the AVP antagonist d(Ch2)5Tyr(Me)AVP (10 Mg/kg estimated BW, iv bolus; ANT) at 0, 30, and 60 min in fetuses at 137-147 days gestation (five studies in two fetuses). E3 Baseline •30 min • • 1 2 0 min
137
142
147
QESTATIONAL AGE (days)
FIG. 4. Fetal plasma ACTH (A) and cortisol (B) concentrations (mean ± SE) before (W) and 30 min (•) and 120 min (•) after AVP antagonist treatment in fetuses at 137 (n = 5), 142 (n = 4), and 147 (n = 3) days gestation. Baseline represents the mean of the hormone values for each fetus immediately preceding antagonist injection at time zero. *, P < 0.05 compared to 137 and 142 days gestation, by ANOVA.
cortisol was not paralleled by changes in the maternal cortisol concentration; daily maternal means were 17 ± 3 ng/ml at 137 days (n = 5) and 17 ± 4 ng/ml at 146 days of pregnancy (n = 3). Therefore, the increases in circulating ACTH and cortisol as the fetus nears term
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
AVP AND BASAL ACTH IN THE OVINE FETUS were not blocked by the regimen of AVP antagonist treatments and were autonomous from those in the pregnant ewe. Discussion The results of this study indicate that basal concentrations of circulating ACTH and cortisol are not altered by selective antagonism of the AVP Vx receptor in fetal sheep during the last 2 weeks of gestation. Since AVP antagonist treatment abolished the pituitary-adrenocortical response to exogenous AVP, the data suggest that endogenous AVP secretion does not drive the progressive increase in basal ACTH and cortisol concentrations that occurs in fetal sheep during late gestation. In younger fetuses at 108-118 days gestation, the same AVP receptor antagonist in a similar treatment protocol was found to block exogenous AVP-induced ACTH release (15). Although the fetal plasma cortisol was not measured, these data in conjunction with the present findings support the hypothesis that AVP is not a principal regulator of basal ACTH secretion in the fetal sheep during the last third of gestation. The role of AVP as a regulator of basal ACTH secretion has been questioned in vivo and in vitro. Episodic and mean concentrations of plasma ACTH and cortisol are maintained in vivo in hypothalamic-pituitary-disconnected sheep compared to those in intact adult sheep (27) in spite of a significant reduction in AVP receptor concentration in the anterior pituitary (28). Immature fetal sheep treated with AVP (either bolus injection or pulses every 4 h for 7 days) had unchanged basal levels of cortisol and ACTH (10, 29). In vitro AVP may not be required for the maintenance of basal ACTH secretion from ovine pituitary corticotropes (9). Human fetal pituitaries spontaneously secrete ACTH in the absence of AVP and/or CRF (30). In addition, adult rat pituitary cells in culture maintain basal ACTH secretion in the absence of AVP and/or CRF (31). However, AVP may indirectly be important for basal ACTH secretion by maintaining stable levels of stored ACTH in the pituitary. When incubated with AVP, the ACTH content in ovine pituitary cells increased (9). Thus, AVP may serve to maintain the response capabilities of pituitary corticotropes to other releasing factors, but basal episodic ACTH secretion appears to be independent of hypothalamic AVP secretion. Findings in hypothalamic-pituitary-disconnected fetal sheep also support the hypothesis that basal ACTH secretion may be independent of hypothalamic releasing factors late in gestation (32). After 138 days gestation, basal plasma ACTH concentrations were the same in the circulation of intact and hypothalamic-pituitary-disconnected fetuses (see 30-min control period in Fig. 5 of Ref.
299
32). However, an intact hypothalamic-pituitary axis is required to mediate the negative feedback regulation of ACTH by cortisol in fetuses after 138 days gestation because cortisol infusion suppressed plasma ACTH levels in intact, but not in hypothalamic-pituitary-disconnected, fetuses (see 240-min cortisol infusion period in Fig. 5 of Ref. 32). Thus, basal ACTH secretion in the fetal sheep near term may not necessarily be dependent upon hypothalamic releasing factors or cortisol negative feedback. The present findings do not obviate a physiological role for AVP in stress-induced or evoked ACTH secretion in sheep. Both hemorrhage (33,34) and hyperinsulinemia (7) induce AVP release and activate the fetal pituitaryadrenal axis in vivo to increase ACTH and cortisol secretion. The ACTH response to such stressors could be regulated by AVP secreted from neurons that either release AVP into the hypophyseal-portal system (6, 7, 12) and/or directly innervate the anterior pituitary in the ovine fetus (13). Alternatively, CRF has been implicated in the stress-induced activation of the pituitaryadrenal axis (6, 7), but not in the maintenance of basal ACTH secretion (29). Therefore, both AVP and CRF regulate physiological functions other than basal episodic ACTH secretion. The present study also confirms findings that AVP can induce ACTH release in the ovine fetus, which is subsequently followed by an increase in plasma cortisol (11, 29). These hormonal changes are independent of maternal plasma cortisol concentrations and emphasize the autonomy of the developing fetus from the ewe late in gestation. The rise in both fetal plasma ACTH and cortisol levels near term is also independent of the decline in maternal cortisol concentrations, thus confirming earlier reports (35, 36). In conclusion, the present results do not support the hypothesis that endogenous AVP modulates developmental increases in basal concentrations of plasma ACTH and cortisol in the ovine fetus during the weeks preceding parturition. This is based in part on the finding that AVP antagonist effectively blocked the fetal ACTH and cortisol response to exogenous AVP while not altering basal secretion. Further, this study has shown that the ovine fetal pituitary-adrenocortical axis during late gestation is capable of robust responses comparable in magnitude and latency to those in the adult. Collectively, the data suggest that basal and stimulated ACTH are under separate regulatory mechanisms. The lack of a role for AVP in the ovine fetus for control of basal ACTH and cortisol secretion does not exclude it as a modulator of stimulated ACTH and cortisol responses during gestation.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
AVP AND BASAL ACTH IN THE OVINE FETUS
300
Acknowledgments We are grateful to Ms. Lela C. Spears for skillful technical assistance, and Ms. Sheila Whitson for typing the manuscript. We thank Dr. Maurice Manning for the AVP antagonist, and Dr. John R. G. Challis for the cortisol antisera used in our studies. We appreciate the thoughtful discussions with Dr. Paul M. Plotsky. Finally, we thank Dr. Grenith Zimmerman for statistical consultation.
References 1. Bassett JM, Thornburn GD 1969 Foetal plasma corticosteroids and the initiation of parturition in sheep. J Endocrinol 44:285-286 2. Magyar DM, Fridshal D, Eisner CW, Glatz T, Eliot JK, Lowe AH, Buster JE, Nathanielsz PW 1980 Time-trend analysis of plasma cortisol concentrations in the fetal sheep in relation to parturition. Endocrinology 107:155-159 3. Rees L, Jack PMB, Thomas A, Nathanielsz PW 1975 Role of fetal adrenocorticotrophin during parturition in sheep. Nature 253:274275
4. Glickman JA, Challis JRG 1980 The changing response pattern of sheep fetal adrenal cells throughout the course of gestation. Endocrinology 106:1371-1376 5. Rose JC, Meis PJ, Urgan RB, Greiss FC 1982 In vivo evidence for increased adrenal sensitivity to adrenocorticotrophin-(1-24) in the lamb fetus late in gestation. Endocrinology 110:80-85 6. Caraty A, Grino M, Locatelli A, Oliver C 1988 Secretion of corticotropin releasing factor (CRF) and vasopressin (AVP) into the hypophysial portal blood of conscious, unrestrained rams. Biochem Biophys Res Commun 155:841-849 7. Engler D, Pham T, Fullerton M, Ooi G, Funder JW, Clarke IJ 1989 Studies on the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. Neuroendocrinology 49:367-381 8. Familari M, Smith AI, Smith R, Funder JW 1989 Arginine vasopressin is a much more potent stimulus to ACTH release from ovine anterior pituitary cells than ovine corticotropin-releasing factor. Neuroendocrinology 50:152-157 9. Liu JP, Robinson PJ, Funder JW, Engler D 1990 The biosynthesis and secretion of adrenocorticotropin by the ovine anterior pituitary is predominantly regulated by arginine vasopressin (AVP). J Biol Chem 265:14136-14142 10. Norman LJ, Challis JRG 1987 Dexamethasone inhibits ovine corticotrophin-releasing factor (oCRF), arginine vasopressin (AVP), and oCRF + AVP stimulated release of ACTH during the last third of pregnancy in the sheep fetus. Can J Physiol Pharmacol 65:1186-1192 11. Pradier P, Davicco MJ, LeFaivre J, Barlet JP, Delost P 1985 Plasma adrenocorticotrophin, cortisol and aldosterone responses to ovine corticotrophin-releasing factor and vasopressin in sheep. Acta Endocrinol (Copenh) 111:93-100 12. Brieu V, Durand P 1989 Adrenocorticotropic hormone released by pituitary cells from ovine fetuses and lambs. Neuroendocrinology 49:300-308 13. Levidiotis M, Oldfield B, Wintour EM 1987 Corticotropin-releasing factor and arginine vasopressin fiber projections to the median eminence of fetal sheep. Neuroendocrinology 46:453-456 14. Hoffman G, McDonald T, Figueroa JP, Nathanielsz PW 1989 Neuropeptide cells and fibers in the hypothalamus and pituitary of the fetal sheep: comparison of oxytocin and arginine vasopressin. Neuroendocrinology 50:633-643 15. Maclsaac RJ, Congiu M, Levidiotis M, McDougall JG, Wintour EM 1989 In vivo regulation of adrenocorticotrophin secretion in the immature ovine fetus. Modulation by ovine corticotropin releasing hormone and arginine vasopressin. J Dev Physiol 12:4147 16. Perks AM, Vizsoly E 1973 Studies of the neurophypohysis in foetal mammals. In: Comline KS, Cross KW, Dawes GS, Nathanielsz PW (eds) Foetal and Neonatal Physiology. Cambridge University
Endo • 1991 Voll29«Nol
Press, Cambridge, pp 430-438 17. Stark RI, Daniel SS, Husain MK, Tropper PJ, James LS 1985 Cerebrospinal fluid and plasma vasopressin in the fetal lamb: basal concentration and the effect of hypoxia. Endocrinology 116:65-72 18. Yellon SM, Longo LD 1987 Melatonin rhythms in fetal and maternal circulation during pregnancy in sheep. Am J Physiol 252:E799-E802 19. Jard S, Gaillard RC, Guillon G, Marie J, Schoenenberg P, Miller AF, Manning M, Sawyer WH 1986 Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol Pharmacol 20:171-177 20. Kruszynski M, Lammek B, Manning M, Seto J, Haldar J, Sawyer WH 1980 [l-(B-Mercapto-BB-cyclopentamethylenepropionicacid), 2-(O-methyl)tyrosine]arginine-vasopressin and [l-(J3-mercapto-BB-cyclopentamethylenepropionic acid) arginine-vasopressin, two highly potent antagonist of the vasopressor response to arginine-vasopressin. J Med Chem 23:364-368 21. Schwartz J, Reid IA 1986 Characteristics of the receptors which mediate the stimulation of ACTH secretion by vasopressin in conscious dogs. Neuroendocrinology 42:93-96 22. Manning MM, Sawyer WH 1990 Discovery, development, and some uses of vasopressin and oxytocin antagonists. J Lab Clin Med 114:617-632 23. Tikanaka T, Alonso JG, Gilbert RD, Siu B, Clemons GK, Longo LD 1989 Fetal responses to long-term hypoxemia in sheep. Am J Physiol 256:R1348-R1354 24. Wood CE, Rudolph AM 1983 Negative feedback regulation of adrenocorticotropin secretion by cortisol in ovine fetuses. Endocrinology 112:1930-1936 25. Challis JRG, Patrick JE, Cross J, Workewych J, Manchester E, Power S 1981 Short-term fluctuations in the concentrations of cortisol and progesterone in fetal plasma, maternal plasma, and amniotic and allantoic fluids from sheep during late pregnancy. Can J Physiol Pharmacol 59:261-267 26. Lottering FK, Gilbert RD, Longo LD 1983 Exercise responses in pregnant sheep: blood gases, temperatures, and fetal cardiovascular system. Am J Physiol 55:842-850 27. Clarke IJ, Clements JA, Cummins JT, Dench F, Smith AI, Robinson PM, Funder JW 1986 Elevated levels of pro-opiomelanocortin derived peptides in sheep following hypothalamo-pituitary disconnection. Neuroendocrinology 44:508-514 28. Shen PJ, Clarke IJ, Canny BJ, Funder JW, Smith AI 1990 Arginine vasopressin and corticotropin releasing factor: binding to ovine anterior pituitary membranes. Endocrinology 127:2085-2089 29. Brooks AN, Power LA, Jones SA, Yang KP, Challis JRG 1989 Controls of corticotrophin-releasing factor output by hypothalamic tissue from fetal sheep in vitro. J Endocrinol 122:15-22 30. Gambacciami M, Liu JH, Swartz WH, Tueros VS, Rasmussen D, Yen SSC 1987 Intrinsic pulsatility of ACTH release from the human pituitary in vitro. Clin Endocrinol (Oxf) 26:557-563 31. Schwartz J, Tham T, Funder JW 1989 Chloroquine decreases adrenocorticotrophin-secretory response to corticotrophin-releasing factor but not to vasopressin in rat pituitary cells. Further evidence for differentially responsive subpopulations. J Endocrinol 2:25-28 32. Ozolins IZ, Young IR, McMillen IC 1990 Effect of cortisol infusion on basal and corticotropin-releasing factor (CRF)-stimulated plasma ACTH concentrations in the sheep fetus after surgical isolation of the pituitary. Endocrinology 127:1833-1840 33. Alexander DP, Bashore RA, Britton HG, Forsling ML 1974 Maternal and fetal arginine vasopressin in the chronically catheterized sheep. Biol Neonate 25:242-248 34. Gomez RA, Meernik JG, Kuehl WD, Robillard JE 1984 Developmental aspects of the renal response to hemorrhage during fetal life. Pediatr Res 18:40-46 35. Hennessey DP, Coghlan JP, Hardy KJ, Scoggins BA, Wintour EM 1980 The origin of cortisol in the blood of fetal sheep. J Endocrinol 95:71-79 36. Jones CT, Luther E, Richie JWK, Worthington D 1975 The clearance of ACTH from the plasma of adult and fetal sheep. Endocrinology 106:1371-1375
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
Vol. 129, No. 1
Printed in U.S.A.
Regulation of Basal Adrenocorticotropin and Cortisol Secretion by Arginine Vasopressin in the Fetal Sheep during Late Gestation* EDE MARIE APOSTOLAKIS, LAWRENCE D. LONGO, AND STEVEN M. YELLON Division of Perinatal Biology, Departments of Physiology, Pediatrics, and Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, California 92350
cortisol remained unchanged after the AVP challenge. To further define the role of endogenous AVP in basal ACTH and cortisol secretion, the AVP antagonist was administered (five studies in two fetuses) at 30-min intervals for a total of three injections per fetus. This extended AVP antagonist regimen also failed to alter fetal circulating concentrations of ACTH or cortisol (P > 0.05). Cortisol in the maternal circulation was not affected by any of the fetal AVP or AVP antagonist treatments. Lambs were born at 146 ± 2 days gestation (n = 5), within the range for the normal duration of pregnancy. These data do not support the hypotheses that AVP is involved in the regulation of basal ACTH secretion in the fetal sheep during the 10 days preceding parturition. Rather, the ability of AVP antagonist to block the AVP-induced rise in plasma ACTH and cortisol in the fetus suggests that basal and stimulated ACTH secretion are under separate regulatory mechanisms. (Endocrinology 129: 295-300, 1991)
ABSTRACT. This study tested the hypothesis that arginine vasopressin (AVP) is involved in the regulation of basal ACTH secretion in the ovine fetus near term. In five fetuses challenged with AVP (1 /ug/ml, iv bolus) plasma ACTH concentrations increased to an 8-fold peak within 10 min of the preceding baseline (55 ± 6 to 403 ± 241 pg/ml). Cortisol in fetal circulation subsequently increased 2-fold (11 ± 1 to 28 ± 5 ng/ml) within 15 min of the AVP injection. The AVP-induced rise in plasma ACTH and cortisol concentrations was blocked when the fetus was pretreated with the AVP V] receptor antagonist d(0h..>)5Tyr(Me)AVP. In a total of seven studies, antagonist (10 Aig/kg estimated BW, iv bolus) was administered to three fetuses, aged 137-147 days gestation, followed 40 min later by the exogenous AVP challenge, as described above. After AVP antagonist treatment, basal ACTH and cortisol concentrations were not significantly different from the preinjection baseline levels (P > 0.05, by analysis of variance). Moreover, plasma ACTH and
I
N THE fetal sheep near term, activation of the pituitary-adrenal axis is part of the endocrine cascade that initiates parturition (1, 2). Cortisol in fetal circulation gradually increases over the last third of gestation, abruptly rising during the final 72 h to peak on the day of birth (~147 days in sheep). During this time period, fetal ACTH secretion also increases, but whether ACTH drives the prepartum cortisol rise is controversial (3-5). The importance of ACTH secretion for stimulation of adrenal cortisol in the ovine fetus has led to investigations of the neuroendocrine mechanism controlling its pituitary release. In adult sheep, arginine vasopressin (AVP) is the predominant regulator of ACTH secretion and biosynthesis (6-9). In fetal sheep, both AVP and
CRF given iv induce fetal ACTH release (10, 11), although the response appears to be greater to AVP than to CRF (10). Both releasing factors are present in the hypothalamus of young fetuses by 90 days gestation, and the AVP content is greater than that of CRF (12). In addition to its presence in hypophyseal-portal vessels (13), AVP from fibers in contact with pituitary corticotropes (14) may influence basal ACTH secretion. Moreover, Maclsaac and colleagues (15) found that AVPstimulated ACTH release was blocked by pretreatment with an AVP receptor antagonist in 106- to 118-day-old fetuses. Whether antagonist treatment affects basal ACTH secretion in fetuses later in gestation, when plasma cortisol, ACTH, and AVP concentrations are increasing in the fetal circulation (16, 17), has not been examined. The goal of the present study was to determine whether AVP is involved in the regulation of basal plasma ACTH release and, subsequently, basal cortisol secretion during the last weeks of gestation in the ovine fetus. The specific AVP Vi receptor antagonist d(CH2)5Tyr(Me)AVP was administered to the fetus during late gestation in an
Received December 4, 1990. Address all correspondence and requests for reprints to: Steven M. Yellon, Ph.D., Division of Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California 92350. * This work was supported by grants from the USPHS (HD-03817) and the Center for Nursing Research (NRSA-06042), and the Pediatrics Department Research Fund, Loma Linda University School of Medicine. Preliminary data from the study were presented at the 1990 Annual Meeting of the Society for Gynecologic Investigation, St. Louis, MO (Abstract 369).
295
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
AVP AND BASAL ACTH IN THE OVINE FETUS
296
attempt to block secretion of basal ACTH and reduce plasma cortisol concentrations in the fetal circulation.
Materials and Methods General Studies were performed in five time-dated pregnant singleton ewes of mixed Western breed (Nebeker Farms, Santa Monica, CA), housed in a controlled access light-locked room with constant humidity, temperature, and photoperiod (10 h of light/ day; lights on at 0700 h, PST). A dim red light remained on at night to facilitate collection of nighttime blood samples. Alfalfa pellets (Nebeker Farms) and water were provided in excess ad libitum. Pregnant ewes were acclimated to the animal care facility for 7 days. At 126 days gestation using sterile surgical procedures, catheters were placed in the brachial artery and cephalic vein of each fetal forelimb, as previously described (18). Catheter tips were advanced to obtain blood from the aortic arch and superior vena cava, respectively. At the same time, maternal catheters were inserted in the left external jugular vein and left pedal artery. All catheters were exteriorized through a left flank stab wound and stored in a nylon pouch sutured to the skin of the ewe. Antibiotics were administered to the ewe for 3 days (2.5 ml Combiotic, im; Pfizer, New York, NY) and to the fetus daily (40 mg Gentamycin, iv; ElkinsSinn, Cherry Hill, NJ). Catheters were flushed each day with heparinized saline (100 U/ml). To assess fetal well-being, arterial blood samples (0.3 ml) were drawn into heparinized syringes every morning (1000 h) for determination of pH, PO2, PCO2, hemoglobin, and percent oxyhemoglobin saturation (ABL300 and OSM2, respectively, Radiometer, Copenhagen, Denmark). Fetal and maternal blood samples (2 and 3 ml, respectively) were drawn daily (~1000 and 2200 h) into chilled EDTA-treated syringes. Blood samples were centrifuged at 4000 rpm for 15 min at 4 C. Plasma was harvested and stored at -70 C in polypropylene microcentrifuge tubes (Fisher Scientific Corp., Pittsburgh, PA) for later RIAs (see below). Experimental protocols The following experiments were conducted in the morning, beginning at 1045 h. A VP challenge. To induce a robust physiological rise in plasma ACTH and cortisol, we administered AVP (1 Mg/ml normal saline, iv bolus; Bachem, Torrance, CA), immediately followed by a 3-ml flush of lactated Ringer's solution (RL) to fetuses at 133 days gestation (n = 5). Beginning 15 min before the AVP injection, fetal (2 ml) and maternal (3 ml) arterial blood samples were taken every 5 min for 45 min, and less frequently thereafter. After each blood sample, RL was infused to replace the volume of withdrawn blood. In addition, fetal and maternal erythrocytes were reconstituted with RL (~3 times the volume) and returned every 20 min to the fetus and ewe, respectively. Arterial blood gases, hemoglobin, and percent oxyhemoglobin saturation were measured every 15-60 min to assess fetal wellbeing. We repeated the same protocol for a total of 75 min at 140 days gestation in two fetuses to test the ability of AVP to stimulate ACTH and cortisol later in gestation.
Endo • 1991 Vol 129 • No 1
AVP antagonist and challenge. To assess the contribution of endogenous AVP in the regulation of basal ACTH release and subsequent cortisol secretion, we administered the AVP Vj receptor antagonist d(CH2)5Tyr(Me)AVP (generously provided by M. Manning, Ph.D., Medical College of Toledo, Toledo, OH), to fetuses at various gestational ages. Antagonism of the AVP V, receptor by d(CH2)5Tyr(Me)AVP blocks AVPstimulated ACTH release in several species (19-22). In a total of seven experiments in three fetuses (137, 142, and 147 days gestation), the AVP antagonist was administered (10 fig/kg estimated BW/ml normal saline, iv bolus), followed immediately by 3 ml RL flush. Based on previous data from our laboratory, fetal weight was estimated to be about 3 kg (23). As before, erythrocytes were reconstituted with RL and returned. As part of this protocol, the efficacy of the receptor antagonism was tested. Each fetus was challenged with AVP (1 Mg/ml; as described above) 40 min after the antagonist injection. Beginning 15 min before the antagonist treatment, fetal (2 ml) and maternal (3 ml) blood samples were taken every 5 min for 115 min, and less frequently thereafter. To assess fetal well-being, arterial blood gases, hemoglobin, and percent oxyhemoglobin saturation were measured every 15-60 min. In another experiment, the duration of the AVP receptor antagonist treatment was extended to further define the role of AVP in the regulation of basal ACTH and cortisol secretion. Five studies were performed in two fetuses beginning at 137 days gestation (n = 2) and repeated at 142 and 147 days gestation (n = 2 and 1, respectively). The AVP antagonist was administered (10 Mg/kg estimated BW) at 30-min intervals (total of three injections). Fetal and maternal blood samples were taken every 5 min for 115 min, beginning 15 min before the first antagonist injection. Again to assess fetal well-being, arterial blood gases, hemoglobin, and percent oxyhemoglobin saturation were measured every 15-60 min. ACTH and cortisol RIAs ACTH was measured with a commercial kit (IncStar Corp., Stillwater, MN), previously validated for use in the fetal sheep (24). Parallelism, accuracy, and repeatability were verified in our laboratory using plasma pools from pregnant and fetal sheep. The intra- and interassay coefficients of variation were 6% and 11%, respectively (n = 12 assays). Assay sensitivity was 1 pg/ml. The cortisol RIA used an antiserum provided by J. R. G. Chain's, Ph.D. (University of Western Ontario, London, Ontario, Canada). Validation and cross-reactivity for this assay as applied to the fetal sheep have been previously described (25). The intra- and interassay coefficients of variation were 7% and 13%, respectively (n = 40 assays). Assay sensitivity was 0.2 ng/ ml. Statistics Statistical analyses were performed on log-transformed data to normalize heterogeneity of variance. To assess differences between fetal age groups, two-way analysis of variance (ANOVA) with repeated measures was used. No statistical differences in ACTH or cortisol concentrations between ages were found; thus, data were pooled and analyzed by one-way ANOVA with repeated measures, with respect to hormone concentration
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
297
AVP AND BASAL ACTH IN THE OVINE FETUS over time. Individual comparisons were then made with Duncan's multiple range test. P < 0.05 was considered significant. At each gestational age, fetuses served as their own control, i.e. the average baseline hormone value (time —15 min to 0 min, four samples) was used as pretreatment control to assess the effect of agonist and/or antagonist treatment in each sheep (see Fig. 1). To account for gestational increases in baseline hormone concentrations in the fetus, data in some figures are plotted as the percent change from the pretreatment baseline. Values are reported as the mean ± SE and/or percent change from baseline. Correlation and linear regression were calculated with fetal cortisol as the dependent variable and maternal cortisol as the independent variable. One-way ANOVA with
repeated measures was used to analyze fetal blood gas data for changes during experiments.
C AVP II
400 m 200
100
Fetuses were born at 146 ± 2 days gestation (n = 5) and weighed 4 ± 0.5 kg; both values are typical for this breed of sheep. Throughout gestation as well as during the AVP treatment, the percent oxyhemoglobin saturation was stable (pH 7.33 ± 0.02; PCO2 46 ± 0.6 Torr; POo 24 ± 0.4 Torr; hemoglobin, 10.5 ± 0.2 dl/g; oxyhemoglobin saturation, 60 ± 1%) and remained within the normal range (26). Maternal blood gases, hemoglobin, and percent oxyhemoglobin saturation also were stable (pH 7.43 ± 0.01; PCO2 37 ± 1 Torr; PO2 109 ± 3 Torr; hemoglobin, 9 ± 0.3 dl/g; oxyhemoglobin saturation, 98 ± 0.7%) and remained within the normal range throughout the experiment (26). Effects of AVP challenge AVP injection in the fetus increased plasma ACTH concentrations within 5 min to peak at 10 min (Fig. 1A, 133 days gestation; n = 5). This was followed by increased plasma cortisol (Fig. IB); a significant rise occurred within 10 min, and the peak concentration was attained by 15 min. In contrast to the rapid decline from the peak in plasma ACTH, cortisol maintained a plateau lasting up to 45 min after AVP injection and then declined. Relative to baseline, plasma ACTH concentrations increased almost 800% within 10 min of AVP injection (Fig. 1C). Circulating cortisol subsequently peaked about 175% over baseline within 15 min of fetal AVP treatment (Fig. ID). The rises in fetal plasma concentrations of ACTH and cortisol after the AVP challenge were significantly correlated (r = 0.82; P < 0.001). Later in gestation, fetal ACTH and cortisol responses to AVP challenge (two fetuses at 140 days gestation) were comparable to those at 133 days. However, one difference was noteworthy. Instead of a rapid decline from the peak that was observed for ACTH at 133 days, both plasma ACTH and cortisol in fetal circulation at 140 days gestation remained at a plateau for upwards of 30 min and at least 45 min in the fetal circulation, respectively, after the
li
•Jt*
AVP 4-
AVP 4-
100
Li
10
0
Results
600 Z
300
1 2 0 TIME RELATIVE TO AGONIST (hours)
1
2
FIG. 1. Plasma ACTH and cortisol are increased by exogenous AVP (1 Mg/ml normal saline, iv bolus injected at time zero) in chronically catheterized fetuses at 133 days gestation (n = 5). Fetal concentrations (mean ± SE) of plasma ACTH (A) and cortisol (B) are from blood samples collected every 5 min for 45 min, and less frequently thereafter. To normalize for the gestational increase in baseline hormone concentrations, data are plotted as the percent change from the pretreatment baseline. Fetal plasma ACTH (C) and cortisol (D) are plotted as the percent change from the baseline average of four samples (20 min) in each sheep immediately preceding the AVP challenge. The percent change was calculated by subtracting the baseline hormone concentration from the sample value after AVP challenge and dividing by the baseline. Thus, in individual sheep the baseline values were used as a control for the experimental effect at each gestational age.
AVP challenge (data not shown). In contrast to the significant changes in fetal cortisol, maternal cortisol did not change during AVP challenge of the fetus. The mean concentration of maternal cortisol after the fetal AVP challenge (5-60 min) was 23 ± 2 ng/ ml; values ranged between 18-29 ng/ml, in no temporal relation to fetal AVP treatment. In addition, fetal plasma cortisol concentrations were not correlated with maternal circulating cortisol (r = 0.21, by Scatchard analysis; P > 0.05, ANOVA). Effects of AVP antagonist and challenge AVP antagonist failed to alter basal concentrations of plasma ACTH or cortisol (data at 137,142, and 147 days gestation are combined in Fig. 2). The mean basal concentrations of fetal ACTH and cortisol (-15 to 0 min) were 61 ± 5 pg/ml and 45 ± 8 ng/ml, respectively. The percent changes in plasma ACTH and cortisol after antagonist treatment were not significantly different from baseline values. Plasma ACTH and cortisol remained unchanged after the AVP challenge (recall that AVP was administered 40 min after antagonist). The mean concentrations of ACTH and cortisol after AVP challenge preceded by antagonist (45-75 min) were 58 ± 5 pg/ml and 53 ± 6 ng/ml, respectively. Thus, antagonist pretreatment blocked the rise in plasma ACTH and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
298
AVP AND BASAL ACTH IN THE OVINE FETUS
ANT
ACTH
AVP
600 -
300
1 l l ANT
Endo«1991 Vol 129 • No 1 i
i
ANT
ANT
i
A ~
E a.
w 400 m
I
2E
O
i
200
i
CORTISOL
AVP
ANT
1
(ng)
ANT
i
cc
ANT
ANT
40
iW
1 p D -
lTTTi
20 I
o o
1
+
d CO
100
-
rtirrflTtTTt
\ Ixjl1
•i
l
i i
A
0
1
2
TIME RELATIVE TO ANTAGONIST (hours)
O
1
2
TIME RELATIVE TO ANTAGONIST (hours)
FIG. 2. Absence of plasma ACTH and cortisol responses (•) to the AVP challenge in fetuses pretreated with the AVP antagonist d(Ch2)6Tyr(Me)AVP (ANT) at 137-147 days gestation (seven treatments in three fetuses). The antagonist (10 ^g/kg estimated BW, iv bolus) was injected at 0 min, and 40 min later AVP was given (1 /ig/ml normal saline, iv bolus). D, Fetal plasma ACTH and cortisol responses to AVP challenge (1 Mg/kg estimated BW, iv bolus) at 133 days gestation, as described in Fig. 1. Data are plotted as the percent change (mean ± SE; seven studies in three fetuses at 137-147 days gestation). See Fig. 1 and Materials and Methods for details.
cortisol that would normally be induced by the AVP challenge. Episodes of increased fetal plasma ACTH were present in individual fetuses, but were not temporally related to antagonist treatment; plasma cortisol was as likely to be increased, decreased, or not changed compared to baseline. There was no relation between fetal and maternal cortisol concentrations (data not shown). Fetuses administered AVP antagonist at 30-min intervals (for a total of three injections) demonstrated no change in plasma concentrations of ACTH or cortisol compared with baseline (Fig. 3). Endogenous episodes of ACTH and cortisol secretion were evident in individual fetuses at all gestational ages during the extended period of antagonist treatment. Thus, basal ACTH and cortisol values were not significantly different after antagonist treatment from baseline hormone concentrations (P > 0.05, by ANOVA). Over the gestational period studied (137-147 days gestation), significant increases in baseline plasma ACTH and cortisol concentrations were observed regardless of treatment (Fig. 4). After antagonist treatment (30 and 120 min), fetal ACTH and cortisol levels were not different from baseline (P > 0.05), but were significantly increased with gestational age at 147 days compared with values at 133 days (P < 0.05). Increased fetal plasma
FIG. 3. Basal plasma ACTH (A) and cortisol (B) concentrations (mean ± SE) are unchanged after administration of the AVP antagonist d(Ch2)5Tyr(Me)AVP (10 Mg/kg estimated BW, iv bolus; ANT) at 0, 30, and 60 min in fetuses at 137-147 days gestation (five studies in two fetuses). E3 Baseline •30 min • • 1 2 0 min
137
142
147
QESTATIONAL AGE (days)
FIG. 4. Fetal plasma ACTH (A) and cortisol (B) concentrations (mean ± SE) before (W) and 30 min (•) and 120 min (•) after AVP antagonist treatment in fetuses at 137 (n = 5), 142 (n = 4), and 147 (n = 3) days gestation. Baseline represents the mean of the hormone values for each fetus immediately preceding antagonist injection at time zero. *, P < 0.05 compared to 137 and 142 days gestation, by ANOVA.
cortisol was not paralleled by changes in the maternal cortisol concentration; daily maternal means were 17 ± 3 ng/ml at 137 days (n = 5) and 17 ± 4 ng/ml at 146 days of pregnancy (n = 3). Therefore, the increases in circulating ACTH and cortisol as the fetus nears term
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
AVP AND BASAL ACTH IN THE OVINE FETUS were not blocked by the regimen of AVP antagonist treatments and were autonomous from those in the pregnant ewe. Discussion The results of this study indicate that basal concentrations of circulating ACTH and cortisol are not altered by selective antagonism of the AVP Vx receptor in fetal sheep during the last 2 weeks of gestation. Since AVP antagonist treatment abolished the pituitary-adrenocortical response to exogenous AVP, the data suggest that endogenous AVP secretion does not drive the progressive increase in basal ACTH and cortisol concentrations that occurs in fetal sheep during late gestation. In younger fetuses at 108-118 days gestation, the same AVP receptor antagonist in a similar treatment protocol was found to block exogenous AVP-induced ACTH release (15). Although the fetal plasma cortisol was not measured, these data in conjunction with the present findings support the hypothesis that AVP is not a principal regulator of basal ACTH secretion in the fetal sheep during the last third of gestation. The role of AVP as a regulator of basal ACTH secretion has been questioned in vivo and in vitro. Episodic and mean concentrations of plasma ACTH and cortisol are maintained in vivo in hypothalamic-pituitary-disconnected sheep compared to those in intact adult sheep (27) in spite of a significant reduction in AVP receptor concentration in the anterior pituitary (28). Immature fetal sheep treated with AVP (either bolus injection or pulses every 4 h for 7 days) had unchanged basal levels of cortisol and ACTH (10, 29). In vitro AVP may not be required for the maintenance of basal ACTH secretion from ovine pituitary corticotropes (9). Human fetal pituitaries spontaneously secrete ACTH in the absence of AVP and/or CRF (30). In addition, adult rat pituitary cells in culture maintain basal ACTH secretion in the absence of AVP and/or CRF (31). However, AVP may indirectly be important for basal ACTH secretion by maintaining stable levels of stored ACTH in the pituitary. When incubated with AVP, the ACTH content in ovine pituitary cells increased (9). Thus, AVP may serve to maintain the response capabilities of pituitary corticotropes to other releasing factors, but basal episodic ACTH secretion appears to be independent of hypothalamic AVP secretion. Findings in hypothalamic-pituitary-disconnected fetal sheep also support the hypothesis that basal ACTH secretion may be independent of hypothalamic releasing factors late in gestation (32). After 138 days gestation, basal plasma ACTH concentrations were the same in the circulation of intact and hypothalamic-pituitary-disconnected fetuses (see 30-min control period in Fig. 5 of Ref.
299
32). However, an intact hypothalamic-pituitary axis is required to mediate the negative feedback regulation of ACTH by cortisol in fetuses after 138 days gestation because cortisol infusion suppressed plasma ACTH levels in intact, but not in hypothalamic-pituitary-disconnected, fetuses (see 240-min cortisol infusion period in Fig. 5 of Ref. 32). Thus, basal ACTH secretion in the fetal sheep near term may not necessarily be dependent upon hypothalamic releasing factors or cortisol negative feedback. The present findings do not obviate a physiological role for AVP in stress-induced or evoked ACTH secretion in sheep. Both hemorrhage (33,34) and hyperinsulinemia (7) induce AVP release and activate the fetal pituitaryadrenal axis in vivo to increase ACTH and cortisol secretion. The ACTH response to such stressors could be regulated by AVP secreted from neurons that either release AVP into the hypophyseal-portal system (6, 7, 12) and/or directly innervate the anterior pituitary in the ovine fetus (13). Alternatively, CRF has been implicated in the stress-induced activation of the pituitaryadrenal axis (6, 7), but not in the maintenance of basal ACTH secretion (29). Therefore, both AVP and CRF regulate physiological functions other than basal episodic ACTH secretion. The present study also confirms findings that AVP can induce ACTH release in the ovine fetus, which is subsequently followed by an increase in plasma cortisol (11, 29). These hormonal changes are independent of maternal plasma cortisol concentrations and emphasize the autonomy of the developing fetus from the ewe late in gestation. The rise in both fetal plasma ACTH and cortisol levels near term is also independent of the decline in maternal cortisol concentrations, thus confirming earlier reports (35, 36). In conclusion, the present results do not support the hypothesis that endogenous AVP modulates developmental increases in basal concentrations of plasma ACTH and cortisol in the ovine fetus during the weeks preceding parturition. This is based in part on the finding that AVP antagonist effectively blocked the fetal ACTH and cortisol response to exogenous AVP while not altering basal secretion. Further, this study has shown that the ovine fetal pituitary-adrenocortical axis during late gestation is capable of robust responses comparable in magnitude and latency to those in the adult. Collectively, the data suggest that basal and stimulated ACTH are under separate regulatory mechanisms. The lack of a role for AVP in the ovine fetus for control of basal ACTH and cortisol secretion does not exclude it as a modulator of stimulated ACTH and cortisol responses during gestation.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
AVP AND BASAL ACTH IN THE OVINE FETUS
300
Acknowledgments We are grateful to Ms. Lela C. Spears for skillful technical assistance, and Ms. Sheila Whitson for typing the manuscript. We thank Dr. Maurice Manning for the AVP antagonist, and Dr. John R. G. Challis for the cortisol antisera used in our studies. We appreciate the thoughtful discussions with Dr. Paul M. Plotsky. Finally, we thank Dr. Grenith Zimmerman for statistical consultation.
References 1. Bassett JM, Thornburn GD 1969 Foetal plasma corticosteroids and the initiation of parturition in sheep. J Endocrinol 44:285-286 2. Magyar DM, Fridshal D, Eisner CW, Glatz T, Eliot JK, Lowe AH, Buster JE, Nathanielsz PW 1980 Time-trend analysis of plasma cortisol concentrations in the fetal sheep in relation to parturition. Endocrinology 107:155-159 3. Rees L, Jack PMB, Thomas A, Nathanielsz PW 1975 Role of fetal adrenocorticotrophin during parturition in sheep. Nature 253:274275
4. Glickman JA, Challis JRG 1980 The changing response pattern of sheep fetal adrenal cells throughout the course of gestation. Endocrinology 106:1371-1376 5. Rose JC, Meis PJ, Urgan RB, Greiss FC 1982 In vivo evidence for increased adrenal sensitivity to adrenocorticotrophin-(1-24) in the lamb fetus late in gestation. Endocrinology 110:80-85 6. Caraty A, Grino M, Locatelli A, Oliver C 1988 Secretion of corticotropin releasing factor (CRF) and vasopressin (AVP) into the hypophysial portal blood of conscious, unrestrained rams. Biochem Biophys Res Commun 155:841-849 7. Engler D, Pham T, Fullerton M, Ooi G, Funder JW, Clarke IJ 1989 Studies on the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. Neuroendocrinology 49:367-381 8. Familari M, Smith AI, Smith R, Funder JW 1989 Arginine vasopressin is a much more potent stimulus to ACTH release from ovine anterior pituitary cells than ovine corticotropin-releasing factor. Neuroendocrinology 50:152-157 9. Liu JP, Robinson PJ, Funder JW, Engler D 1990 The biosynthesis and secretion of adrenocorticotropin by the ovine anterior pituitary is predominantly regulated by arginine vasopressin (AVP). J Biol Chem 265:14136-14142 10. Norman LJ, Challis JRG 1987 Dexamethasone inhibits ovine corticotrophin-releasing factor (oCRF), arginine vasopressin (AVP), and oCRF + AVP stimulated release of ACTH during the last third of pregnancy in the sheep fetus. Can J Physiol Pharmacol 65:1186-1192 11. Pradier P, Davicco MJ, LeFaivre J, Barlet JP, Delost P 1985 Plasma adrenocorticotrophin, cortisol and aldosterone responses to ovine corticotrophin-releasing factor and vasopressin in sheep. Acta Endocrinol (Copenh) 111:93-100 12. Brieu V, Durand P 1989 Adrenocorticotropic hormone released by pituitary cells from ovine fetuses and lambs. Neuroendocrinology 49:300-308 13. Levidiotis M, Oldfield B, Wintour EM 1987 Corticotropin-releasing factor and arginine vasopressin fiber projections to the median eminence of fetal sheep. Neuroendocrinology 46:453-456 14. Hoffman G, McDonald T, Figueroa JP, Nathanielsz PW 1989 Neuropeptide cells and fibers in the hypothalamus and pituitary of the fetal sheep: comparison of oxytocin and arginine vasopressin. Neuroendocrinology 50:633-643 15. Maclsaac RJ, Congiu M, Levidiotis M, McDougall JG, Wintour EM 1989 In vivo regulation of adrenocorticotrophin secretion in the immature ovine fetus. Modulation by ovine corticotropin releasing hormone and arginine vasopressin. J Dev Physiol 12:4147 16. Perks AM, Vizsoly E 1973 Studies of the neurophypohysis in foetal mammals. In: Comline KS, Cross KW, Dawes GS, Nathanielsz PW (eds) Foetal and Neonatal Physiology. Cambridge University
Endo • 1991 Voll29«Nol
Press, Cambridge, pp 430-438 17. Stark RI, Daniel SS, Husain MK, Tropper PJ, James LS 1985 Cerebrospinal fluid and plasma vasopressin in the fetal lamb: basal concentration and the effect of hypoxia. Endocrinology 116:65-72 18. Yellon SM, Longo LD 1987 Melatonin rhythms in fetal and maternal circulation during pregnancy in sheep. Am J Physiol 252:E799-E802 19. Jard S, Gaillard RC, Guillon G, Marie J, Schoenenberg P, Miller AF, Manning M, Sawyer WH 1986 Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol Pharmacol 20:171-177 20. Kruszynski M, Lammek B, Manning M, Seto J, Haldar J, Sawyer WH 1980 [l-(B-Mercapto-BB-cyclopentamethylenepropionicacid), 2-(O-methyl)tyrosine]arginine-vasopressin and [l-(J3-mercapto-BB-cyclopentamethylenepropionic acid) arginine-vasopressin, two highly potent antagonist of the vasopressor response to arginine-vasopressin. J Med Chem 23:364-368 21. Schwartz J, Reid IA 1986 Characteristics of the receptors which mediate the stimulation of ACTH secretion by vasopressin in conscious dogs. Neuroendocrinology 42:93-96 22. Manning MM, Sawyer WH 1990 Discovery, development, and some uses of vasopressin and oxytocin antagonists. J Lab Clin Med 114:617-632 23. Tikanaka T, Alonso JG, Gilbert RD, Siu B, Clemons GK, Longo LD 1989 Fetal responses to long-term hypoxemia in sheep. Am J Physiol 256:R1348-R1354 24. Wood CE, Rudolph AM 1983 Negative feedback regulation of adrenocorticotropin secretion by cortisol in ovine fetuses. Endocrinology 112:1930-1936 25. Challis JRG, Patrick JE, Cross J, Workewych J, Manchester E, Power S 1981 Short-term fluctuations in the concentrations of cortisol and progesterone in fetal plasma, maternal plasma, and amniotic and allantoic fluids from sheep during late pregnancy. Can J Physiol Pharmacol 59:261-267 26. Lottering FK, Gilbert RD, Longo LD 1983 Exercise responses in pregnant sheep: blood gases, temperatures, and fetal cardiovascular system. Am J Physiol 55:842-850 27. Clarke IJ, Clements JA, Cummins JT, Dench F, Smith AI, Robinson PM, Funder JW 1986 Elevated levels of pro-opiomelanocortin derived peptides in sheep following hypothalamo-pituitary disconnection. Neuroendocrinology 44:508-514 28. Shen PJ, Clarke IJ, Canny BJ, Funder JW, Smith AI 1990 Arginine vasopressin and corticotropin releasing factor: binding to ovine anterior pituitary membranes. Endocrinology 127:2085-2089 29. Brooks AN, Power LA, Jones SA, Yang KP, Challis JRG 1989 Controls of corticotrophin-releasing factor output by hypothalamic tissue from fetal sheep in vitro. J Endocrinol 122:15-22 30. Gambacciami M, Liu JH, Swartz WH, Tueros VS, Rasmussen D, Yen SSC 1987 Intrinsic pulsatility of ACTH release from the human pituitary in vitro. Clin Endocrinol (Oxf) 26:557-563 31. Schwartz J, Tham T, Funder JW 1989 Chloroquine decreases adrenocorticotrophin-secretory response to corticotrophin-releasing factor but not to vasopressin in rat pituitary cells. Further evidence for differentially responsive subpopulations. J Endocrinol 2:25-28 32. Ozolins IZ, Young IR, McMillen IC 1990 Effect of cortisol infusion on basal and corticotropin-releasing factor (CRF)-stimulated plasma ACTH concentrations in the sheep fetus after surgical isolation of the pituitary. Endocrinology 127:1833-1840 33. Alexander DP, Bashore RA, Britton HG, Forsling ML 1974 Maternal and fetal arginine vasopressin in the chronically catheterized sheep. Biol Neonate 25:242-248 34. Gomez RA, Meernik JG, Kuehl WD, Robillard JE 1984 Developmental aspects of the renal response to hemorrhage during fetal life. Pediatr Res 18:40-46 35. Hennessey DP, Coghlan JP, Hardy KJ, Scoggins BA, Wintour EM 1980 The origin of cortisol in the blood of fetal sheep. J Endocrinol 95:71-79 36. Jones CT, Luther E, Richie JWK, Worthington D 1975 The clearance of ACTH from the plasma of adult and fetal sheep. Endocrinology 106:1371-1375
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:47 For personal use only. No other uses without permission. . All rights reserved.
