Developmental aspects of ovine adrenal adrenocorticotropic hormone receptor expression.

OBJECTIVE: Maturation of adrenocortical function is important because a prepartum increase in fetal plasma glucocorticoids is required for survival after birth. Adrenal maturation may include alterations in the regulation of adrenocorticotropic hormone (ACTH) receptor expression. Therefore, we quantitated ACTH receptor expression in the ovine adrenal cortex during development and after manipulations to better understand the regulation of the adrenal receptor in vivo. METHODS: For the ontogeny study, adrenals were obtained from fetuses at different stages of development, and the cortical tissue was stored at -80C until total RNA was extracted. The ACTH binding studies were done on adrenal membranes obtained from fetuses at two different ages using I125 (Phe-2, Nle-4) ACTH as the ligand. Plasma ACTH was measured by two-site immunoradiometric assay, and cortisol was measured by radioimmunoassay. ACTH receptor mRNA was quantitated by ribonuclease protection assay. The data were analyzed by analyses of variance. RESULTS: ACTH receptor mRNA level progressively increased in fetal life; relative changes in receptor mRNA and binding were similar (3.0-fold and 2.4-fold, respectively). Physiologic increases in fetal plasma cortisol decreased adrenal ACTH receptor mRNA concentration, and there was a strong (r2=0.76, P<.002) linear relationship between fetal plasma ACTH concentration and receptor mRNA levels. Receptor mRNA stability increased in development, and message half-life was greater in adulthood than in fetal life. CONCLUSION: The data suggest that developmental increases in receptor expression are part of the maturation process in the fetal adrenal and that plasma ACTH concentration plays a major role in regulating ACTH receptor mRNA levels in vivo.

Renal mRNA response to reduced perfusion pressure conserved despite denervation in mature ovine fetuses.

We hypothesized that renal denervation in mature ovine fetuses reduces renin mRNA response to 24 h of reduced renal perfusion pressure (RPP). Seven occluder (O) (132.4 +/- 1.2 days gestation) and six control (C) (131.5 +/- 1.2 days gestation) fetuses underwent left renal denervation. Postoperatively, O fetuses experienced 24 h of reduced RPP by suprarenal aortic occlusion. Femoral arterial blood pressure (FAB) and plasma active renin (pARC) and prorenin (pPRC) concentrations were obtained hourly for 6 h and at h 23 and 24. Renin mRNA was measured by RNase protection assay. We quantitated renin containing glomeruli by immunocytochemistry. Variables were compared by ANOVA. Mean O group FAB reduction from baseline was -6.60 +/- 0.41 mmHg. pARC and pPRC increased with occlusion, renal ARC and renal PRC did not increase with occlusion. No effect in renin mRNA or number of positive glomeruli was noted with denervation in the basal state; however, significant increases were noted in response to RPP irrespective of innervation status. In conclusion, 24 h or reduced RPP in mature ovine fetus increases renal renin mRNA and the immunocytochemical expression of renin. This response is conserved despite denervation.

Effect of renal denervation on renin gene expression, concentration, and secretion in mature ovine fetus.

To determine the role of the renal nerves on renin secretion and expression in the mature ovine fetus, we performed bilateral renal denervation on eight fetuses of time-dated pregnant ewes (126.8 +/- 0.6 days gestation) and compared renin in them to seven fetuses that underwent sham denervation (126.7 +/- 0.6 days gestation). Fetal arterial and venous catheters were implanted, and after 5-7 days of recovery isoproterenol was infused. Plasma active renin was lower in denervated animals than in intact animals under basal conditions and at each dose of isoproterenol. Plasma prorenin levels were lower in denervated fetuses but unaffected by isoproterenol. Denervation did not change renal renin, prorenin, or renin mRNA, but it did block isoproterenol-induced increases in renin mRNA in renocortical cells in vitro. We conclude that the renal nerves are required for renin secretory mechanisms and responsiveness of renin mRNA to beta-adrenergic stimulation but not for the expression of renin in the fetal kidney. We propose that one or more of the factors that maintain renin expression in the perinatal period may be absent or may be replaced by the renal nerves in the adult.

Active renin, prorenin, and renin gene expression after reduced renal perfusion pressure in term ovine fetuses.

We studied the pattern of plasma active renin concentration (ARC), prorenin concentration (PRC), renal renin concentration, and the renin mRNA levels in ovine fetuses subjected for 24 h to reduced renal perfusion pressure (RPP). The results obtained in five animals (133.8 +/- 1.4 days of gestation) in which RPP was reduced by 10 mmHg were compared with those in seven control fetuses (130.3 +/- 0. 8 days of gestation) without pressure reduction. Plasma samples were obtained before and at intervals of 24 h after initiating reduced RPP. The plasma ARC increased within 60 min of reduced RPP, reaching a maximum (13.0 +/- 4.7 vs. 115.7 +/- 23.8, P < 0.01) at 3 h. The ARC then declined toward control values. In contrast, plasma PRC did not increase consistently until 4 h into reduced RPP, with maximal levels at 24 h (8.2 +/- 2.4 vs. 87.7 +/- 21.9, P = 0.016). Within the kidney PRC, but not ARC, increased significantly, by 2.5-fold. Reduced RPP also increased renal renin mRNA levels (P = 0.004). We conclude that a chronic reduction in RPP in the near-term ovine fetus increases renal PRC and is associated with increased plasma prorenin levels. The data suggest that the conversion of prorenin to active renin is an important regulation point of the renin ANG system during development.

Cortisol infusion depresses the ratio of bioactive to immunoreactive ACTH in adrenalectomized sheep fetuses.

We examined the effects of exogenous cortisol on plasma immunoreactive adrenocorticotropic hormone (iACTH), bioactive ACTH (bACTH), and ACTH-(1-39) in nine adrenalectomized fetuses at 126-130 and 136-140 days of gestation. Fetuses received 4 h of cortisol (2 micrograms.kg-1.min-1) or saline infusions on consecutive days. Blood was obtained before and at intervals during infusions. Arterial blood gases and hematocrits were normal and did not change with age. Plasma cortisol did not change during saline infusions but increased significantly (range 30-70 ng/ml) during cortisol infusions. Basal plasma iACTH, bACTH, ACTH-(1-39), and bACTH-to-iACTH and ACTH-(1-39)-to-iACTH ratios were significantly higher in the older fetuses. Cortisol infusions decreased plasma iACTH, bACTH, and ACTH-(1-39) in both groups, and the suppression as a percent of the baseline was similar. The bACTH-to-iACTH ratio declined to the same level at 126-130 (0.201 +/- 0.040 to 0.051 +/- 0.002) and 136-140 (0.389 +/- 0.088 to 0.046 +/- 0.002) days of gestation. These data suggest that physiological concentrations of cortisol selectively inhibit bACTH secretion, and the ACTH response to cortisol inhibition is not different between 126 and 140 days of gestation in adrenalectomized sheep fetuses.

ACTH-like bioactivity and immunoactivity in fetal lamb pituitaries at 0.65 and 0.95 gestation.

We wished to determine if the concentration of bioactive ACTH-like activity increased during development and if there was heterogeneity in ovine fetal anterior pituitary ACTH activity as measured by bioassay and radioimmunoassay (RIA). We obtained anterior pituitaries from eight sheep fetuses (four at 0.65 and four at 0.95 gestation; term 145 +/- 5 days) and extracted and homogenized them in ice-cold 5N acetic acid, 0.3% phenylmethanesulfonyl fluoride (PMSF) and 0.2% BSA. Fractionation of each pituitary extract was performed by size-exclusion chromatography using Sepadex G-50. The ACTH-like immunoactivity (ALI) profile for each pituitary showed two well-defined peaks. One eluted with human ACTH1-39 and the other eluted with the high molecular weight fraction in the void volume. Four fractions from the first peak representing the high molecular weight forms of ACTH activity and four fractions from the second peak representing the low molecular weight forms of ACTH activity were pooled separately. These two pools were subjected to reverse-phase chromatography (RPC) on a C-8 column using a linear gradient of 70% acetonitrile in 0.8% trifluoroacetic acid over a 60 min period. Based upon the RIA, the high molecular weight forms of ACTH from the G-50 column were resolved into three main fractions, one eluting similar to the standard ACTH1-39 and the remaining two eluting after that. The low molecular weight forms of ACTH from the G-50 column were resolved into three peaks, before, with, and after the standard. We used collagenase-dispersed rat adrenal cells to test the ACTH-like bioactivity (ALB) of the crude extracts and of the different fractions obtained from the RPC of the high and low molecular weight material. The concentration of ACTH-like bioactivity in the crude extracts was similar at the two stages of gestation. However, there was a trend for the low molecular weight peak to have more peptide eluting with human ACTH1-39 and higher ratios of ALB/ALI than did the high molecular weight peak. These results suggest that multiple ACTH molecular forms with different ALB/ALI ratios are present in the ovine fetal pituitary and that there is no selective increase in ACTH1-39 concentration in the fetal pituitary in late gestation.

Regulation of bioactive and immunoreactive ACTH secretion by CRF and AVP in sheep fetuses.

The purpose of this study was to determine the effects of corticotropin-releasing factor (CRF) or arginine vasopressin (AVP) on the secretion of bioactive adrenocorticotropic hormone (bACTH) and immunoreactive ACTH (iACTH), the latter being measured by radioimmunoassay and separate two-site immunoradiometric assays for ACTH-(1-39) and ACTH precursors. Experiments were performed on chronically catheterized fetal sheep at 0.70 (n = 9) and 0.90 (n = 8) gestation. Each fetus received a 15-min infusion of CRF, AVP, or saline on 3 consecutive days. Blood was obtained before and 15 and 60 min after the infusion began. CRF significantly increased iACTH at 15 (younger group) and 60 min (both groups). CRF significantly increased bACTH and the bACTH-to-iACTH ratio (bACTH/iACTH) in both groups at 15 and 60 min. AVP significantly increased iACTH, bACTH, and bACTH/iACTH in both groups at 15 min. In two subgroups (n = 4/subgroup), CRF significantly increased ACTH-(1-39) and ACTH precursors at 15 and 60 min. CRF increased the ratio of ACTH-(1-39) to ACTH precursors [ACTH-(1-39)/ACTH precursors] at 15 (younger group) and 60 min (both groups). AVP increased ACTH-(1-39), ACTH precursors, and ACTH-(1-39)/ACTH precursors in both groups at 15 min. These findings show that both CRF and AVP can stimulate the secretion of bACTH, ACTH-(1-39), and ACTH precursors at 0.70 and 0.90 gestation. The proportional increments in bACTH/iACTH and ACTH-(1-39)/ACTH precursors suggest that CRF and AVP evoke selective increases in bACTH and ACTH-(1-39).

The effects of chronically elevated plasma cortisol on the bioactive and immunoreactive corticotropin secretory responses to hemorrhage in the fetal sheep at 0.70 gestation.

OBJECTIVE: Our purpose was to test the hypothesis that a long-term physiologic elevation of plasma cortisol would not alter the basal plasma concentrations of immunoreactive and bioactive corticotropin, yet it would evoke an increase in the ratio of bioactive to immunoreactive corticotropin secreted during stress. We define immunoreactive corticotropin (i.e., immunoreactive corticotropin-like activity) as that material obtained from plasma that displaces the binding of tracer quantities of iodine 125-labeled corticotropin from antisera directed toward the 6-24 portion of the corticotropin (1-39) molecule, whereas bioactive corticotropin (i.e., bioactive corticotropin-like activity) is defined as that material obtained from plasma that stimulates the secretion of corticosterone from dispersed rat adrenal cells in comparison to known concentrations of synthetic corticotropin (1-39). STUDY DESIGN: We studied the plasma immunoreactive and bioactive corticotropin response to hemorrhage in eight vehicle- and 10 cortisol-treated fetal sheep at 101 +/- 1 days of gestation. At 94 +/- 1 days of gestation each fetus began receiving a 6-day continuous infusion of either vehicle or cortisol. During the last 10 minutes of the infusion about 30% of the estimated blood volume was withdrawn from the fetuses. RESULTS: Basal plasma cortisol was significantly higher in the cortisol group (20.6 +/- 2.3 ng/ml vs 2.7 +/- 0.3 ng/ml). Basal plasma immunoreactive ACTH and bioactive corticotropin were not significantly different between groups. In both groups the plasma immunoreactive corticotropin significantly increased during hemorrhage, although the increase in the cortisol group (32 +/- 8 to 40 +/- 8 pg/ml) was significantly less than that in the vehicle group (45 +/- 14 to 86 +/- 28 pg/ml). In contrast, plasma bioactive corticotropin increased significantly during hemorrhage in the vehicle group (10 +/- 1 to 16 +/- 3 pg/ml) alone. CONCLUSIONS: In the early-gestation fetal sheep a chronic elevation of plasma cortisol does not significantly lower basal plasma immunoreactive and bioactive corticotropin. Nevertheless, it (1) attenuates the immunoreactive corticotropin response and (2) abolishes the bioactive corticotropin response to hemorrhage. It is possible that some of the negative feedback effects of plasma cortisol on corticotropin release occur at the posttranslational level.

Bioactive-to-immunoreactive ACTH activity changes with severity of stress in late-gestation ovine fetus.

The late-gestation ovine fetus (> 130 days, 0.90 gestation, full term 145 days) is capable of increased pituitary-adrenal activity following a variety of stressors. To examine the relationship between plasma immunoreactive adrenocorticotropic hormone (iACTH) and bioactive ACTH-like activity (bACTH), late-gestation fetuses were studied under nonstress (group I, chronically catheterized), "mild" stress (group II, acutely exteriorized), or marked stress (group III, acutely exteriorized and hemorrhaged 30% of blood volume). Plasma iACTH was determined by standard radioimmunoassay. Plasma bACTH was determined by a bioassay that utilized dispersed rat adrenal cells. Plasma iACTH was lower in group I than in groups II or III (78.4 +/- 16.3 vs. 320 +/- 116 and 622 +/- 144 pg/ml, respectively, mean +/- SE, P < 0.05 group I vs. III). Plasma bACTH was significantly lower in groups I and II than in group III (13.8 +/- 2.7 and 52 +/- 22 vs. 601 +/- 106 pg/ml, respectively, P < 0.05). The ratio of bACTH to iACTH was low in groups I and II but elevated in group III (0.188 +/- 0.028 and 0.091 +/- 0.07 vs. 0.996 +/- 0.122, P < 0.05). We conclude that the amount of bACTH compared with iACTH in the late-gestation fetus is low under nonstress states or mild stress and that bACTH increases disproportionately compared with iACTH after marked stress. This suggests that a change in ACTH processing and/or secretion is associated with stimulus intensity in the late-gestation ovine fetus.

Corticotropin and cortisol responses to corticotropin-releasing factor in the chronically hypoxemic ovine fetus.

OBJECTIVE: The purpose of this study was to determine if mild hypoxemia (approximately 25% below normal) of at least 5 days' duration alters corticotropin and cortisol responses to corticotropin-releasing factor. STUDY DESIGN: We studied 14 (hypoxemic, n = 5; normoxemic, n = 9) fetuses of 135 +/- 1 (mean +/- SEM) days' gestational age. Fetuses were placed in the experimental group if arterial PO2 was < or = 16 mm Hg for 5 days. In normoxemic animals arterial PO2 was > or = 17 mm Hg. Plasma hormone responses were compared by analysis of variance. RESULTS: Resting corticotropin levels were not different (hypoxemic 26 +/- 5 pg/ml, normoxemic 29 +/- 12 pg/ml), and corticotropin-releasing factor (530 +/- 30 ng/kg) increased (p = 0.01) corticotropin levels similarly in both groups. Basal plasma cortisol levels (hypoxemic 20 +/- 10 ng/ml, normoxemic, 30 +/- 7 ng/ml) were not significantly different. Both groups had similarly increased (p < 0.01) plasma cortisol levels after corticotropin-releasing factor administration. CONCLUSION: Mild hypoxemia lasting 5 days does not significantly alter corticotropin and cortisol responses to corticotropin-releasing factor in the late-gestation ovine fetus.

The ratio of plasma bioactive to immunoreactive ACTH-like activity increases with gestational age in the fetal lamb.

The fetal ovine pituitary-adrenal axis plays an important role in the timing of parturition, in fetal lung maturation, and in fetal and neonatal responses to stress. While the ovine pituitary during the last third of gestation (term = 145 days) is capable of secreting immunoreactive ACTH (iACTH) in response to various stimuli, plasma cortisol levels frequently do not reflect the rise in plasma ACTH. Therefore, we examined the relationship between plasma iACTH and steroidogenic ACTH-like activity (bACTH) in a group of immature fetal lambs (Group I: gestational age = 97 +/- 2 days, mean +/- SEM, n = 16) and a group of near-term fetuses (Group II: gestational age = 136 +/- 1 days, n = 13) following acute exteriorization. Plasma iACTH was determined by RIA. Plasma bACTH was determined by the ability of glass-extracted material to stimulate corticosterone (B) production in an acutely dispersed rat adrenal bioassay. Plasma iACTH and bACTH levels varied among animals within age groups, with iACTH tending to be higher in immature fetal lambs (Group I) than near-term lambs (Group II) and bACTH being higher (P < 0.05) near term than earlier (Group I: iACTH = 807 +/- 273 pg/ml, bACTH = 173 +/- 44 pg/ml; Group II: iACTH = 405 +/- 85 pg/ml, bACTH = 371 +/- 96 pg/ml). The proportion of iACTH that had biologic activity (e.g. B/I ratio) was significantly greater in the older than in the younger fetuses (Group II: B/I = 0.862 +/- 0.109; Group I: B/I = 0.462 +/- 0.105 P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

ACTH and cortisol responses to hypotension in fetal sheep after a prior CRF injection.

To determine whether an ovine corticotropin-releasing factor (oCRF) injection modifies adrenocorticotropic hormone (ACTH) and cortisol responses to hypotension and whether the effect of any interactions between these stimuli changes across gestation, we studied chronically cannulated fetal lambs of 103-113 ("immature") and 133-139 days gestation ("mature"). Experimental groups received 500 ng/kg oCRF injections and 6 h later had arterial pressure reduced 20% for 10 min with nitroprusside. Blood samples were obtained before and after each manipulation. Controls received vehicle instead of oCRF. The oCRF increased plasma cortisol levels from 2.1 +/- 0.4 to 14.2 +/- 4.7 (SE) ng/ml in immature and 44.9 +/- 2.2 to 102.8 +/- 15 ng/ml in mature animals. In mature fetuses the oCRF did not alter plasma ACTH and cortisol increases due to hypotension. In immature animals ACTH increases were normal but cortisol increases were eliminated. This suggests that the CRF caused maximal stimulation of the adrenal gland. In older fetuses, it appears that the action of ACTH-releasing factors, secreted in response to arterial hypotension, can overcome the negative feedback effects of elevations in endogenous cortisol.

Cardiovascular effects of dynorphin A-(1-13) and arginine vasopressin in fetal lambs.

The cardiovascular effects of the kappa-opioid receptor agonist, dynorphin A-(1-13) (D13), were compared with those of arginine vasopressin (AVP) in intact and bilaterally vagotomized (VGX) fetal lambs. Intravenous injection of AVP (114 ng/kg) produced a significant rise in mean arterial pressure (MAP) lasting 15-20 min in both intact and VGX lambs. AVP produced a bradycardia in intact fetuses concurrently with the MAP response and had no effect on heart rate (HR) in VGX fetuses. D13 (500 micrograms/kg) also produced significant increases in MAP in intact and VGX fetuses that lasted 45-60 min. D13 produced a bradycardia in intact fetal lambs but, unlike AVP, significantly increased HR in VGX fetuses. HR responses to D13 had time courses similar to MAP responses in both intact and VGX fetal lambs. Pretreatment with a vasopressin V1-receptor antagonist significantly attenuated pressor responses to AVP in both treatment groups and to D13 in intact fetuses and abolished the D13 pressor response in VGX fetuses. The antagonist also completely blocked HR responses to AVP in intact and to D13 in VGX fetuses and attenuated the D13 HR response in intact fetuses. Therefore the effects of D13 on ovine fetal cardiovascular function appear to be mediated in part through AVP pathways. D13 also appears to have a positive chronotropic effect on the heart that is normally masked by inhibitory vagal activity.

Comparison of high-dose opioid antagonist effects on ovine fetal cardiovascular function.

The opioid antagonists, naloxone (NOX) and naltrexone (NTX), were found to produce dose-dependent increases in fetal mean arterial pressure over a dose range of 5-80 mg/kg. There was a concomitant decrease in fetal heart rate up to 40 mg/kg. Above this dose, NOX and NTX caused an increase in heart rate as well as blood pressure. NTX produced similar effects in maternal ewes, although at lower doses (mg/kg) than those needed for fetal lambs. There were no age-related differences in antagonist effects in two fetal age groups studied (100-116 and 124-144 days of gestation). The partial antagonist, levallorphan (LVL), produced effects which were qualitatively similar to those produced by NOX and NTX in doses up to 20 mg/kg. These effects were not stereospecific, as the enantiomer of LVL, dextrallorphan, produced similar effects at equal doses. Pretreatment with the alpha 1-adrenoreceptor antagonist, prazosin, abolished the opioid antagonist effects on fetal blood pressure. We postulate that high doses of opioid antagonists activate sympathetic systems to increase fetal blood pressure through mechanisms which do not involve interactions with mu, delta or kappa opioid receptors.

Rat adrenal zona fasciculata cell electrophysiological responses to ACTH and gamma-MSH vary with age.

A comparison was made of the resting membrane potential (MP) as well as the MP responses to K+, ACTH and gamma-MSH of superfused adrenocortical, zona fasciculata cells from young (Y) and old (O) male rats. The resting MP of these cells did not vary with age. However, the MP responses to altered extracellular [K+] varied significantly with age. A prolonged biphasic depolarization was observed following ACTH administration; these responses were significantly reduced in O cells. In contrast, stimulation with gamma-MSH did not consistently elicit depolarization in the Y but caused a consistent and prominent depolarization in O cells. These findings suggest that aging is associated with changes in primary membrane events which could explain the reduced ACTH-stimulated steroidogenesis associated with age. Elevated cellular responses to gamma-MSH may contribute to a maintenance of, or increase in, circulating corticosterone levels in aged rats.