Ovine fetal adrenal synthesis of cortisol: regulation by adrenocorticotropin, angiotensin II and transforming growth factor-beta.

An increase in cortisol production by the fetal adrenal cortex is an important prepartum event. The increase in ovine fetal adrenal synthesis of cortisol appears to rely in part on the ACTH induction of 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) which occurs before parturition. In the present study we examined the effect of ACTH treatment on cortisol production and P-450(17) alpha expression using primary cultures of ovine fetal adrenal cells. In addition, we examined the effects of angiotensin II (A-II) and transforming growth factor-beta (TGF beta) on ACTH-treated cells. We have demonstrated previously that these factors modulate P-450(17 alpha) levels in adult ovine and bovine adrenal cells. Fetal ovine adrenal cells were isolated from 126- to 130-day fetuses (term = 144 +/- 3 days) and placed in monolayer cell culture. After 1 day in culture the cells were treated with ACTH (10 nM) with or without A-II (0.1-100 nM) or TGF beta (1-100 pM). Medium content of cortisol was low under basal conditions, whereas ACTH-stimulated cortisol production by 10- to 100-fold. A-II and TGF beta inhibited ACTH-induced cortisol production by 70-90%. In addition, 3 days of treatment with ACTH caused a greater than 10-fold induction of P-45017 alpha enzyme activity in fetal adrenal cells. A-II and TGF beta inhibited the ability of ACTH to induce P-45017 alpha activity by at least 75%. Using an antibody to P-45017 and immunoblotting techniques the effects of ACTH, A-II, and TGF beta on enzyme activity were observed to correspond to cellular levels of P-45017 alpha protein. The inhibitory effects of TGF beta and A-II could not be overcome by the cAMP analog (Bu)2cAMP. Interestingly, the expression of the enzyme 3 beta-hydroxysteroid dehydrogenase was much less sensitive to inhibition by A-II or TGF beta. The ability of A-II and TGF beta to suppress P-45017 alpha expression could play a role in determining the pathway of steroidogenesis and specifically the amount of cortisol produced by fetal adrenocortical cells in vivo.     

Adenosine 3'5'-cyclic monophosphate dependent protein kinase in human adrenocortical tumors.

Protein kinase activity has been studied in four human adrenocortical tumors and compared to the one of the normal human adrenal. In two cases where the lack of action of ACTH was related to an anomaly of ACTH receptor, the protein kinase activity was normal. In the other two cases the ACTH receptor was normal, but the protein kinase activity was different from that of the normal adrenal. In one of these cases where the steroidogenesis response of isolated tumor cells to ACTH and DcAMP was higher than in normal adrenal, basal and cAMP stimulated protein kinase activities were significantly higher than those of the normal adrenal, but the activation constants of both nucleotides were similar to those of the normal gland. In the other case, the basal and the cAMP stimulated protein kinase activities were significantly lower, as well as the activation constant of cAMP. However, the binding affinity of 3H-cAMP was normal. Normal adrenal cytosol contains three protein kinases, as resolved by DEAE-cellulose, two of which designated I and II, are cAMP-dependent. The DEAE-cellulose chromatography of the last tumor showed a loss of isoenzyme II. In addition, the protein kinase eluted at the same molarity as that of isoenzyme I of the normal adrenal was not activated by cAMP. Therefore, the lack of response to ACTH of some adrenocortical human tumors may be attributed either to an anomaly of the ACTH receptor or to some defect of the cAMP-dependent protein kinase.     

Effects of fetal hypophysectomy and the in vitro treatment by pituitary extracts on the maturation of cultured ovine fetal adrenal cells

In the first series of experiments, the ability of cultured adrenal cells from 113-day-old ovine fetuses to produce both cAMP and corticosteroids in response to ACTH-(1-24) or to fetal (FPE) or newborn acidic pituitary extracts (NPE) was investigated daily. Basal cAMP output did not change during the culture period. When cells from 124-day-old ovine fetuses, or from 5-day-old lambs, were repeatedly stimulated (2 h/day for 4 days) by ACTH-(1-24) or by pituitary extracts, the cAMP responses increased with the same pattern. Outputs on day 4 were 7-fold higher than those on day 1 for ACTH-(1-24)-matured cells or for cells matured by FPE or NPE. The steroid output induced by ACTH-(1-24) or by FPE or NPE developed identically during the experiment to become, on day 4, more than 40-fold higher than on day 1. The response to ACTH-(1-24) on day 5 was also identical both in cAMP and corticosteroids whether the cells had been previously treated with ACTH-(1-24) or with FPE or NPE, In the second set of experiments, adrenal cells from 124-day-old-ovine fetuses either intact of hypophysectomized (Hx) at 118 days of gestation were cultured for 6 days in the absence or presence of ACTH-(1-24). ACTH-(1-24) treatment resulted in a development of cAMP and corticosteroid responses to the hormone which was slower for cells from Hx than cells from control fetuses during the first 3 days of culture. Likewise both cAMP and corticosteroids responses to ACTH-(1-24) of adrenal cells from Hx fetuses cultured for 1 to 3 days in the absence of ACTH were lower than those of cells from control fetuses cultured under the same conditions. These results demonstrate that the pituitary from 124-day-old ovine fetuses contains trophic (and steroidogenic) substances in a sufficient amount to allow in vitro adrenal maturation. Moreover, it appears that high mol wt forms of ACTH, which are most probably extracted by the method we used, did not prevent the in vitro development of the response to ACTH-(1-24). Finally, they show that removal of pituitary hormones in vivo resulted in a decreased potency of fetal adrenal cells to respond in vitro to ACTH-(1-24).     

Adenosine 3',5'-monophosphate-dependent protein kinase from normal human adrenal.

The protein kinase of normal human adrenal cytosol has been resolved by DEAE-cellulose chromatography into two major components, the protein kinases I and II, which are both adenosine 3',5'-monophosphate (cAMP) dependent. Both enzymes have similar substrate specificities, cAMP-dependency, and sensitivity to the stimulation by this nucleotide, but differ in their states of activation after preincubation with histone. The DEAE--cellulose charomatography of dissociated cytosol protein kinase reveals only one peak of kinase activity and two peaks of cAMP binding activity (A and B). Both binding proteins are able to inhibit the kinase activity of the catalytic subunit. Recombination experiments suggest that the regulatory subunit A originated from protein kinase I and subunit B from protein kinase II. The phosphorylation of histone by adrenal protein kinases is inhibited by a heat-stable protein inhibitor isolated from human fetal brain and human adult adrenal.     

Two pathways for thyroxine 5'-monodeiodination in brown adipose tissue in fetal sheep: ontogenesis and divergent responses to hypothyroidism and 3,5,3'-triiodothyronine replacement

Thermogenesis in rat brown adipose tissue (BAT) is thyroid hormone responsive. Rat BAT expresses a type II 5'-iodothyronine monodeiodinase (5'MDI) which mediates local T3 production from T4. Earlier studies show that BAT from fetal and newborn sheep contains a high Km type I, instead of type II, 5'MDI. To better characterize the 5'MDI of ovine fetal BAT, we studied the in vitro monodeiodination of [125I]T4 at a low substrate concentration (2 nM) and in the presence of 1 mM propylthiouracil in BAT homogenates of control and thyroidectomized fetuses at different gestational ages as well as in newborn lambs. Thyroidectomies were performed at three gestational ages: 99-107 days (group 1), 129-132 days (group 2), and 115-117 days (group 3A). Animals were studied 8-13 days after surgery. A significant increase in the activity of a low Km T4 5'MDI was noted in BAT from hypothyroid fetuses at all three gestational ages. This low Km activity was similar to the type II enzyme in rat BAT and brain in that the activity was also T3 resistant. A gradual rise in BAT type II 5'MDI activity was measured between 99 days gestation and term (150 days). These results indicate that ovine BAT contains two distinct iodothyronine 5'-monodeiodinating activities, one with a high Km and another with a low Km. The latter, resembling the type II 5'MDI in rat brain and BAT, is increased in ovine hypothyroid BAT. The former predominates in euthyroid tissue and is similar to the type I 5'MDI characterized in rat liver, kidney, and thyroid. We speculate that BAT type II 5'MDI may be important for neonatal BAT thermogenesis, while the type I enzyme may play a significant role in the increase in serum T3 concentration that occurs at birth.     

Thyroid hormone increases type I adenosine 3', 5'-monophosphate-dependent protein kinase and casein kinase activities in rat liver cytosol: analysis of protein kinases by polyacrylamide disc gel electrophoresis

We investigated the action of thyroid hormone on each protein kinase in rat liver cytosol. Kinases were analyzed by polyacrylamide disc gel electrophoresis and isoelectric focusing in polyacrylamide gel. Polyacrylamide disc gel electrophoresis separated cAMP-dependent protein kinase type I (Rf = 0.35), type II (Rf = 0.44), their catalytic subunit (Rf = 0.26), and cAMP-independent protein kinase (Rf = 0.50). Casein kinase was detected at Rf = 0.37. In addition to the catalytic subunit with Rf = 0.26, another catalytic subunit was found at Rf = 0.44 when the cytosol was preincubated with cAMP. The administration of T3 (20 micrograms/100 g BW for 3 days) to hypothyroid rats increased enzyme activities of type I holoenzyme and casein kinase by 48%. Free catalytic subunit, separated from holoenzyme, had the same level of enzyme activity in both groups, suggesting greater endogenous dissociation of type I holoenzyme in hypothyroid rats. When heat-inactivated rat liver cytosol was used as substrate in the assay of protein kinase activity, the peak enzyme active in phosphorylating the cytosol corresponded to the casein kinase peak. Our data indicate that casein kinase is the main enzyme that mediates phosphorylation of endogenous proteins in rat liver cytosol, and that T3 treatment increases the activity of casein kinase and of type I cAMP-dependent protein kinase.     

Responses of rat fetal adrenals to synthetic adrenocorticotrophic hormone and alpha-melanocyte-stimulating hormone in-vivo and in-vitro studies

The response of the adrenals from rat fetuses at 16, 18 and 20 days of gestation to 1-24 ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH) was studied in vitro. The response to 1-24 ACTH increased as gestation progressed. By the end of fetal life, corticosterone release induced by ACTH from whole adrenals was greater than that observed with adrenal tissue from non-pregnant adult female rats. High doses of alpha-MSH also stimulated adrenal activity but the response to ACTH was always higher than that to alpha-MSH. The effect of 1-24 ACTH and alpha-MSH on fetal adrenal growth was also compared in vivo. The adrenal atrophy induced by fetal hypophysectomy on day 17 of gestation could be prevented by i.m. administration of 10 microgram 1-24 ACTH or alpha-MSH. However, the adrenal growth was greater in ACTH-treated fetuses than in alpha-MSH treated ones. Later in gestation, between days 19 and 20, 1-24 ACTH but not alpha-MSH was able to prevent atrophy induced by fetal hypophysectomy. These findings are discussed in relation to the literature on levels of ACTH and alpha-MSH in the plasma and pituitary glands of the rat throughout the last third of gestation. High levels of ACTH in the fetal circulation contrast sharply with very weak or undetectable concentrations of alpha-MSH. Since the present data suggest that both trophic and steroidogenic activities of ACTH were greater than those of alpha-MSH, it may be concluded that ACTH but not alpha-MSH plays a major physiological role during gestation in the regulation of both fetal adrenal growth and function in the rat.     

Somatostatin inhibits corticotropin-releasing factor-stimulated adrenocorticotropin release, adenylate cyclase, and activation of adenosine 3',5'-monophosphate-dependent protein kinase isoenzymes in AtT20 cells

The mechanisms by which somatostatin (SRIF) inhibits CRF-induced ACTH secretion from AtT20 cells were characterized by comparing the effects of SRIF on cAMP production, adenylate cyclase activity, and activation of cAMP-dependent protein kinase isoenzymes with its effects on ACTH release. In isolated membranes, CRF (100 nM) stimulated adenylate cyclase activity 4- to 5-fold. SRIF inhibited CRF-stimulated adenylate cyclase in a concentration-dependent manner. However, maximal inhibition was 50%. SRIF did not inhibit basal adenylate cyclase or forskolin-stimulated cyclase in the absence of guanine nucleotides and had only small effects on forskolin-stimulated cyclase when assayed in the presence of guanine nucleotides. CRF (100 nM) induced small rises (2-fold) in intracellular cAMP levels which produced maximal ACTH release. SRIF inhibited basal and CRF-stimulated ACTH release in a concentration-dependent manner, and there was a good correlation between inhibition of ACTH release and inhibition of the activation of cAMP-dependent protein kinases in these cells. Thus, the effect of SRIF on CRF-induced ACTH release appeared to result from its effect on inhibition of adenylate cyclase. In the presence of 3-methylisobutylxanthine (MIX), CRF increased cAMP levels 20-fold and activated a greater proportion of cAMP-dependent protein kinase, but did not stimulate ACTH release more than CRF alone. Under these conditions, SRIF (100 nM) inhibited cAMP accumulation by 90%. ACTH release was also inhibited, but higher concentrations of SRIF were required to block ACTH release compared to cells incubated in the absence of MIX. Sufficient cAMP levels were achieved so that activation of cAMP-dependent protein kinases was only partially blocked. There was still sufficient cAMP to activate cAMP-dependent protein kinase to an extent equal to that seen with CRF without MIX. Similar effects of SRIF on cAMP accumulation and protein kinase activation were seen when cells were stimulated with forskolin. Our results demonstrate that SRIF inhibits ACTH release from AtT20 cells by inhibiting hormone-sensitive adenylate cyclase and thereby prevents the activation of cAMP-dependent protein kinases. However, under conditions where cAMP-dependent protein kinases are still sufficiently active to induce ACTH secretion, high concentrations of SRIF can inhibit ACTH release by a mechanism independent of cAMP-dependent protein kinase.     

Hormonal effects on the immunocytochemical location of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in rat tissues

Homogeneous preparations of type I and type II regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinase (cAMP kinase) were utilized as antigens to obtain isozyme specific antisera. Injections of pure catalytic subunit (C) from the type I isozyme resulted in antisera that reacted with C subunit obtained from either isozyme type. Cross-reactivity of the antisera raised against isolated subunits of the kinase was assessed by immunodiffusion analysis and by measuring the cAMP binding and phosphotransferase activities of the subunits after immunoprecipitation. These antisera were used to localize subunits of type I and type II cAMP kinases in rat skeletal muscle, liver, and adrenal by using indirect immunofluorescence and immunoperoxidase techniques. Specificity of the immunofluorescence was shown by absorption of the antisera with pure homologous antigens. In skeletal muscle, both R and C subunits of the type I and type II cAMP kinases were localized in the area of the sarcoplasmic reticulum and in periodic crossbands. Specific fluorescence for these components was observed in both isotropic and anisotropic band regions of the sarcomere. Densitometric determinations of immunoperoxidase staining revealed a larger amount of RI, RII, and C subunits in the isotropic band than in the anisotropic band regions. In liver, C, RI, and RII subunits were distributed both in cytoplasmic and nuclear areas and along plasma membranes of hepatocytes; however, there were qualitative differences observed among these various subcellular sites. With each antiserum, fluorescence was blocked by prior absorption with homologous antigen. After treatment of rats with glucagon, dramatic changes in the relative distribution patterns of C and RII were noted in the nucleus. In the adrenal gland, RI, RII, and C subunits were localized in both cytoplasmic and nuclear areas, and an apparent redistribution of these subunits occurred after treatment of (dexamethasone-suppressed) rats with ACTH. The application of this immunocytochemical approach provides a tool for examining and monitoring the subcellular distribution of these components of cAMP kinase in biological systems.     

In vivo adrenocorticotropin (1-24)-induced accumulation of cyclic adenosine monophosphate by ovine fetal adrenal cells is inhibited by concomitant infusion of metopirone

Activation of the ovine fetal adrenal gland after pulse ACTH (P-ACTH) administration is associated with an increase in plasma cortisol levels. We have investigated whether cortisol may play a role in this adrenal activation process. The ability of fetal adrenal cells to accumulate cAMP in response to ACTH in vitro was compared in fetuses (day 132 of gestation) that had received infusions (100 h) in utero of 1) saline or saline + tartrate (0.5 ml for 15 min/2 h) (n = 4); 2) P-ACTH (66.6 ng/min for 15 min/2 h) (n = 4); 3) P-ACTH + metopirone (31.3 mg/h) (n = 4), to inhibit 11 beta-hydroxylase activity. Control fetuses showed no significant increase in plasma cortisol levels throughout the infusion and there was no significant accumulation of cAMP by fetal adrenal cells in vitro in response to ACTH. There was a significant (P less than 0.05) increase in plasma cortisol concentrations from 2.21 +/- 0.47 (mean +/- SEM) ng/ml at 0 h to 35.7 +/- 11.6 ng/ml at 96 h in fetuses receiving P-ACTH in vivo. In these fetuses there was a significant (P less than 0.05) accumulation of cAMP after addition of ACTH by fetal adrenal cells in vitro (mean increment delta = 48 pmol). This rise in plasma cortisol was prevented in fetuses receiving P-ACTH + metopirone. Further, metopirone treatment prevented the increase in fetal adrenal weight and accumulation of cAMP after in vitro ACTH that normally followed ACTH treatment in vivo. This effect was not overcome by further addition of guanylylimido-diphosphate. These experiments raise the possibility that cortisol might mediate the increase in cAMP accumulation resultant upon in vivo P-ACTH treatment.     

Evidence that cortisol inhibits basal adrenocorticotropin secretion in the sheep fetus by 0.70 gestation

To ascertain if reductions in fetal plasma cortisol cause increases in fetal plasma ACTH, we treated pregnant ewes or their fetuses with aminoglutethimide (10 mg/kg BW) and metyrapone (20 mg/kg BW) and measured the hormonal responses with RIAs. When given to fetuses (n = 9) at 0.90 +/- 0.01 gestation (term-145 days), the steroid synthesis inhibitors reduced fetal plasma cortisol from 35.1 +/- 11.9 to 18.5 +/- 6.2 ng/ml (P less than 0.01) and plasma ACTH increased from 37 +/- 7 to 189 +/- 74 pg/ml (P less than 0.02). Thus, late in gestation cortisol from the fetal adrenal suppresses basal fetal ACTH secretion. Blockade of steroid biosynthesis in pregnant ewes carrying intact fetuses at 0.76 +/- 0.02 gestation (n = 11) or adrenalectomized fetuses at 0.81 +/- 0.01 gestation (n = 6) also reduced cortisol and increased ACTH in fetal plasma. In intact fetuses cortisol declined from 9.4 +/- 2.0 to 3.6 +/- 0.9 ng/ml (P less than 0.05), and ACTH increased from 46 +/- 8 to 183 +/- 67 (P less than 0.01); cortisol declined in adrenalectomized fetuses from 2.1 +/- 0.4 to 1.1 +/- 0.3 ng/ml (P less than 0.01), and ACTH increased from 106 +/- 13 to 400 +/- 104 pg/ml (P less than 0.01). Cortisol infusions into intact and adrenalectomized fetuses prevented both the decline in steroid concentration caused by the biosynthesis inhibitors given to the ewe and the increase in fetal plasma ACTH concentration. These data indicate that reductions in plasma cortisol in adrenalectomized fetuses or intact fetuses at a time in development when the fetal adrenal produces little cortisol cause compensatory increases in fetal plasma ACTH concentration. The simplest explanation for these observations is that from approximately 0.70 gestation, basal fetal ACTH secretion is tonically inhibited by cortisol circulating in fetal plasma. This cortisol can originate from sources other than the fetal adrenal.     

Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells

Activins and inhibins are glycoprotein hormones produced mainly in gonads but also in other organs. They are believed to be important para/autocrine regulators of various cell functions. We investigated activin/inhibin receptor and binding protein gene expression and the regulation of activin/inhibin secretion in human adrenal cells. RT-PCR revealed inhibin/activin alpha-, betaA/B-subunit, follistatin, activin type I/II receptor, and inhibin receptor (betaglycan and inhibin-binding protein) mRNA expression in fetal and adult adrenals and cultured adrenocortical cells. Cultured cells secreted activin A and inhibin A/B as determined by specific ELISAs. ACTH stimulated inhibin A/B secretion in fetal (1.8- and 1.8-fold of control, respectively) and in adult cells (3.4- and 1.7-fold of control, respectively) without significant effect on activin A. 8-bromoadenosine cAMP (protein kinase A activator) increased activin A and inhibin A/B secretion in the human adrenocortical NCI-H295R cell line (32-, 17-, and 3-fold of control, respectively). 12-O-tetradecanoyl phorbol-13-acetate (protein kinase C activator) stimulated both activin A and inhibin A secretion (764- and 32-fold of control, respectively), and activin treatment increased inhibin B secretion in these cells (25-fold of control). In conclusion, human adrenocortical cells produce dimeric activins and inhibins. ACTH stimulates inhibin secretion and decreases activin/inhibin secretion ratio, probably via the protein kinase A signal transduction pathway. This, together with the adrenocortical activin/ inhibin receptor and binding protein expression, suggests a physiological role for activins and inhibins in the human adrenal gland.     

Specific loss of protein kinase activities in senescent erythrocytes

Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity technique and the activity of protein kinases and other enzymes was analyzed in cytosols and membranes from the isolated cells. The activities of cytosolic protein kinase C (PKC), cAMP-dependent kinase (PKA), and casein kinase type I and II (CKI and II) were all found to undergo an age-dependent decrease of twofold to fourfold over the 8-week lifespan of the cells. Membrane-associated tyrosine kinase showed little or no decrease, but membrane-associated CKI showed a dramatic eightfold decrease over the 8-week period. By contrast, various cytosolic enzymes, including lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and acid phosphatase, showed no change in activity over the same time period. Density-separated human erythrocytes showed qualitatively similar decreases in cytosolic protein kinase activities in the densest fractions, which contain the oldest cells. Our results show that aging erythrocytes undergo progressive loss of protein kinases that may adversely affect various cellular processes. The age-dependent loss of kinase activity reported here is one of the most striking manifestations of erythrocyte senescence yet to be reported.     

Hormonal regulation of cAMP-independent protein phosphokinase activities: thyroxine and cortisol control of enzymes from rat liver cytosol.

Thyroxine control of cAMP-independent histone and casein phosphokinase activities was studied in thyroidectomized rats treated with thyroxine. All activities were evaluated in the presence of a thermostable inhibitor of cAMP-dependent enzymes. Cytosol enzymes can be resolved by sucrose gradient ultracentrifugation into three peaks of histone kinase activity (3.2S, 5S and 7.2S) and two peaks of casein kinases (3.6S and 7.1S). Neither thyroidectomy nor subsequent treatment of operated animals with thyroxine modifies the total histone kinase activity estimated, either in total cytosol or after its fractionation by the sucrose gradient ultracentrifugation. The activity ratios of different peaks were, however, changed. Casein kinase activity was significantly decreased after thyroidectomy (about 50%). Subsequent treatment with thyroxine restored this activity to its initial value. Sucrose gradient ultracentrifugation analysis showed that thyroxine action on the casein kinase activity is very specific. Only molecules that sediment in the 9S region were significantly stimulated by the hormone. Cortisol action on the casein kinase activity was studied in adrenalectomized animals treated with hormone for 24 h. Cortisol decreases the total casein kinase activity by about 30%. Sucrose gradient ultracentrifugation analysis showed that the population of molecules sedimenting at about 9S was the most sensitive to cortisol. The above data show that both thyroxine and cortisol control, in a selective way, the activities of cAMP-independent protein kinases. The same kinase molecules can be under double control by two different hormones that have opposite effects.     

Effect of fetal adrenalectomy on messenger ribonucleic acid for proopiomelanocortin in the anterior pituitary and for corticotropin-releasing hormone in the paraventricular nucleus of the ovine fetus

In the ovine fetus, adrenalectomy at 90-120 days gestational age (dGA) results in a gradual increase in basal concentrations of fetal plasma ACTH beginning at approximately 122 dGA. Bilateral adrenalectomy at 116-119 dGA also results in an increase in POMC mRNA in the fetal pituitary. It is not known whether both the paraventricular nuclei (PVN) of the hypothalamus and the anterior pituitary of the ovine fetus are responsive in late gestation to the removal of cortisol negative feedback. The purpose of this study was to determine the subsequent effect of fetal adrenalectomy at 118-121 dGA on the CRH mRNA content in fetal PVN and on POMC mRNA in the fetal anterior pituitary at 134 dGA. Mature Rambouellet-Columbia cross-bred ewes (n = 10), bred on a single occasion only and carrying fetuses of known gestational ages, were used. Both fetal adrenal glands were exposed via a retroperitoneal approach and removed [adrenalectomized (ADX); n = 5]. In control fetuses (CONT; n = 5) adrenal glands were exposed and isolated, but not removed. At 134 dGA, fetal plasma cortisol concentrations were significantly greater in CONT fetuses (7.2 +/- 2.5 ng/ml) than in ADX fetuses (mean +/- SD, 1.97 +/- 0.9 ng/ml; P less than 0.025). At 134 dGA the fetal PVN was removed by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was prepared by the guanidium isothiocyanate-cesium chloride method and subjected to Northern analysis using specific cDNA probes to CRH and POMC. After autoradiography, quantification of mRNA was performed by scanning densitometry. Quantities of specific hybridization signal for POMC and CRH were normalized to the content of actin mRNA in each individual sample. RNA prepared from PVN exhibited a single specifically hybridizing band for CRH of approximately 1300 nucleotides. RNA prepared from anterior pituitary exhibited a single specifically hybridizing band for POMC at approximately 1300 nucleotides. Anterior pituitary POMC mRNA was significantly increased (P less than 0.025) in ADX fetuses (236 +/- 32% of CONT). CRH mRNA in PVN was greater in ADX fetuses than in CONT fetuses (P less than 0.05; mean +/- SEM, 179 +/- 21% of CONT). Adrenalectomy in fetal sheep significantly increased expression of CRH and POMC. We conclude that the increased levels of mRNA for CRH and POMC indicate that both the fetal PVN (CRH) and the anterior pituitary (POMC) are responsive to removal of the primary source of circulating glucocorticoid at this gestational age.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergism between systemic corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin release in vivo varies as a function of gestational age in the ovine fetus

In sheep, parturition is associated with maturation of fetal pituitary-adrenal function, and with rises in the concentrations of ACTH and cortisol (F) in fetal plasma. We examined the hypothesis that pituitary ACTH output in response to arginine vasopressin (AVP) and CRF separately and together might change during late pregnancy as a function of fetal age. Fetal sheep were chronically catheterized, and bolus iv injections of equimolar AVP, CRF, AVP plus CRF, or saline (controls) were given on days 110-115, 125-130, and 135-140 of gestation. AVP evoked significant rises in plasma ACTH on days 110-115 and 125-130, but not on days 135-140. After AVP, the peak plasma concentrations of ACTH were attained at 5-10 min, and basal (preinjection) values were reestablished by 30-60 min. After CRF treatment, plasma ACTH rose progressively throughout the 240 min of the study. Evidence was obtained in support of an increase in pituitary responsiveness to CRF between days 110-115 and 125-130 and a decrease in response on days 135-140, when basal F concentrations were higher. The ACTH response to AVP, relative to that to CRF, was greatest in the youngest fetuses. On days 110-115 only, CRF and AVP showed a synergistic response in ACTH output, especially during the first 30 min after agonist injection. Plasma F rose in response to the changes in endogenously released ACTH in a manner consistent with progressive fetal adrenal maturation between days 110-140 of pregnancy. We conclude that in vivo the ovine fetal pituitary responds separately and synergistically to AVP and CRF on days 110-115 of gestation, but the relative role of AVP in stimulating ACTH release decreases with progressive gestational age.     

Different roles for the pituitary and adrenal cortex in the control of enkephalin peptide localization and cortico-medullary interaction in the sheep adrenal during development.

We have investigated the effect of adrenocorticotrophic hormone (ACTH) replacement after fetal hypophysectomy on the pattern of localization of enkephalin-containing peptides (enkephalins) and phenylethanolamine N-methyltransferase (PNMT) in the fetal sheep adrenal. We have also investigated the relative roles of the fetal pituitary and adrenal cortex in determining the extent of the interdigitation of the peripheral adrenaline (AD)-containing cells of the adrenal medulla with the inner zones of the adrenal cortex in the late gestation fetus. Fetal hypophysectomy (Hx; n = 12) or sham operations (n = 8) were performed at 109-118d. At 138 or 139d, ACTH (1-24) (10.5 micrograms/h) was infused intravenously for 72 h into 4 Hx fetuses (Hx + ACTH group). Saline was infused for 72 h into 4 Hx fetuses (Hx + Sal) and into 4 sham-operated fetal sheep (Intact + Sal). Fetal adrenal glands were collected at autopsy from 141/2d Intact + Sal, Hx + Sal and Hx + ACTH groups, from 4 intact fetal sheep at 145-147d gestation (145/7d Intact group) and 4 Hx fetal sheep at 147-164d gestation (147/64d Hx group). Adrenals were also collected from 4 newborn lambs at 10-12d after birth (10/12d Newborn group). Using the peroxidase-antiperoxidase immunocytochemical staining method, sections of adrenal glands (10-12 microns) from all groups were stained anti-PNMT. Sections of adrenal glands from the 141/2d groups were also stained separately with anti-dopamine-beta-hydroxylase (anti-D beta H) and anti-enkephalin (anti-ENK).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of basal adrenocorticotropin and cortisol secretion by arginine vasopressin in the fetal sheep during late gestation.

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 microgram/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 V1 receptor antagonist d(Ch2)5Tyr(Me)AVP. In a total of seven studies, antagonist (10 micrograms/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 greater than 0.05, by analysis of variance). Moreover, plasma ACTH and 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 greater than 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.