Immunoreactive ACTH Research Articles

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).

Development of adrenocorticotropin-(1-39) and precursor peptide secretory responses in the fetal sheep during the last third of gestation

Although it is known that concentrations of immunoreactive ACTH increase during late gestation in fetal sheep plasma, the nature of the ACTH has not been well characterized. We used two-site immunoradiometric assays to separately measure high mol wt ACTH precursors (POMC and pro-ACTH) and ACTH-(1-39) in plasma of fetal sheep with chronic arterial and venous catheters. We compared the ratio of these peptides as a function of gestational age under basal conditions and in response to exogenous vasopressin and/or corticotropin-releasing hormone. Under basal conditions, the concentration of precursors was not changed throughout the last third of gestation; however, ACTH-(1-39) increased significantly approaching term. The molar ratio of precursors to ACTH-(1-39), therefore, decreased from 15.8 +/- 1.0 at 110 days to 7.9 +/- 0.6 at 140 days gestation. At all gestational ages, vasopressin and corticotropin-releasing hormone increased ACTH-(1-39) and precursors, albeit with different time courses. At 120 days gestation, arginine vasopressin plus CRH produced synergistic increases in ACTH-(1-39) and precursors, whereas the response was only additive at other ages. The present results indicate that the elevation in the resting plasma immunoreactive ACTH concentration that occurs near term is constituted by an increase in the concentration of ACTH-(1-39) relative to those of POMC and pro-ACTH, which may have further physiological significance. Also, CRH and AVP are potent stimulators of both ACTH-(1-39) and ACTH precursors.

Development of adrenocorticotropin-(1-39) and precursor peptide secretory responses in the fetal sheep during the last third of gestation.

Although it is known that concentrations of immunoreactive ACTH increase during late gestation in fetal sheep plasma, the nature of the ACTH has not been well characterized. We used two-site immunoradiometric assays to separately measure high mol wt ACTH precursors (POMC and pro-ACTH) and ACTH-(1-39) in plasma of fetal sheep with chronic arterial and venous catheters. We compared the ratio of these peptides as a function of gestational age under basal conditions and in response to exogenous vasopressin and/or corticotropin-releasing hormone. Under basal conditions, the concentration of precursors was not changed throughout the last third of gestation; however, ACTH-(1-39) increased significantly approaching term. The molar ratio of precursors to ACTH-(1-39), therefore, decreased from 15.8 +/- 1.0 at 110 days to 7.9 +/- 0.6 at 140 days gestation. At all gestational ages, vasopressin and corticotropin-releasing hormone increased ACTH-(1-39) and precursors, albeit with different time courses. At 120 days gestation, arginine vasopressin plus CRH produced synergistic increases in ACTH-(1-39) and precursors, whereas the response was only additive at other ages. The present results indicate that the elevation in the resting plasma immunoreactive ACTH concentration that occurs near term is constituted by an increase in the concentration of ACTH-(1-39) relative to those of POMC and pro-ACTH, which may have further physiological significance. Also, CRH and AVP are potent stimulators of both ACTH-(1-39) and ACTH precursors.

Cellular localization of the growth hormone receptor/binding protein in the human anterior pituitary gland.

The increasing use of GH therapy has led to the description of its target cells in human tissues, but no data are yet available on the localization of the GH receptor in the human pituitary. In the present study, we used immunocytochemistry to detect the presence of GH receptor/binding protein (GHR/BP), and we examined its distribution among the different types of human anterior pituitary cells. Human pituitaries were taken from autopsies and were processed for embedding in paraffin wax. Immunocytochemistry was performed by using monoclonal antibody 263 raised against purified rat and rabbit GHR/BP, which cross-reacts with the human GH receptor. In order to determine the types of cells that display immunoreactivity for GHR/BP, adjacent pituitary sections were used to detect immunoreactivity for GH, PRL, ACTH, TSH, LH, and FSH. Several controls were carried out to verify the specificity of the immunostaining. Receptor immunoreactivity was found in the cytoplasm and nuclei of the somatotrophs, lactotrophs, and gonadotrophs but not in the thyrotrophs or corticotrophs. In order to demonstrate that the detected GHR/BP immunoreactivity was not caused solely by a cellular capture, we also investigated the cellular distribution of GHR gene expression. This was performed by in situ hybridization with use of complementary oligonucleotide radioactive probes encoding distinct domains of the GHR. Several tests were carried out to validate the detection of gene expression. In situ hybridization demonstrated the presence of GHR messenger RNAs in the anterior lobe of human pituitary, and examination of the signal strengthened the cell-specificity of GHR gene expression. These results demonstrate the presence of GHR/BP in discrete human pituitary cells and indicate a paracrine, autocrine, or intracrine role for GH in the pituitary.

Effect of arginine vasopressin on regional adrenal blood flow and plasma cortisol concentration in fetal sheep

Objective: To determine whether arginine vasopressin (AVP) at plasma concentrations measured during moderate hypoxemia affects adrenal blood flow. Study design: Regional blood flows were measured in 5 unanesthetized normoxemic fetuses (124–128 days' gestation) during a 24-h intravenous infusion of aVP in isotonic saline solution. Another 5 fetuses received an infusion of vehicle. Blood flows were determined before the infusion and at 2 h and 24 h from its onset using radionuclide-labeled microspheres. Results: At 2 h and 24 h of AVP infusion, fetal plasma concentrations of IR-AVP had risen from 4.7 ± 0.9 pg/ml to 9.8 ± 1.1 pg/l and 9.4 ± 0.7 pg/ml, respectively. Thus we achieved plasma concentrations of IR-AVP comparable to those previously reported during moderate hypoxemia. There was no significant effect of treatment on fetal plasma concentrations of immunoreactive adrenocorticotropic hormone (ACTH) or cortisol. AVP infusion significantly decreased fetal heart rate and raised cotyledonary blood flow from 198 ± 18 ml/min per 100 g to 235 ± 17 ml/min and 218 ± 10 ml/min per 100 g at 2 h and 24 h, respectively, from the start of the AVP infusion. Basal values for adrenal medullary and cortical blood flows were similar in the AVP and saline groups, and did not change significantly during the infusions. Conclusion: These findings suggest that the rise in adrenal blood flow seen after hypoxemia is not due to a direct action of systemic AVP, but is attributable to other influences, likely including changes in circulating ACTH.

Inhibition of ACTH secretion blocks hypoxia-induced increase of adrenal cortical blood flow in fetal sheep

To examine the role of endogenous adrenocorticotropic hormone (ACTH) in adrenal blood flow responses to hypoxia, we studied unanesthetized ovine fetuses during an intravenous infusion of cortisol or vehicle. Fetal hypoxia was induced after 5 h of cortisol or vehicle infusion. Control fetuses were not made hypoxic. Blood flows were determined before and at three time points during the infusions. At 2 and 6 h of hypoxia, in vehicle-infused fetuses, fetal plasma concentrations of immunoreactive ACTH (irACTH) had risen from 9 +/- 3 (SE) pg/ml to 68 +/- 25 and 127 +/- 37 pg/ml, respectively. No significant change in fetal plasma irACTH occurred in the other groups. Adrenal cortical blood flow rose three- to fourfold during hypoxia in vehicle-infused fetuses but did not change from prehypoxia levels in cortisol-infused fetuses (P < 0.005). Medullary flow rose with hypoxemia, and this was not affected by concurrent cortisol infusion. Adrenal blood flows did not change in the control groups. Thus prior infusion of cortisol suppressed the rise in fetal plasma ACTH during hypoxia and selectively blocked the increase in adrenal cortical blood flow.

Effects of Corticosterone In Vivo and In Vitro on Adrenocorticotropic Hormone Production by Corticotropin Releasing Factor-Stimulated Leukocytes

Corticotropin releasing factor (CRF) and corticosterone have been shown to affect immune cell function. Previously, we have shown that CRF stimulates immunoreactive adrenocorticotropic hormone (ACTH) production by leukocytes. In this study, splenic leukocytes from corticosterone-injected chickens failed to show a CRF-induced increase in ACTH production. In addition, corticosterone in vitro inhibited the production of leukocyte ACTH as well as the stimulatory effect of CRF on splenic leukocyte ACTH production. These findings show that, as with anterior pituitary ACTH production, CRF-stimulated leukocyte ACTH production is inhibited by glucocorticoids.

Central diabetes insipidus in a dog with a pro-opiomelanocortin-producing pituitary tumor not causing hyperadrenocorticism.

Central diabetes insipidus was diagnosed by vasopressin measurements during hypertonic stimulation in a 9-year-old male giant Schnauzer with polyuria and polydipsia. The impaired release of vasopressin was believed to be caused by a large pituitary tumor, which was visualized by computed tomography. Studies of the function of the anterior lobe and the pars intermedia of the pituitary gland were conducted, and high concentrations of ACTH and alpha-melanotrophic hormone (alpha-MSH) were found without concomitant hyperadrenocorticism. Studies of the molecular size of the immunoreactive ACTH in plasma by gel filtration revealed that most of the circulating immunoreactivity was not ACTH but its precursor pro-opiomelanocortin (POMC) and low-molecular-weight POMC-derived peptides. The pituitary tumor of this dog probably originated from melanotrophic cells of the pars intermedia. The sensitivity of the pituitary-adrenocortical system for the suppressive effect of dexamethasone was unaffected.

Responses of immunoreactive ACTH and bioactive ACTH to large hemorrhage and resuscitation in conscious dogs.

We studied the effect of fluid resuscitation on immunoreactive adrenocorticotropic hormone (irACTH) and bioactive ACTH (bioACTH) after hemorrhage in conscious dogs. Animals (n = 7) were bled 30% (approximately 25 ml/kg) over 3 min and 30 min later were either resuscitated [43.3 ml/kg 0.9% NaCl (1.8 times hemorrhage volume) over 10 min] or not. Blood was reinfused after 210 min. Animals had both treatments (> 4 days apart). irACTH, bioACTH, cortisol, angiotensin II, and aldosterone increased rapidly after hemorrhage. Resuscitation increased blood volume and cardiac output to resting values, but arterial hypotension persisted. bioACTH and irACTH decreased 40-90 min after hemorrhage in both groups, but each decreased more rapidly after resuscitation. The elimination half-life of bioACTH was shorter than that of irACTH, but neither was affected by resuscitation. The ratio of bioACTH to irACTH followed the same pattern with or without resuscitation. Angiotensin II and aldosterone remained increased without resuscitation but decreased promptly after resuscitation. In conclusion, 1) saline infusion at 1.8 x hemorrhage volume provides effective cardiovascular resuscitation, with resolution of hormonal responses to hemorrhage; 2) although ACTH responses resolved with or without resuscitation, resuscitation produced more rapid resolution without changing the parameters of ACTH elimination; 3) the dynamics of the resolution of the ACTH response to hemorrhage are similar whether induced by stimulus removal or feedback inhibition.

Changes in ACTH and β-endorphin immunoreactivity in immune tissues during a chronic inflammatory stress are not correlated with changes in corticotropin-releasing hormone and arginine vasopressin

We have previously demonstrated that the chronic inflammatory stress of adjuvant-induced arthritis in rats can alter levels of the neuropeptides adrenocorticotropin (ACTH), β-endorphin, arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH) in tissues of the immune system. We now present data showing that the patterns of these changes in the spleens and thymuses of Piebald-Viral-Glaxo rats are quite dissimilar throughout the course of the disease. Immunoreactive (ir)-CRH, AVP, ACTH and β-endorphin were measured by radioimmunoassays in spleen and thymic extracts taken at days 3, 7, 11 and 14 following injection of adjuvant. AVP was increased in the spleen at day 14 compared to the controls (79.4 ± 4.4 and 60.0 ± 9.0 fmol/g tissue respectively), but no change occurred in the thymus. CRH contents were increased in the spleen at day 14 (33.4 ± 3.5) compared to controls (22.1 ±2.4 fmol/g tissue), and in the thymus at day 11 (24.0 ±2.3) compared to controls (14.1 ± 2.5 fmol/g tissue). Increases in ACTH content were observed in spleens from arthritic rats at days 3 (365 ± 23), 11 (359 ± 32) and 14 (355 ± 45 fmol/g tissue) compared to controls (198 ± 37 fmol/g tissue). In the thymus, however, ACTH was elevated only at day 14. β-Endorphin levels in the spleen were elevated only at day 14 (289 ± 41) compared to controls (97 ± 22 fmol/g tissue). We conclude that the responses of ir-CRH, AVP, ACTH and β-endorphin to an inflammatory stress in the spleen and thymus are activated at different time-points and by different events which occur during the course of a chronic stress. It is also apparent, at least in the spleen, that unlike the hypothalamo-pituitary axis, immune POMC activation is not necessarily consequent to increases in immune CRH or AVP.

Effect of interleukin-1 beta on plasma ACTH, beta-endorphin, and corticosterone levels in infant and prepubertal rats.

IL-1 beta is known to enhance ACTH release from the anterior pituitary in the adult rat, mainly by simulating the hypothalamic ACTH-releasing hormone (CRH) release, but it seems to have a direct effect on the pituitary and on the adrenal hormone secretion, too. The effect of IL-1 beta on the beta-endorphin (beta E) secretion from the intermediate lobe is less well studied. There is very little information on the effect of IL-1 beta on the hypothalamic-pituitary-adrenal axis (HPAA) in the postnatal rat, which is a special period, because the reactivity of the HPAA is blunted. The effect of IL-1 beta in this period seemed to be of special interest, because neither the immune nor the endocrine system is fully developed. In the present study we tested the 30- and 120-min effect of intraperitoneally administered 0.5 and 100 ng/g body weight IL-1 beta on the plasma immunoreactive (ir) ACTH, beta E, and corticosterone (CS) levels in the 10-d-old (infant) and 30-d-old (prepubertal) rat. Generally, the ir-ACTH, ir-beta E, and ir-CS levels were significantly higher in prepubertal than in infant rats. Hormone levels were more enhanced by the higher dose of IL-1 beta, and changes were more pronounced at 120 min than at 30 min. The relative increase of ir-ACTH and ir-beta E was smaller in the infant than in the prepubertal rat. In contrast, the relative increase of ir-CS was more pronounced in the infant rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion and clearance of immunoreactive ACTH by fetal lung.

We have previously reported the presence of immunoreactive adrenocorticotropic hormone (irACTH) in fetal and adult ovine lung and secretion of the irACTH into the pulmonary venous blood during surgical stress. The aim of the present experiments was to determine whether the fetal lung secretes irACTH during less stressful conditions. Seven fetal sheep were chronically prepared with systemic, pulmonary arterial, and pulmonary venous catheters. After recovery from surgery, paired samples were obtained from the pulmonary arterial and venous catheters. In 51 pairs of samples, we quantified the arteriovenous (A-V) difference in plasma irACTH concentration. Pulmonary A-V difference in plasma irACTH was significantly related to the arterial plasma concentration of irACTH but not to fetal blood gases, pH, or gestational age. Regression analysis revealed that, at arterial plasma concentrations of irACTH less than approximately 200 pg/ml, the lung releases irACTH into plasma and that, at higher concentrations, the lung clears irACTH from the plasma. We conclude that the lung independently secretes and clears irACTH into and from plasma.

Dissociation of plasma adrenocorticotropin and cortisol levels in critically ill patients: possible role of endothelin and atrial natriuretic hormone.

The regulatory mechanisms of the hypothalamo-pituitary-adrenal system were studied in critically ill, intensive care unit patients. Serial measurements of immunoreactive ACTH-(1-39) (ACTHi), cortisol, endothelin-1 (ETi), and atrial natriuretic hormone (ANHi) were performed in blood samples of 18 patients with clinically defined sepsis, 12 critically ill patients after multiple trauma, and 15 hospitalized matched control subjects without acute illness for 8 consecutive days after admission. On admission, plasma levels of cortisol and ACTHi were significantly elevated in patients with sepsis (1.32 +/- 0.21 mumol/L and 130.0 +/- 38.2 pmol/L, mean +/- SD) and with multiple trauma (1.23 +/- 0.28 mumol/L and 123.7 +/- 41.3 pmol/L) compared to those in the control subjects (0.37 +/- 0.08 mumol/L and 15.6 +/- 5.8 pmol/L, respectively). The plasma cortisol levels of critically ill patients remained high (> 0.8 mumol/L) during the whole observation period. In contrast, plasma ACTHi levels decreased between days 3-5, reaching significantly lower levels on day 5 compared to those in the control group and remained below 5.0 pmol/L during the rest of the observation period. Plasma levels of ETi and ANHi were significantly elevated during the whole period in both patient groups (ETi, > 10 ng/L; ANHi, > 250 ng/L) compared to those in control subjects (< 5 and < 50 ng/L, respectively). The high plasma concentration of ETi observed in our patients may stimulate the steroid secretion of the adrenal cortex directly or potentiate the adrenal effect of ACTH. On the other hand, the increased concentration of ANHi found in critically ill patients together with the increased plasma cortisol level may explain the inhibition of ACTH secretion. Accordingly, we speculate that the high ET level exerts a positive drive on the adrenocortical level, that the high ANH level has an inhibitory effect on the hypothalamo-pituitary level, and that both mechanisms play a role in regulation of the hypothalamo-pituitary-adrenal axis during critical illness.

Prostaglandin E2 administered to fetal sheep increases the plasma concentration of adrenocorticotropin (ACTH) and the proportion of ACTH in low molecular weight forms.

This study investigated the effects of repeated short term (2-h) intrafetal infusions of prostaglandin E2 (PGE2) on ACTH and cortisol release in fetal sheep during late gestation (119-144 days). We compared the effects of administration of PGE2 (2 micrograms/min) into the fetal carotid artery or jugular vein. PGE2 infusion significantly (P < 0.001) increased fetal plasma immunoreactive (ir-) ACTH and cortisol concentrations regardless of the vessel used for administration. Saline infusion did not alter the concentrations of ir-ACTH or cortisol for the duration of the experiment. To compare the responses of fetal ir-ACTH and cortisol to repeated intracarotid infusions of PGE2, the hormone data were grouped into five gestational age ranges (119-125, 126-130, 131-135, 136-140, and 141-145 days). Fetal ir-ACTH was stimulated by PGE2 infusion at all gestational ages studied; the greatest response was achieved at the earliest gestational age range, 119-125 days. PGE2 infusion preferentially stimulated the release of low mol wt ACTH [ACTH-(1-39); 60 min from the start of infusion] at all gestational ages (P < 0.01), but basal low mol wt ACTH did not increase with gestational age until after 140 days. Cortisol concentrations were increased within 30 min of infusion at all gestational ages studied. These results suggest that PGE2 may play a role in maintaining elevated ir-ACTH concentrations in the face of high levels of cortisol in fetal sheep before parturition.

Validation of an Assay to Measure Adrenocorticotropin in Plasma and from Chicken Leukocytes

A RIA for mammalian adrenocorticotropic hormone (ACTH) was modified and validated to measure chicken ACTH. The assay was capable of detecting an increase in chicken plasma ACTH following treatments known to increase plasma ACTH. Both splenic and peripheral blood leukocytes stimulated with corticotropin-releasing factor (CRF) showed a significant increase in ACTH production compared with unstimulated leukocytes. This finding supports the conclusion that the substance produced by leukocytes previously shown in our laboratory to stimulate adrenal cells to secrete corticosterone is immunoreactive ACTH.

The hypothalamic-pituitary-adrenal axis in rat pregnancy and lactation: circadian variation and interrelationship of plasma adrenocorticotropin and corticosterone

The circadian variation in immunoreactive (I-) ACTH and corticosterone was studied at several stages throughout rat pregnancy and compared with those before pregnancy and during lactation. Serial blood samples were obtained from chronically cannulated, conscious rats at 2- to 3-h intervals beginning at 0800 h at diestrus of the cycle; on days 2, 6, 10, 14, 18, and 22 of pregnancy (term = day 23); and on day 4 of lactation. Plasma I-ACTH and corticosterone were determined in all samples, and indices of their circadian variation (acrophase, mesor, and amplitude) were derived by cosinor analyses within each rat. A circadian variation in corticosterone was clearly evident in all groups, with individual cosinor r2 values being consistently high. Plasma I-ACTH also exhibited distinct circadian variation up to day 14 of pregnancy, but the cosinor r2 value then fell (P < 0.05, by analysis of variance) and remained low during lactation. Mesor levels (midpoint of the derived circadian range) of I-ACTH fell (P < 0.05) by 34% to 20.6 +/- 2.4 pg/ml by day 2 of pregnancy; corticosterone also fell (P < 0.05) by 34% to 141 +/- 27 ng/ml, and both changes were due primarily to reductions in peak levels. I-ACTH mesors then remained effectively unchanged for the remainder of pregnancy, but mesor corticosterone increased progressively after day 10 to reach a maximum of 286 +/- 28 ng/ml by day 22, and this rise was due to elevations in both trough and peak corticosterone levels. Further changes occurred after parturition, with mesor I-ACTH increasing more than 2-fold between day 22 of pregnancy and day 4 of lactation, whereas mesor corticosterone declined by more than half over the same period. Plasma corticosterone and I-ACTH were positively associated within rats in all groups (P < 0.01), but the common slope of this association increased considerably after midgestation, indicative of higher corticosterone levels for a given concentration of I-ACTH. In summary, this study identifies marked changes in the hypothalamic-pituitary-adrenal axis during rat pregnancy and lactation. Although a circadian variation in corticosterone was observed throughout, that in plasma I-ACTH was evident early in pregnancy but declined after midgestation. Absolute concentrations of plasma I-ACTH and corticosterone fell in parallel early in pregnancy, but corticosterone subsequently increased without any change in I-ACTH.

Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus).

The role of the posterior hypothalamus in the development of the epithelial hypophysis was studied in Bufo embryos. In animals from which the central part of the neural plate (NP) had been surgically removed at the open neurula stage, the infundibulum did not develop, and the epithelial hypophysis was formed away from the normal site without morphological connection with the brain. Immunoreactive MSH cells and ACTH cells, i.e., the pituitary POMC cells, were not detected in any of the surgically treated animals, while other types of secretory cells (PRL, GH, TSH and GTH cells) were invariably present. In view of the fact that POMC cells originate in the anterior neural ridge, and not in the neural plate, the embryonic brain seems to exert an inductive influence upon the primordial pituitary POMC cells. Since these cells differentiate in a tail graft, isolated from the brain at a later stage (tail-bud stage), the inductive stimuli must be conveyed from/via the posterior hypothalamus to the pituitary anlage between the open neurula and the tail-bud stages.

The hypothalamic-pituitary-adrenal axis in rat pregnancy and lactation: circadian variation and interrelationship of plasma adrenocorticotropin and corticosterone.

The circadian variation in immunoreactive (I-) ACTH and corticosterone was studied at several stages throughout rat pregnancy and compared with those before pregnancy and during lactation. Serial blood samples were obtained from chronically cannulated, conscious rats at 2- to 3-h intervals beginning at 0800 h at diestrus of the cycle; on days 2, 6, 10, 14, 18, and 22 of pregnancy (term = day 23); and on day 4 of lactation. Plasma I-ACTH and corticosterone were determined in all samples, and indices of their circadian variation (acrophase, mesor, and amplitude) were derived by cosinor analyses within each rat. A circadian variation in corticosterone was clearly evident in all groups, with individual cosinor r2 values being consistently high. Plasma I-ACTH also exhibited distinct circadian variation up to day 14 of pregnancy, but the cosinor r2 value then fell (P < 0.05, by analysis of variance) and remained low during lactation. Mesor levels (midpoint of the derived circadian range) of I-ACTH fell (P < 0.05) by 34% to 20.6 +/- 2.4 pg/ml by day 2 of pregnancy; corticosterone also fell (P < 0.05) by 34% to 141 +/- 27 ng/ml, and both changes were due primarily to reductions in peak levels. I-ACTH mesors then remained effectively unchanged for the remainder of pregnancy, but mesor corticosterone increased progressively after day 10 to reach a maximum of 286 +/- 28 ng/ml by day 22, and this rise was due to elevations in both trough and peak corticosterone levels. Further changes occurred after parturition, with mesor I-ACTH increasing more than 2-fold between day 22 of pregnancy and day 4 of lactation, whereas mesor corticosterone declined by more than half over the same period. Plasma corticosterone and I-ACTH were positively associated within rats in all groups (P < 0.01), but the common slope of this association increased considerably after midgestation, indicative of higher corticosterone levels for a given concentration of I-ACTH. In summary, this study identifies marked changes in the hypothalamic-pituitary-adrenal axis during rat pregnancy and lactation. Although a circadian variation in corticosterone was observed throughout, that in plasma I-ACTH was evident early in pregnancy but declined after midgestation. Absolute concentrations of plasma I-ACTH and corticosterone fell in parallel early in pregnancy, but corticosterone subsequently increased without any change in I-ACTH.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative response of the immature and mature ovine fetus to corticotrophin-releasing hormone (CRH)

The aim of this study was to address the possibility that the low concentrations of adrenocorticotrophin (ACTH) seen in the ovine fetus between 90 and 120 days of gestation could be attributed to an alteration in the sensitivity or responsiveness of the fetal pituitary to corticotrophin-releasing hormone (CRH), a key regulator of ACTH secretion. Chronically cannulated ovine fetuses at Days 104-108 (n = 11, representing fetuses from this 90-120-day period) and Days 138-142 (n = 6) of pregnancy received graded doses of ovine CRH (0.8, 1.6, 3.8 and 7.6 micrograms h-1 for 60 min each, given consecutively and in ascending order) or isotonic saline (n = 4 at both age groups studied). Arterial blood samples were taken concurrently for analysis of plasma immunoreactive CRH, ACTH and cortisol throughout the infusion to assess the pituitary-adrenal response. Regression lines describing the relationship between log.PCRH and log.PACTH were calculated for both age group studied. A significant (P < 0.001) rightward shift in the log.PCRH/log.PACTH regression line for the Day 104-108 group was found, suggesting that the ovine fetus at this age is less sensitive or responsive to exogenous oCRH than the mature Day 138-142 fetus. This decreased responsiveness could explain the low concentrations of endogenous ACTH seen during the 90-120-day period.

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

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). 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. 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. 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.