Ectopic pro-opiolipomelanocortin: sequence of cDNA coding for beta-melanocyte-stimulating hormone and beta-endorphin.

A recombinant bacterial plasmid, pMS1, was constructed that contains 318 nucleotides complementary to a portion of pro-opiolipomelanocortin (proOLMC) messenger RNA from an ectopic adrenocorticotropin-producing tumor. The cloned complementary DNA insert, which contains the sequence that codes for all of the beta-melanocyte-stimulating hormone and beta-endorphin portions of proOLMC, as well as the 3' nontranslated section, is identical to the genomic sequence. Hybridization of tumor proOLMC complementary DNA to RNA subjected to electrophoresis and transferred to a nitrocellulose filter revealed two proOLMC messenger RNA species in the tumor polyadenylated RNA, but only one in pituitary polyadenylated RNA. At least one of the tumor proOLMC messenger RNA's is similar, if not identical, to human pituitary proOLMC messenger RNA.

Effect of synthetic ovine corticotropin-releasing factor. Dose response of plasma adrenocorticotropin and cortisol.

Synthetic ovine corticotropin-releasing factor (CRF) was administered to normal male volunteer subjects as an intravenous bolus or 30-s infusion. Doses of CRF ranging from 0.001 to 30 micrograms/kg body wt were administered, and plasma immunoreactive (IR)-ACTH and IR-cortisol concentrations were measured. The threshold dose appeared to be 0.01-0.03 micrograms/kg, the half-maximal dose 0.3-1 micrograms/kg, and the maximally effective dose 3-10 micrograms/kg. Basal concentrations of IR-ACTH and IR-cortisol were 14 +/- 7.6 pg/ml (mean +/- SD) and 5.6 +/- 2.2 micrograms/dl, respectively. IR-ACTH rose as early as 2 min after CRF injection, reached peak levels in 10-15 min, and declined slowly thereafter. IR-cortisol rose at 10 min or later and reached peak levels in 30-60 min. At a dose of 30 micrograms/kg, neither IR-ACTH nor IR-cortisol fell from peak levels of 82 +/- 21 pg/ml (mean +/- SE) and 23 +/- 1.4 micrograms/dl, respectively, during the 2-h course of the experiment, indicating that CRF has a sustained effect on ACTH release and/or a prolonged circulating plasma half-life. There was little or no increase in the levels of other anterior pituitary hormones. At doses of 1 microgram/kg and higher, facial flushing, tachycardia, and, in some subjects, a 15-29-mmHg decline in systemic arterial blood pressure were observed, even though blood volume was replaced and the subjects remained supine. These data indicate that synthetic ovine CRF is a very potent and specific ACTH secretagogue in man. Administered with caution until its vasomotor effects are more fully defined, CRF promises to be a safe and very useful investigative, diagnostic, and, possibly, therapeutic agent in man.

Arginine vasopressin potentiates adrenocorticotropin release induced by ovine corticotropin-releasing factor.

Arginine vasopressin (AVP) stimulates ACTH release in man and acts synergistically with synthetic ovine corticotropin-releasing factor (oCRF) in vitro. This study was designed to examine in man the combined effects of synthetic AVP (10 U intramuscularly) and oCRF (1 micrograms/kg intravenously) on ACTH release. Five normal male volunteers participated in five separate experiments: (a) AVP alone; (b) oCRF alone; (c) AVP followed by oCRF 15 min later; (d) simultaneous AVP and oCRF; and (e) insulin-induced hypoglycemia. Plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol were measured for 4 h after injection of each hormone; basal levels for all subjects were less than or equal to 9 +/- 1.2 pg/ml and 4.9 +/- 0.4 micrograms/dl (mean +/- SE), respectively. AVP and oCRF, when given individually, caused rapid rises in IR-ACTH to similar peak levels of 25 +/- 6.6 and 33 +/- 4.6 pg/ml, respectively. AVP given 15 min before oCRF caused a 2.6-fold potentiation of the oCRF response, with a peak IR-ACTH of 85 +/- 4.6 pg/ml. AVP given at the same time as oCRF produced a fourfold potentiation of the peak IR-ACTH response to 132 +/- 11 pg/ml. These ACTH responses were far greater than those previously observed after 30-fold greater doses of oCRF alone. By way of comparison, insulin-induced hypoglycemia caused a peak IR-ACTH of 169 +/- 20 pg/ml. IR-ACTH returned to base line at 60-90 min after AVP alone, whereas the prolonged effect of oCRF was apparent whether it was given alone or in combination with AVP. The mean peak IR-cortisol responses to AVP, oCRF, and AVP given 15 min before oCRF were similar (16.5 +/- 0.9, 16.4 +/- 2.3, and 18.5 +/- 0.8 micrograms/dl, respectively), but the peak IR-cortisol responses to AVP and oCRF given simultaneously and to insulin-induced hypoglycemia were 1.5 and 1.7 times greater, respectively. IR-cortisol returned to base line within 2-3 h after AVP alone, but remained elevated for at least 4 h after oCRF alone or in combination with AVP. These results indicate that AVP acts synergistically with oCRF to release ACTH in man and suggest that AVP may play a physiologic role in modulating the ACTH response mediated by corticotropin-releasing factor.

Proopiomelanocortin gene is expressed in many normal human tissues and in tumors not associated with ectopic adrenocorticotropin syndrome.

Immunoreactive (IR) POMC peptides have been detected in several human nonpituitary tissues and most pheochromocytomas and lung cancers, including those not associated with ectopic ACTH syndrome. We found IR-ACTH, IR-gamma MSH, IR-beta-endorphin (beta END), and IR-lipotropin in extracts from the following 10 normal human tissues, listed in order of decreasing POMC peptide concentrations: adrenal, testis, spleen, kidney, ovary, lung, thyroid, liver, colon, and duodenum. IR-ACTH, IR-gamma MSH, and IR-beta END were detected in all six pheochromocytomas and all 12 lung tumors (six squamous cell carcinomas, five adenocarcinomas, and one small cell carcinoma) we examined, as well as in a squamous cell carcinoma of the larynx. None of the patients had clinical evidence of ectopic ACTH syndrome. To determine whether these nonpituitary tissues and tumors actually synthesize POMC, rather than simply absorb POMC peptides from plasma, we examined poly(A) RNA prepared from these tissues and total RNA from pituitary by Northern blot hybridization for the presence of POMC-like mRNA with an exon 3 riboprobe. Pituitary contained a single POMC mRNA species of about 1150 bases. A short POMC-like mRNA of about 900 bases was found in all normal nonpituitary tissues, three of five pheochromocytomas, eight of nine lung cancers, and the laryngeal squamous cell tumor. In addition, larger POMC-like mRNA species between 1200 to 1500 bases were detected in adrenal, testis, ovary, placenta, two pheochromocytomas, and three squamous cell lung tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid receptor structure and function in an adrenocorticotropin-secreting small cell lung cancer.

ACTH secretion by tumors of nonpituitary origin is characteristically resistant to negative feedback regulation by glucocorticoids. One possible mechanism for the phenomenon could be a structural defect in the intracellular glucocorticoid receptor (GR). We studied the GR in DMS-79 cells derived from a human ACTH-secreting small cell lung cancer. Compared with control cells, DMS-79 cells were found to have greatly diminished GR ligand-binding activity and immunoreactive 94-kilodalton (kDa) GR content. Northern blot analysis revealed expression of GR transcripts that appeared to be slightly larger than those in control cells. A DMS-79 cell GR cDNA was cloned by reverse transcription/polymerase chain reaction amplification of mRNA using primers specific for full-length normal GR. The derived sequence of this full-length GR differed from the reported sequence by a single altered codon (G to A; Asn to Ser at codon 363) outside the steroid-binding domain. This N363S DMS-79 GR functioned normally to activate a target gene [mouse mammary tumor virus-chloramphenicol acetyl transferase (MMTV-CAT)] in transient transfection experiments in COS cells. Evidence for expression of a second type of GR mRNA was obtained by screening a DMS-79 cell cDNA library. This GR cDNA contained normal GR sequence up to nucleotide 2155, corresponding exactly to the end of exon 7 in the normal GR gene. The sequence appended to the GR sequences was not matched by any known sequence in DNA databases and included an in-frame termination codon after only 6 bases. The predicted truncated GR protein product (GR delta) has a mol wt of 73,740 and lacks most of the ligand-binding domain. Transient transfection of the GR delta form into COS cells did not reveal any dominant negative effect on the function of a cotransfected normal GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive proopiomelanocortin (POMC) peptides and POMC-like messenger ribonucleic acid are present in many rat nonpituitary tissues.

Immunoreactive (IR) POMC peptides have been found in several rat nonpituitary tissues. We found IR-ACTH, IR-beta-endorphin (beta END), and IR-gamma MSH in extracts from the following eight rat nonpituitary tissues, listed in order of decreasing POMC peptide concentrations: testis, duodenum, kidney, colon, liver, lung, stomach, and spleen, but not in adrenal or muscle extracts. Concentrations were very low and ranged from less than 0.00003% to 0.0005% of pituitary levels. In testis, duodenum, and colon, IR-gamma MSH and IR-beta END concentrations were only 5-37% of IR-ACTH levels. Gel filtration chromatography showed that IR-ACTH and IR-beta END coeluted in a major peak of 15,000 daltons, which is slightly larger than expected for a C-terminal peptide containing rat ACTH and beta-lipotropin. There were also a minor higher mol wt peak of IR-ACTH and IR-beta END and a minor IR-beta END peak that eluted in the position of mature beta END. There was no peak of IR-ACTH that corresponded to the size of mature ACTH. To determine whether these nonpituitary tissues also contained a POMC-like mRNA, which would confirm that the peptides were synthesized locally within the tissues, we examined poly(A) RNA prepared from 10 nonpituitary tissues and total RNA from pituitary by Northern blot hybridization for the presence of a POMC-like mRNA with an exon 3 riboprobe. Pituitary contained a single POMC mRNA species of about 1000 nucleotides. A short POMC-like mRNA of about 800 bases was found in all nonpituitary tissues, except spleen and muscle. Compared to POMC mRNA levels in pituitary, the concentration of POMC-like mRNA was 0.5% in testis and 0.03-0.07% in the other tissues. The ratio of POMC-like mRNA to IR-POMC peptide concentrations in nonpituitary tissues was at least 1000 times greater than that in the pituitary. We conclude that the POMC gene is expressed in many nonpituitary tissues and that either the short POMC-like mRNA is translated much less efficiently or POMC peptides are released or degraded much more rapidly in nonpituitary tissues than in the pituitary.

Lymphocyte-derived adrenocorticotropin is insufficient to stimulate adrenal steroidogenesis in hypophysectomized rats.

Cells of the immune system can produce and respond to peptide hormones associated with the endocrine system. However, the physiological significance of these endocrine-immune interactions is not known. It has been postulated that cells of the immune system, when stimulated with viruses that induce interferon-alpha, produce sufficient levels of ACTH to stimulate adrenal steroidogenesis and, thus, function as an auxiliary source of ACTH that may have a role in the response to stress. However, we have confirmed that levels of ACTH-related peptides produced by immunocompetent cells are far lower than those produced by the pituitary, raising questions about the ability of lymphocyte-derived ACTH to stimulate adrenal function. Furthermore, we have rigorously examined this issue using intact and hypophysectomized rats treated with Newcastle disease virus. Although high levels of interferon-alpha were produced by both intact and hypophysectomized rats, and the plasma corticosterone concentration increased dramatically in intact animals, corticosterone remained undetectable in hypophysectomized rats. The lack of a corticosterone response in these animals was not due to adrenal insensitivity to ACTH, as shown by a normal rise in corticosterone following Cosyntropin injection 8 h after hypophysectomy. The findings demonstrate that levels of ACTH produced by nonpituitary sources in response to viral infection are not sufficient to stimulate adrenal steroidogenesis.

Effects of short and long duration hypothyroidism and hyperthyroidism on the plasma adrenocorticotropin and corticosterone responses to ovine corticotropin-releasing hormone in rats.

We report here a study of the plasma ACTH and corticosterone responses to synthetic ovine CRH (oCRH) in hypothyroid and hyperthyroid rats studied 7, 15, and 60 days after either thyroidectomy or the administration of pharmacological doses of T4. The purpose of this study was to further clarify the time-dependent effects of alterations in thyroid status on the functional integrity of the hypothalamic-pituitary-adrenal axis and to aid in the interpretation of the oCRH stimulation test in hypo- and hyperthyroid states. Our data demonstrate that hypothyroid rats have a significant reduction in the cerebrospinal fluid (CSF) levels of corticosterone and a significant decrease in adrenal weight in association with significant increases in the plasma ACTH response to oCRH. On the other hand, the corticosterone response to the ACTH released during the oCRH stimulation test was significantly reduced in hypothyroidism. With increasing duration of thyroidectomy-induced hypothyroidism, there was a progressive fall in CSF corticosterone levels, a progressive increase in the plasma ACTH response to oCRH, and a gradual normalization of the corticosterone responses to the ACTH released during oCRH stimulation. Our findings in hyperthyroid rats were generally the converse of those seen in hypothyroidism. Hence, there was a significant increase in the CSF levels of corticosterone and a significant increase in adrenal weight in association with an initial slight decrease in the ACTH response to oCRH. On the other hand, the corticosterone response to the ACTH released during oCRH stimulation was significantly increased. There was a gradual increase in the magnitude of the rise in CSF corticosterone levels with time, as well as a gradual normalization of adrenocortical responses during oCRH stimulation. The ACTH plasma clearance rates were similar in hypo-, hyper-, and euthyroid rats. Our data do not permit definitive identification of the precise locus in the hypothalamic-pituitary-adrenal axis that is principally affected by experimentally induced alterations in thyroid status. However, these data are most compatible with a subtle hypothyroid-induced centrally mediated adrenal insufficiency and a subtle hyperthyroid-induced centrally mediated hypercortisolism. These data also suggest that alterations in hypothalamic-pituitary-adrenal function in states of disturbed thyroid function become somewhat more pronounced as the duration of thyroid dysfunction increases. The fact that pituitary-adrenal responses to oCRH are consistently altered in states of thyroid dysfunction may be relevant to the clinical interpretation of oCRH stimulation tests.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of synthetic ovine corticotropin-releasing factor: prolonged duration of action and biphasic response of plasma adrenocorticotropin and cortisol.

The duration of the response to synthetic ovine corticotropin-releasing factor (CRF) was studied in 13 healthy male volunteer subjects. Placebo or CRF (0.3, 3, or 30 micrograms/kg BW) was administered as an iv bolus or, in the case of the largest dose, a 30-sec infusion in single blind fashion in the late afternoon. Basal plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol were 10.8 +/- 7.7 pg/ml and 5.0 +/- 1.8 micrograms/dl (mean +/- SD), respectively. IR-ACTH rose rapidly after CRF, reached an initial peak at 15 min, fell rapidly until 1.5 h after CRF, and then either fell more slowly (after the lowest dose) or rose to a second major peak at 2-3 h before falling back to baseline. After 0.3, 3, and 30 micrograms/kg CRF, IR-ACTH remained elevated for 4, 7, and 8 h, respectively. The effect on plasma IR-cortisol was similar, but more prolonged. The magnitude of both peaks of IR-ACTH, the duration of the response, and the area under the curve all appeared dose dependent. The same was true for IR-cortisol, except that the first peak height was similar after all three doses. The duration of CRF's action is probably due to its long circulating half-life. The biphasic response curve may reflect initial secretion of a readily releasable pool of ACTH, followed by later secretion of a second pool of newly synthesized and/or matured peptide. The next morning's normal circadian rise in both IR-ACTH and IR-cortisol was delayed and diminished after 3 micrograms/kg CRF; there was no increase in IR-ACTH after 30 micrograms/kg CRF, and the IR-cortisol level was diminished. Inhibition of the normal circadian rise may reflect inhibition of ACTH secretion by the sustained high plasma cortisol levels.

Plasma distribution, disappearance half-time, metabolic clearance rate, and degradation of synthetic ovine corticotropin-releasing factor in man.

The plasma distribution, disappearance half-time, MCR, and degradation of corticotropin-releasing factor (CRF) were studied in normal men who received a pulse injection of synthetic ovine CRF (oCRF). Graded iv doses of oCRF produced a linear increase in plasma immunoreactive oCRF (IR-oCRF). The calculated total plasma content of IR-oCRF 2 min after injection represented 41.7 +/- 2.5% (mean +/- SE) of the injected dose. The disappearance of IR-oCRF from plasma was characterized by a biexponential decay curve, with initial distribution and subsequent metabolic t 1/2 values of 6.1 +/- 0.5 and 55 +/- 3.8 min (mean +/- SE), respectively. In two subjects who were studied for 14-16 h after being given the largest dose of oCRF, there was third phase of disappearance, with a t 1/2 of 198 +/- 54 min. The MCR of IR-oCRF was 2.4 +/- 0.2 ml/min . kg (146 +/- 12 l/m2 . day) and was relatively constant over a 3000-fold dose range. The volume of distribution of IR-oCRF was 6.2 +/- 0.6 liters. The plasma IR-oCRF component, examined at increasing intervals after injection, was indistinguishable from the injected oCRF in that its apparent molecular size had not been altered, nor had its biological activity been attenuated. The continued circulation of apparently intact, biologically active oCRF for at least 90 min after injection was associated with sustained release of ACTH into the plasma. Thus, the clearance of oCRF from circulating human plasma is prolonged and appears to be responsible for the sustained release of ACTH that occurs after injection of this hormone-releasing factor.

Adrenocorticotropin-independent bilateral macronodular adrenal hyperplasia: an unusual cause of Cushing's syndrome.

Inappropriate ACTH secretion with bilateral diffuse or macronodular adrenal hyperplasia is the most common cause of Cushing's syndrome. This report describes a patient with Cushing's syndrome and feminization due to ACTH-independent bilateral macronodular adrenal hyperplasia. A 47-yr-old black man presented with Cushingoid features, diabetes mellitus, hypertension, impotence, and gynecomastia. Urinary cortisol and 17-hydroxycorticosteroid excretion were 94 nmol/mmol creatinine (normal, less than 32) and 5.8 mumol/mmol creatinine (normal, 0.6-3.6), respectively. Both decreased by less than 30% after administration of dexamethasone (8 and 16 mg/day), and urinary 17-hydroxycorticosteroid excretion did not increase after metyrapone (750 mg, orally, every 4 h for six doses). Plasma ACTH was undetectable (less than 1 pmol/L) and was not stimulated by administration of metyrapone or ovine CRH. Serum testosterone was 5.2 nmol/L (normal, 7-30), FSH was 5 U/L (normal, 3-18), LH was 2.8 U/L (normal, 1.5-9.2), and estrone was 767 pmol/L (normal, 55-240). Both adrenal glands were enlarged, with a total weight of 86 g (normal, 8-10), and contained multiple nodules (diameter, greater than 0.5 cm) composed of two active cell types, one of which was also observed between the nodules. Cushing's syndrome with feminization due to ACTH-independent bilateral macronodular adrenal hyperplasia is an unusual process of unknown etiology that should be included with the other known causes of Cushing's syndrome.

Effects of ovine corticotropin-releasing hormone on adrenocorticotropin secretion in the absence of glucocorticoid feedback inhibition in man.

We studied 1) the nature of the plasma ACTH response to ovine CRH (oCRH) in the absence of normal glucocorticoid negative feedback inhibition and 2) the cause of the diminished circadian peak in plasma ACTH in normal men the morning after 3-30 micrograms/kg BW doses of oCRH. Placebo or oCRH (3 micrograms/kg BW, iv) was administered as iv injections to five normal men given metyrapone to produce acute glucocorticoid deficiency. Four studies were performed: 1) placebo oCRH plus placebo hydrocortisone (HC), 2) oCRH plus placebo HC, 3) placebo oCRH plus HC, and 4) oCRH plus HC. HC was given as a variable rate iv infusion to mimic the plasma cortisol response to the same dose of oCRH in normal men. Plasma cortisol levels rose only slightly after oCRH, indicating nearly complete blockade of cortisol biosynthesis. Plasma cortisol levels during the HC infusion were similar to those in normal men given 3 micrograms/kg oCRH. There was an exaggerated rise in both the first and second peaks of the plasma ACTH response to oCRH in the metyrapone-treated men. HC infusion did not alter the plasma ACTH response during the first 60 min after oCRH, but markedly attenuated the response thereafter; however, it did not affect the timing of the second peak. This inhibitory effect continued for up to 11 h, which was 2-3 h longer than the period that plasma cortisol levels were increased. Thus, cortisol secreted in response to ACTH released by oCRH modulates, after about a 60-min delay, the continuing release of ACTH. Despite the greater oCRH-induced release of pituitary ACTH in the metyrapone-treated men, the magnitude of their next morning's circadian plasma ACTH peak was similar to that after they received placebo oCRH. Thus, depletion of pituitary ACTH did not appear to explain the diminished circadian peak. Its magnitude was reduced by the combination of oCRH and HC, but not by HC alone. Administration of oCRH, alone or in combination with HC, delayed the onset of the circadian rise, while oCRH, HC, or the combination thereof delayed the time of the circadian peak. Thus, it appears that both the glucocorticoid response to oCRH and direct or indirect effect(s) of oCRH are required to produce these two phenomena.

Pituitary microadenomas causing Cushing's disease respond to corticotropin-releasing factor.

Corticotropin-releasing factor (CRF) was administered as an iv bolus to two young women with mild Cushing's disease shortly before and one week after successful transsphenoidal microadenomectomy. The dose of CRF (1 microgram/kg body weight) had previously been shown to stimulate increased plasma ACTH and cortisol in normal subjects. In the first patient, prior to surgery, there were brisk increases in ACTH and cortisol that exceeded those observed in normal subjects. ACTH rose by 2 min and reached a peak between 15-30 min. Cortisol increased by 10 min and peaked between 45-60 min. After surgery, at a time when plasma cortisol was maintained at similar levels with exogenous hydrocortisone, there was no plasma ACTH or LH, TSH and prolactin increased after administration of LRH and TRH, and GH increased in response to insulin-induced hypoglycemia. The second patient had higher basal plasma ACTH and cortisol than the first patient. CRF-induced increments in ACTH and cortisol were much less, but the time course was similar and peak levels attained were still higher than those in normal subjects. After surgery, at a time when plasma cortisol was maintained at a much lower level with exogenous hydrocortisone, there was no plasma ACTH or cortisol response. She had mild, transient diabetes insipidus. Basal levels of all other anterior pituitary hormones were normal. These results demonstrate that two microadenomas causing Cushing's disease were responsive to CRF in situ and suggest that CRF may be involved in the etiology and/or the responses to changes in plasma glucocorticoid concentrations observed in patients with Cushing's disease.

Single subcutaneous doses of a luteinizing hormone-releasing hormone antagonist suppress serum gonadotropin and testosterone levels in normal men.

The ability of single doses of a LHRH antagonist [Ac-delta 3Pro1, 4F-D-Phe2, D-Trp3,6]LHRH (4F-antagonist) to suppress serum gonadotropin and testosterone levels was studied in six normal men. The 4F-antagonist was given sc at four doses: 40, 80, 160, and 320 micrograms/kg body weight. Serum immunoreactive LH, FSH, and testosterone and bioactive LH were measured at intervals for the subsequent 18 h. Serum LH decreased rapidly by (mean +/- SE) 39.7 +/- 2.7%, 41.6 +/- 5.4%, 45.5 +/- 4.7%, and 45.3 +/- 5.4% after each of the four doses. The mean number of LH pulses and their amplitude decreased after each dose and remained suppressed for at least 6 h. After each of the four doses, mean serum FSH levels decreased by 20.0 +/- 4.1%, 33.8 +/- 6.8%, 25.8 +/- 3.6%, and 33.3 +/- 5.7%, and mean serum testosterone levels decreased by 47.7 +/- 7.3%, 55.6 +/- 10.5%, 58.2 +/- 10.8%, and 76.0 +/- 6.0%. Serum testosterone remained low for at least 18 h after the two higher doses. LH bioactivity and the ratio of bioactive LH to immunoreactive LH decreased in all subjects, especially after higher doses of the 4F-antagonist. No side effects or adverse reactions occurred after 4F-antagonist administration, and toxicology studies were negative. These results demonstrate that a single sc injection of this potent LHRH antagonist inhibits the pituitary-gonadal axis in normal men.

Rapid sequential intravenous administration of four hypothalamic releasing hormones as a combined anterior pituitary function test in normal subjects.

Normal subjects were studied to test the feasibility of a combined anterior pituitary function test using iv administration of four hypothalamic releasing hormones: ovine corticotropin-releasing hormone, human GH-releasing hormone, GnRH, and TRH. Initially, nine normal men were studied with various combinations of these four hormones to exclude the possibility that they might inhibit or synergize with each other in releasing the individual anterior pituitary hormones. When given in combination, the releasing hormones were administered as sequential 20-sec iv infusions in the following order and doses: ovine corticotropin-releasing hormone, 1 microgram/kg; GnRH, 100 micrograms; human GH-releasing hormone, 1 microgram/kg; and TRH, 200 micrograms. Plasma or serum samples were assayed for ACTH, cortisol, GH, PRL, FSH, LH, and TSH at multiple times for 120 min after injection. Compared to individual administration, combined administration of these four hypothalamic releasing hormones caused no apparent inhibition or synergism with respect to the individual hormone responses of these normal subjects. Side-effects of the combined test were the same as those observed with individual hormone administration. No unusual or dangerous side-effects were observed. Having confirmed the efficacy of combined administration of the four releasing hormones, we administered the combination to five additional normal men and 12 normal women. Anterior pituitary hormone and cortisol responses were the same in men and women, except for a lower LH and a greater PRL response in women. There was a rapid increase in all hormones, with peak levels usually reached by 60 min. Adequate assessment of individual hormone responses can be achieved by assaying a basal and only 2 (or 3 in the case of ACTH and GH) postinfusion samples. A rapid, safe, and useful test of combined anterior pituitary function appears to be feasible using these four hypothalamic releasing hormones.

Effect of altered thyroid hormone levels on hypothalamic-pituitary-adrenal function.

To determine whether alterations in serum thyroid hormone levels affect hypothalamic-pituitary-adrenal function, we measured the plasma immunoreactive (IR) ACTH and IR-cortisol responses to 1 microgram/kg BW ovine CRH (oCRH) given iv in the late afternoon and the plasma IR-ACTH, IR-cortisol, and IR-11-deoxycortisol responses to 2 g metyrapone given orally at midnight in 10 athyreotic patients during T4 treatment and 1 month after stopping T4 when they were biochemically, but not clinically, hypothyroid. Mean serum TSH increased from 0.7 +/- 0.9 (+/- SD) mU/L (normal range 0.5-4.9 mU/L) during T4 therapy to 107 +/- 82 mU/L after stopping T4. The serum total T4 level and free T4 index fell from 165 +/- 37 nmol/L and 1.9 +/- 0.4, respectively (normal range, 59-154 nmol/L and 0.9-2.5, respectively), to 19 +/- 9 and 0.2 +/- 0.1, respectively, after stopping T4. Basal plasma IR-ACTH and IR-cortisol levels at 0800 and 1630 h were similar during and after stopping T4 therapy. Peak plasma IR-ACTH and IR-cortisol levels after oCRH were significantly greater after stopping T4 (20 +/- 9.2 pmol/L and 880 +/- 260 nmol/L, respectively) than during T4 therapy (9.7 +/- 4.7 pmol/L and 720 +/- 190 nmol/L; P less than 0.01 and P less than 0.05, respectively). The mean integrated plasma IR-ACTH and IR-cortisol responses to oCRH were also significantly greater P less than 0.01 and P less than 0.05, respectively) after stopping T4 than during T4 therapy. Plasma IR-ACTH the morning after metyrapone was slightly (1.6-fold) but not significantly greater during therapy than after stopping T4 therapy (100 +/- 86 vs. 65 +/- 54 pmol/L, respectively). The plasma IR-11-deoxycortisol responses to metyrapone during and after stopping T4 therapy were similar (720 +/- 250 and 750 +/- 330 nmol/L, respectively), presumably because plasma IR-ACTH concentrations were maximally stimulating in both instances. These results indicate that thyroid hormone deficiency of short duration 1) increases corticotroph sensitivity to oCRH, 2) may diminish the plasma ACTH response to metyrapone-induced hypocortisolemia, and 3) has no apparent effect on the acute adrenal response to ACTH. These data together with those of previous studies that have shown reduced responses of the hypothalamic-pituitary-adrenal axis to metyrapone and hypoglycemia in hypothyroid patients suggest that the release of hypothalamic CRH and/or other ACTH secretagogues may be decreased in hypothyroidism.

Effect of ovine corticotropin-releasing hormone administered during insulin-induced hypoglycemia on plasma adrenocorticotropin and cortisol.

The factors that mediate the hypothalamic-pituitary response to hypoglycemia in man are unknown. To investigate the role of CRH in the plasma ACTH response to hypoglycemia, two different doses of ovine CRH (oCRH) were given to normal men during insulin-induced hypoglycemia. We hypothesized that if the endogenous CRH response to hypoglycemia were less than maximally stimulating, administration of oCRH during hypoglycemia would result in a greater peak plasma immunoreactive (IR) ACTH response. Six normal men were given 1) 0.15 U/kg regular insulin, iv; 2) insulin plus 1 microgram/kg oCRH, iv, 5 min after serum glucose fell to 40 mg/dL or less; and 3) oCRH alone. The degree and duration of hypoglycemia were the same when insulin was given alone or with oCRH. Plasma IR-ACTH after insulin alone and insulin plus oCRH rose at the same rate to similar peaks of 226 +/- 37 (mean +/- SEM) and 213 +/- 53 pg/mL, respectively, both of which were greater (P less than 0.05) than the peak plasma IR-ACTH after oCRH alone (61 +/- 19 pg/mL). The peak plasma IR-cortisol levels after insulin alone (24 +/- 4 micrograms/dL), insulin plus oCRH (27 +/- 3 micrograms/dL), and oCRH alone (18 +/- 2 micrograms/dL) were not significantly different. In a second study, six normal men were given 0.15 U/kg regular insulin, iv; insulin plus 10 micrograms/kg oCRH, iv; and 10 micrograms/kg oCRH alone. Administration of oCRH 5 min after serum glucose fell to 40 mg/dL or less did not affect the degree or duration of hypoglycemia. Plasma IR-ACTH after insulin alone and insulin plus oCRH rose at the same rate to similar peaks of 258 +/- 14 and 290 +/- 33 pg/mL, respectively, both of which were greater (P less than 0.01) than the peak (54 +/- 6 pg/mL) after oCRH alone. After insulin alone, plasma IR-ACTH declined to baseline by 3 h. However, after insulin plus oCRH, plasma IR-ACTH fell gradually until 2 h, rose to a second peak at 2.5-3 h, and remained greater (P less than 0.01) than after insulin or oCRH alone for the 4-h duration of the study. The mean peak plasma IR-cortisol level after insulin plus oCRH (33 +/- 4 micrograms/dL) was similar to that after insulin alone (28 +/- 3 micrograms/dL), but was greater (P less than 0.05) than that after oCRH alone (18 +/- 2 micrograms/dL).(ABSTRACT TRUNCATED AT 400 WORDS)

Synthetic ovine corticotropin-releasing hormone: simultaneous release of proopiolipomelanocortin peptides in man.

The response of plasma proopiolipomelanocortin-derived peptide levels to synthetic ovine corticotropin-releasing hormone (CRH) was studied in six normal men. CRH was given as a 30-sec iv injection of 30 micrograms/kg body weight in the late afternoon, and blood samples were drawn for up to 16 h thereafter. Low levels of immunoreactive (IR)-ACTH, IR-beta-endorphin and IR-lipotropins (LPH) were measured before CRH administration. All subjects had prompt, concomitant, biphasic, and prolonged release of all of these proopiolipomelanocortin-derived peptides. The plasma levels of these IR-peptides rose in all subjects by 5 min after CRH, reached a first peak at 10-15 min, fell until 90 min, rose to a second peak at 2-4 h, and then gradually declined over several hours. The molar concentrations of the IR-peptides closely paralleled one another at all times, especially during the first 90 min after CRH administration. Later, IR-LPH increased slightly more and remained slightly higher than did the other IR-peptides, although the difference was not significant. This observation probably reflects the longer plasma disappearance half-life of IR-LPH. The maximum change (mean +/- SEM) in the concentration of these IR-peptides was similar: IR-ACTH, 18.0 +/- 4.0; IR-LPH, 20.5 +/- 4.0; and IR-beta-endorphin, 16.9 +/- 3.2 fmol/ml. The next morning's circadian rise in IR-peptides was blocked, presumably due to negative feedback inhibition of the hypothalamic-pituitary-adrenal axis by the prolonged high plasma cortisol levels stimulated by CRH the previous evening.

Effect of subcutaneous and intranasal administration of ovine corticotropin-releasing hormone in man: comparison with intravenous administration.

Long term use of ovine corticotropin-releasing hormone (oCRH) requires a convenient route of administration. The effects of 0.3, 3, and 30 micrograms/kg BW synthetic oCRH given as a sc injection and of 10 and 30 micrograms/kg given as an intranasal spray were studied in 10 normal men in the late afternoon. Basal plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol levels were 14 +/- 1.9 pg/ml and 4.3 +/- 0.4 microgram/dl (mean +/- SEM). Peak IR-ACTH levels (mean +/- SEM) were 43 +/- 5.5, 53 +/- 8.1, and 64 +/- 8.9 pg/ml after the 0.3, 3, and 30 micrograms/kg doses of oCRH given sc, respectively, and 23 +/- 4.3 and 36 +/- 4.8 pg/ml after the 10 and 30 micrograms/kg doses of oCRH given intranasally, respectively. The lowest sc dose and both intranasal doses caused only single IR-ACTH peaks. After 3 and 30 micrograms/kg sc oCRH, IR-ACTH rose by 15 min, reached an initial peak at 45-60 min, fell rapidly until 90-120 min, and rose to a second peak at 3-5 h. This biphasic response is similar to that previously found after iv administration. IR-ACTH levels remained elevated for 4, 10, and at least 16 h after 0.3, 3, and 30 micrograms/kg sc oCRH, respectively, and for 1.5 and 3 h after 10 and 30 micrograms/kg intranasal oCRH respectively. The effect on IR-cortisol was similar, but more prolonged. Compared to the iv route, sc oCRH produced similar mean peak IR-ACTH and IR-cortisol levels and had a slightly longer duration of action. Intranasal oCRH was only about 1% as effective. Peak plasma IR-oCRH levels in 2 subjects receiving 3 micrograms/kg sc oCRH were 13 and 17 ng/ml at 90 min. These peaks were lower than those after iv administration of the same dose, but the levels remained elevated longer, probably accounting for the longer duration of action of sc oCRH. Peak plasma IR-oCRH levels in 4 subjects given 10 microgram/kg intranasal oCRH were only 64-122 pg/ml, presumably reflecting poor absorption through the nasal mucosa. These results demonstrate that sc injection of oCRH is at least as effective as the iv route with respect to plasma IR-ACTH and IR-cortisol responses. The convenience of this route of administration and the prolonged duration of action of oCRH suggest the feasibility of long term oCRH use.

Diurnal variation in the response of plasma adrenocorticotropin and cortisol to intravenous ovine corticotropin-releasing hormone.

To determine whether the plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol responses to ovine corticotropin-releasing hormone (oCRH) depend on the time of day, we administered 1 microgram/kg BW synthetic oCRH as an iv bolus dose to five normal men at their usual time of awakening between 0530-0740 h, at 1600 h, and at 2300 h. Mean basal plasma IR-ACTH and IR-cortisol levels were highest upon awakening, intermediate at 1600 h, and lowest at 2300 h, reflecting the diurnal rhythm of ACTH secretion. There was no significant difference in the plasma IR-ACTH response to oCRH at different times of the day. In contrast, the mean maximum plasma IR-cortisol increment and mean integrated response were 2- and 2.6-fold greater (P less than 0.05), respectively, at 2300 h than upon awakening. In another study, oCRH was given in the morning (0700-0900 h) to 22 normal men and in the late afternoon (1600-1800 h) to 24 normal men. Mean basal plasma IR-ACTH and IR-cortisol levels were significantly higher (P less than 0.001) in the morning [24 +/- 3 pg/ml (mean +/- SEM) and 10.6 +/- 0.8 micrograms/dl, respectively] than in the afternoon (13 +/- 2 pg/ml and 5.6 +/- 0.6 micrograms/dl, respectively). Mean peak plasma IR-ACTH was slightly greater in the morning (60 +/- 5.5 pg/ml) than in the afternoon (47 +/- 5.5 pg/ml), the mean maximum plasma IR-ACTH increments were the same (35 +/- 4 and 34 +/- 5 pg/ml, respectively), and the mean integrated IR-ACTH response was slightly less in the morning (2036 +/- 414 vs. 2365 +/- 358 pg . min/ml), but none of these differences was statistically significant. Mean peak plasma IR-cortisol concentrations in the morning and afternoon were similar (18.7 +/- 0.7 and 17.3 +/- 0.9 micrograms/dl, respectively), but the mean maximum plasma IR-cortisol increments (8.1 +/- 0.8 and 11.7 +/- 0.9 micrograms/dl, respectively; P less than 0.005), and the mean integrated IR-cortisol responses (588 +/- 115 and 976 +/- 95 micrograms . min/dl, respectively; P less than 0.01) were greater in the afternoon. There was an inverse correlation between basal plasma IR-cortisol concentration and the integrated IR-ACTH response (P less than 0.05), the maximum IR-cortisol increment (P less than 0.001), and the integrated IR-cortisol response (P less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)