Serum thyrotropin concentrations and bioactivity during sleep deprivation in depression.

BACKGROUND: One night of sleep deprivation induces a brief remission in about half of depressed patients. Subclinical hypothyroidism may be associated with depression, and changes in hypothalamic-pituitary-thyroid function may affect the mood response to sleep deprivation. We wished to define precisely the status of the hypothalamic-pituitary-thyroid axis of depressed patients during sleep deprivation and the possible relationship of hypothalamic-pituitary-thyroid function to the mood response. METHODS: We studied 18 patients with major depressive disorder and 10 normal volunteers. We assessed mood before and after sleep. We measured serum thyrotropin every 15 minutes during the night of sleep deprivation, thyrotropin bioactivity, the thyrotropin response to protirelin the next afternoon, and other indexes of hypothalamic-pituitary-thyroid function. To determine if the changes were limited to the hypothalamic-pituitary-thyroid axis, we measured serum cortisol, which also has a circadian secretory pattern. RESULTS: Nocturnal serum thyrotropin concentrations were consistently higher in responders, entirely because of elevated levels in the women reponders. Responders had exaggerated responses to protirelin the next afternoon. The bioactivity of thyrotropin in nonresponders was significantly greater than in responders (F(1,8. 99) = 7.52; P =.02). Other thyroid indexes and serum cortisol concentrations were similar among groups. CONCLUSIONS: Depressed patients have mild compensated thyroid resistance to thyrotropin action, not subclinical autoimmune primary hypothyroidism. Sleep deprivation responders compensate by secreting more thyrotropin with normal bioactivity; nonresponders compensate by secreting thyrotropin with increased bioactivity.

Insulin-like growth factor I receptor mRNA and protein expression in pig corpora lutea.

Insulin-like growth factor I (IGF-I) is believed to play a luteotrophic role in the pig corpus luteum during the oestrous cycle. Since the actions of IGF-I in target tissues are mediated by the type I IGF receptor, the concentrations of IGF-I receptor mRNA and protein were examined in pig corpora lutea at different stages of the oestrous cycle. Corpora lutea were collected from normally cyclic gilts on days 4, 7, 10, 13, 15 and 16 of the oestrous cycle (n = 4 animals per day). Corpora lutea on days 7, 10 and 13 were dissociated with collagenase, and large and small luteal cell sub-populations were separated by elutriation. Northern and slot blots were used to examine mRNA, and western blots were used to measure the concentrations of IGF-I receptor protein in the pig corpus luteum. On northern blots, luteal IGF-I receptor mRNA was present as a single 11 kb transcript. The slot blots showed that the steady state expression of IGF-I receptor mRNA increased significantly (P < 0.05) from its lowest value on day 4, to reach a maximum on days 13-16. IGF-I receptor mRNA was also expressed to a greater extent in large compared with small luteal cells (P < 0.05). On western blots, IGF-I receptor appeared as a 95 kDa protein band (beta-subunit) and IGF-I receptor protein concentrations were significantly higher (P < 0.05) on days 4-10 than on days 13-16. Finally, large luteal cells appeared to contain more IGF-I receptor protein than the small luteal cells. In conclusion, since IGF-I receptor was detected in the pig corpus luteum, it is a likely target tissue for IGF-I, especially during the early luteal phase. Furthermore, IGF-I receptor was localized primarily on large luteal cells, thus it is hypothesized that IGF-I may play a paracrine role in the pig corpus luteum.

Intra- and inter-individual correlations between cholecystokinin and corticotropin-releasing hormone concentrations in human cerebrospinal fluid.

Despite strong evidence of a physiologic relationship between cholecystokinin (CCK) and corticotropin-releasing hormone (CRH) in the rat central nervous system (CNS), evidence of such a relationship between the two hormones in the human CNS is lacking. A post hoc analysis of serial concentrations of immunoreactive CCK and CRH, obtained every ten minutes from CSF continuously collected over six hours, was performed. A total of 30 subjects were studied: 15 normal volunteers, 10 patients with major depression, and 5 recently-abstinent, alcohol-dependent patients. Overall, we observed an average intra-subject correlation of +.273 (P < 0.001) between CSF CRH and CCK. Inter-subject correlations between mean CSF levels of CRH and CCK were +.948 (P = 0.0001) and +.959 (P = 0.005) in the depressed and abstinent alcoholic patients, respectively. These inter-individual correlations were significantly greater than that seen within the group of normal volunteers (r = +.318, n.s.). The present data suggest that interactions between CCK and CRH are significant in the human CNS, particularly perhaps in depressed and alcoholic patients, and that CSF samples may be used to assess elements of the relationship between these hormones.

Serial CSF corticotropin-releasing hormone levels and adrenocortical activity in combat veterans with posttraumatic stress disorder.

OBJECTIVE: The authors sought to carefully test, by using a technique of continuous CSF sampling, the hypothesis that basal elevations in CSF corticotropin-releasing hormone (CRH) concentrations exist in patients with posttraumatic stress disorder (PTSD). They also sought to assess the relationship among PTSD symptoms, adrenocortical activity, and CSF CRH levels. METHOD: CSF was withdrawn by means of a flexible, indwelling subarachnoid catheter over a 6-hour period, and hourly CSF concentrations of CRH were determined for 11 well-characterized combat veterans with PTSD and 12 matched normal volunteers. Twenty-four-hour urinary-free cortisol excretion was also determined. PTSD and depressive symptoms were correlated with the neuroendocrine data. RESULTS: Mean CSF CRH levels were significantly greater in PTSD patients than in normal subjects (55.2 [SD = 16.4] versus 42.3 pg/ml [SD = 15.6]). No correlation was found between CSF CRH concentrations and PTSD symptoms. While there was no significant difference between groups in 24-hour urinary-free cortisol excretion, the correlation between 24-hour urinary-free cortisol excretion and PTSD symptoms was negative and significant. CONCLUSIONS: By using a serial CSF sampling technique, the authors found high basal CSF CRH concentrations and normal 24-hour urinary-free cortisol excretion in combat veterans with PTSD, a combination that appears to be unique among psychiatric conditions studied to date.

Cerebrospinal fluid and plasma beta-endorphin in combat veterans with post-traumatic stress disorder.

Opioid-mediated analgesia develops in experimental animals following traumatic stress and increased opioid-mediated analgesia has been observed in combat veterans with post-traumatic stress disorder (PTSD). These observations have led to the hypothesis that increased central nervous system (CNS) opioidergic activity exists in patients with PTSD. However, direct CNS data on opioid peptide concentrations and dynamics in patients with PTSD are lacking. We withdrew cerebrospinal fluid (CSF) via a flexible, indwelling subarachnoid catheter over a 6-h period and determined hourly CSF concentrations of immunoreactive beta-endorphin (ir beta END) in 10 well-characterized combat veterans with PTSD and nine matched normal volunteers. Blood was simultaneously withdrawn to obtain plasma for ir beta END. PTSD symptom clusters, as measured by the CAPS, were correlated with neuroendocrine data. Mean CSF ir beta END was significantly greater in patients with PTSD compared with normals and there was a negative correlation between the ir beta END and PTSD intrusive and avoidant symptoms of PTSD. No intergroup difference between plasma ir beta END was found, nor was there a significant correlation between CSF and plasma ir beta END. Immunoreactive beta-lipotropin (ir beta LPH) and pro-opiomelanocortin (irPOMC), both precursors of beta END, were much more plentiful in human CSF than was beta-endorphin itself, as has been previously reported. It remains to be determined whether the increased CNS opioid concentrations predate traumatic stress, thereby conferring a vulnerability to dissociative states and PTSD itself, or result from the trauma. The negative correlation between CSF ir beta END and avoidant and intrusive symptoms suggests that CNS hypersecretion of opioids might constitute an adaptive response to traumatic experience. Poor correlation between CSF and plasma ir beta END limits use of plasma measures to assess CNS opioid activity.

Plasma corticotropin and cortisol responses to ovine corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), CRH plus AVP, and CRH plus metyrapone in patients with Cushing's disease.

The CRH test may sometimes be useful in the differential diagnosis of Cushing's syndrome, because most patients with pituitary ACTH-dependent Cushing's syndrome (Cushing's disease) respond to CRH, but those with other causes of Cushing's syndrome usually do not. However, about 10% of Cushing's disease patients fail to respond to CRH. We wondered if we could eliminate these false negative results either by exploiting the potential additive or synergistic effects of another ACTH secretagogue or by reducing glucocorticoid inhibition of CRH's ACTH-releasing effect. We compared the effect on plasma ACTH and cortisol in 51 patients with Cushing's disease of administering ovine CRH (1 microgram/kg BW, i.v.) alone, arginine vasopressin (AVP; 10 U, i.m.) alone, the combination of CRH and AVP, and CRH after pretreatment with metyrapone (1 g, orally, every 4 h for three doses; CRH + MET). The rates of nonresponse (ACTH increment, < 35%; cortisol increment, < 20%) to AVP and CRH alone were 26% and 8%, respectively; all patients responded to CRH + AVP. The lack of response was not due to improper administration or rapid metabolism of the agonist, because plasma CRH and AVP concentrations were similar in responders and nonresponders. A synergistic ACTH response to CRH + AVP occurred in 65% of the patients. MET pretreatment increased basal plasma ACTH levels in most patients and induced the greatest mean peak ACTH response to CRH, but 8% of the patients did not respond to CRH + MET with an ACTH increment of 35% or more. Because all of the Cushing's disease patients tested in this study responded to the combination of CRH + AVP, whereas 8% failed to respond to CRH alone, we conclude that CRH + AVP administration may provide a more reliable test for the differential diagnosis of ACTH-dependent Cushing's syndrome than administration of CRH alone. Whether this improved sensitivity is accompanied by unaltered specificity for Cushing's disease must be tested in patients with chronic ectopic ACTH syndrome.

Preprothyrotropin-releasing hormone-(178-199) does not inhibit corticotropin release.

Pituitary ACTH synthesis and secretion are positively regulated by hypothalamic factors and negatively regulated by adrenal corticosteroids. Negative hypothalamic regulation of pituitary ACTH synthesis and secretion has been postulated, but not proved. The search for a hypothalamic corticotropin release-inhibiting factor has recently focused on peptides derived from the prepro-TRH precursor of TRH. One of the peptides, prepro-TRH-(178-199), was reported to suppress basal and stimulated ACTH release. We examined the effects of prepro-TRH-(178-199) alone and in combination with CRH and corticosterone, two known physiologic regulators of ACTH secretion. Prepro-TRH-(178-199) had no effect on basal, stimulated, or attenuated ACTH release from cells that responded normally to CRH and/or corticosterone. These results indicate that prepro-TRH-(178-199) is not a corticotropin release-inhibiting factor.

Factitious hypercortisoluria.

A case of factitious hypercortisoluria due to the presumed addition of glucocorticoid to the urine collections is presented. The discrepancy between urine and blood steroid hormone levels first suggested that the patient had tampered with the urine collections. Plasma steroid hormone levels were normal, whereas the urinary free cortisol level fluctuated in a totally random fashion. Urinary 17-hydroxycorticosteroid and urinary free corticosterone levels were normal. Partition chromatography of the urine indicated that the cortisol immunoactivity coeluted with authentic cortisol. This study illustrates the value of multiple urinary and plasma steroid determinations, especially urinary or plasma corticosterone measurements, in suspected cases of factitious Cushing's syndrome.

Concentrations of corticotropin-releasing hormone, norepinephrine, MHPG, 5-hydroxyindoleacetic acid, and tryptophan in the cerebrospinal fluid of alcoholic patients: serial sampling studies.

Abnormalities in corticotropin-releasing hormone (CRH) secretion, noradrenergic neurotransmission, and serotonergic activity in the central nervous system (CNS) have all been hypothesized to exist in alcoholic patients, as have abnormalities in hypothalamic-pituitary adrenal function. To test these hypotheses, we continuously sampled cerebrospinal fluid (CSF) from alcoholic patients after 38-124 days of abstinence and from normal volunteers via a flexible, indwelling lumbar subarachnoid catheter and measured CRH, norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), tryptophan, and 5-hydroxyindoleacetic acid (5-HIAA) concentrations at 10-min intervals, from 11:00 through 17:00 h. The spinal canal catheter was inserted at approximately 08:00 h. Serial plasma ACTH, cortisol, and NE concentrations were also measured. A mixed liquid meal was consumed at 13:00 h. CSF CRH concentrations were lower in alcoholic patients than in normal volunteers (26 +/- 15 vs. 60 +/- 30 pg/ml, respectively, p < 0.05 by ANOVA), as were CSF NE levels (0.33 +/- 0.09 vs. 1.15 +/- 0.51 pmol/ml, respectively, p < 0.01). Plasma NE and CSF MHPG levels were normal in the alcoholic patients. CSF tryptophan and 5-HIAA and plasma ACTH and cortisol concentrations did not differ between the groups. These studies extend our finding of reduced spinal canal CSF CRH concentrations in depressed patients to abstinent chronic alcoholics.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive radioimmunoassay for fludrocortisone in human plasma.

Fludrocortisone has been a mainstay of therapy for orthostatic hypotension for many years. Clinical experience suggests that there exists a substantial interindividual variation in responsiveness to the drug. To assess this, we have developed an assay that permits measurement of the low concentrations of fludrocortisone found in human plasma. Fludrocortisone was detected by radioimmunoassay. A polyclonal rabbit antibody, raised against dexamethasone which cross-reacts strongly with fludrocortisone, was reacted with either standard or unknown samples in the presence of [125I]fludrocortisone-3-TyrNH2 (synthesized by coupling tyrosine amide to fludrocortisone-3-oxime and iodinating with chloramine T oxidation). The ED10, ED50, and ED80 were 0.34, 5.0, and 30 ng/mL of plasma, respectively. The cross reactivity with other 9-fluorinated steroids was found as follows: dexamethasone, 340%; betamethasone, 230%; and triamicinolone, 8%. To preclude an erroneous result, subjects who were pregnant or receiving any steroid medication were excluded from the study. The percent cross-reactivity with the main naturally occurring steroids was as follows: 11-desoxycortisol 3.2%, cortisol 1.1%, DOC 0.3%, pregnenolone 0.1%, corticosterone 0.06%, progesterone 0.05%, and aldosterone < 0.05%. The only compound with potential for interference, because of its high level in the circulation in the early morning, was cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin in human cerebrospinal fluid: concentrations, dynamics, molecular forms and relationship to fasting and feeding in health, depression and alcoholism.

Very little is known about the physiologic significance of the gut-brain hormone cholecystokinin (CCK) in the human central nervous system, although the hormone has been hypothesized to be involved in the regulation of both appetite and anxiety. We continuously collected lumbar cerebrospinal fluid (CSF) via indwelling subarachnoid catheters in ten normal volunteers, ten patients with major depression and five abstinent alcoholic humans, while fasting and after eating. Five other healthy subjects were fasted throughout the experiment. We quantified CSF immunoreactive cholecystokinin (IR-CCK) and glucose concentrations at 10-min intervals from 11.00 to 17.00 h. No difference in CSF IR-CCK concentration, half-life or rhythm was observed between normal volunteers and either depressed or alcoholic patients. Fasting CSF IR-CCK concentrations were 1.3 +/- 0.18, 1.3 +/- 0.21 and 1.2 +/- 0.21 fmol/ml (mean +/- S.E.M.) in normal volunteers, depressed patients and alcoholic patients, respectively. After eating, CSF IR-CCK concentrations rose to 1.5 +/- 0.21, 1.5 +/- 0.24 and 1.4 +/- 0.26 fmol/ml, respectively. Normal volunteers who did not eat had similar basal CSF IR-CCK concentrations (1.1 +/- 0.1 fmol/ml) which similarly rose to 1.4 +/- 0.13 fmol/ml during the sampling interval. In contrast, CSF glucose concentrations rose only in the subjects who ate, beginning to rise after about 1 h and remaining elevated for at least 3 h after eating. These data suggest the existence of a diurnal rhythm of IR-CCK release into CSF, as opposed to a response to feeding. The disappearance half-time of CCK in human CSF is less than 13 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of protein kinase-C depletion on inositol trisphosphate-mediated and cyclic adenosine 3',5'-monophosphate-dependent protein kinase-mediated adrenocorticotropin secretion.

We studied the effect of Ca2+/phospholipid-dependent protein kinase-C (protein kinase-C) down-regulation by chronic exposure to phorbol 12-myristate 13-acetate (PMA) on ACTH secretion by dispersed male rat anterior pituitary cells in a microperifusion system. Preincubation for 24 h and preperifusion for 3 h with 0.1 and 1 microM PMA significantly inhibited (by 85% and 91%, respectively) the specific cell binding of [3H]phorbol 12,13-dibutyrate, an index of protein kinase-C concentration, and significantly reduced (by 101% and 20%, respectively) the sustained plateau (final 15-min) phase of the ACTH response to arginine vasopressin (AVP) and (by 56% and 54%, respectively) the sustained (full 20-min) response to dioctanoylglycerol (DOG), both of which are mediated by protein kinase-C activation. In contrast, the spike (initial 5-min) phase of the response to AVP, which is mediated by intracellular Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores, was significantly increased (by 112% and 99%, respectively), but the spike-type response to ionomycin, which releases intracellular Ca2+ by an InsP3-independent mechanism, was unaffected. AVP significantly stimulated inositol bisphosphate and InsP3, but not inositol monophosphate, accumulation, and PMA pretreatment significantly enhanced their AVP-stimulated accumulation (by 86%, 34%, and 78%, respectively), an effect that was abolished by simultaneous preperifusion with PMA and cycloheximide to inhibit new protein synthesis. Enhancement of the spike phase response to AVP and AVP-stimulated InsP3 accumulation were lost within 1 h of PMA removal, but [3H]phorbol 12,13-dibutyrate binding and the sustained responses to AVP and DOG remained suppressed after 3 h. Pretreatment with 0.1 and 1 microM PMA slightly reduced the sustained responses to CRF (by 29% and 16%, respectively) and 8-bromo-cAMP (by 8% and 12%, respectively), which are mediated by protein kinase-A activation and extracellular Ca2+ influx via L-type voltage-sensitive Ca2+ channels, but not the response to KCl, which is mediated by extracellular Ca2+ influx via all types of voltage-sensitive Ca2+ channels. The sustained response to CRF was still suppressed 1 h after PMA removal, but returned to the control level by 3 h. When new protein synthesis was inhibited by preperifusion with cycloheximide alone for 3 h after 24-h PMA pretreatment, recovery from the effects of PMA was abolished. Three-hour exposure to cycloheximide without PMA pretreatment inhibited the sustained responses to CRF, AVP, and DOG, but not the spite response to AVP.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromogranin A does not mediate glucocorticoid inhibition of adrenocorticotropin secretion.

It has recently been proposed that chromogranin A (CgA), a 50-kilodalton acidic glycoprotein that is costored and cosecreted with hormones and neurotransmitters in a variety of tissues, mediates glucocorticoid inhibition of ACTH secretion from AtT20/D16v mouse anterior pituitary corticotroph tumor cells by an undefined autocrine mechanism. We used AtT20/D16v cells, RIAs for murine CgA and ACTH, complementary DNA probes for CgA and POMC, the precursor of ACTH, antiserum that reacts with murine CgA, highly purified bovine CgA, and synthetic rat and porcine pancreastatin, a bioactive cleavage product of CgA in some systems, to study the kinetics of the effect of glucocorticoids on CgA and ACTH synthesis and secretion and of CgA's subsequent effects on ACTH secretion. Exposure to 100 nM dexamethasone (DEX) did not alter the size of CgA or POMC messenger RNA (mRNA) transcripts but increased cell CgA mRNA content 42% by 3 h and 192% by 48 h. DEX decreased cell POMC mRNA content 22% by 6 h and 57% by 48 h. These divergent effects of DEX on steady state mRNA levels were accompanied by similar divergent effects on the production of CgA and ACTH protein. Thirty-minute exposure to 10 nM ovine CRF increased CgA and ACTH release to 300% and 360% of basal levels, respectively. One-hour DEX pretreatment inhibited CRF-stimulated CgA and ACTH release 58% and 49% at 30 min and 67% and 66% at 60 min, respectively. There was a positive correlation between CgA and ACTH release under all conditions at both times (r = 0.976 and 0.964, respectively, P < 0.001), consistent with costorage and cosecretion of the two proteins. The ratio of secreted ACTH to CgA decreased progressively with DEX treatment. Purified bovine CgA (100 nM) had little or no effect on basal or CRF-stimulated ACTH secretion from AtT20/D16v cells, 100 nM synthetic pancreastatin had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells or dispersed normal male rat anterior pituitary cells, and anti-CgA sera had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells. These results indicate: 1) that DEX stimulates CgA synthesis, whereas it inhibits POMC synthesis; 2) that CgA and ACTH are cosecreted; 3) that DEX increases CgA secretion relative to ACTH secretion, but decreases the absolute secretion of both proteins; and 4) that neither CgA nor its proteolytic product, pancreastatin, inhibits ACTH secretion. Thus, CgA does not mediate the inhibitory effect of DEX on ACTH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Adrenocorticotropin stimulation test: effects of basal cortisol level, time of day, and suggested new sensitive low dose test.

Adrenal response to iv administration of 1-24 ACTH (250 micrograms) was examined in normal volunteers under various conditions. The effect of basal cortisol levels was examined by performing the tests at 0800 h with and without pretreatment with dexamethasone. The effect of time of day was evaluated by performing the tests at 0800 h and at 1600 h, eliminating possible basal cortisol influence by pretreatment with dexamethasone. In the first set of tests, despite significantly different baseline levels, 30-min cortisol levels were not different (618 +/- 50 vs. 590 +/- 52 nmol/L). Afternoon cortisol levels in response to ACTH were found to be significantly higher than morning levels at 5 min (254 +/- 50 vs. 144 +/- 36 nmol/L, p less than 0.01) and at 15 min (541 +/- 61 vs. 433 +/- 52 nmol/L, p less than 0.02). This difference in response was no longer notable at 30 min (629 +/- 52 and 591 +/- 52 nmol/L). We tried also to determine the lowest ACTH dose which will elicit a maximal cortisol response. No difference was found in cortisol levels at 30 and 60 min in response to 250 and 5 micrograms 1-24 ACTH. Using 1 micrograms ACTH, the 30-min response did not differ from that to 250 micrograms (704 +/- 72 vs. 718 +/- 55 nmol/L, respectively). However, the 60-min response to 1 microgram was significantly lower (549 +/- 61 vs. 842 +/- 110 nmol/L, p less than 0.01). Using this low dose ACTH test (1 microgram, measuring 30-min cortisol level), we were able to develop a much more sensitive ACTH test, which enabled us to differentiate a subgroup of patients on long-term steroid treatment who responded normally to the regular 250 micrograms test, but had a reduced response to 1 microgram. The stability of 1-24 ACTH in saline solution, kept at 4 C, was checked. ACTH was found to be fully stable after 2 hs in a concentration of 5 micrograms/ml in glass tube and 0.5 micrograms/ml in plastic tube. It was also found to be fully stable, both immunologically and biologically, for 4 months, under these conditions. We conclude that the 30-min cortisol response to ACTH is constant, unrelated to basal cortisol level or time of day. It is therefore the best criterion for measuring adrenal response in the short ACTH test. The higher afternoon responses at 5 and 15 min suggest greater adrenal sensitivity in the afternoon, but further studies are needed to clarify this issue.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of protein kinase-C in the adrenocorticotropin secretory response to arginine vasopressin (AVP) and the synergistic response to AVP and corticotropin-releasing factor by perifused rat anterior pituitary cells.

Arginine vasopressin (AVP) stimulates biphasic release of ACTH from anterior pituitary corticotrophs. The response consists of an initial transient spike phase lasting less than 3 min and a subsequent sustained plateau phase that persists for as long as AVP is present. AVP also acts synergistically with CRF on ACTH release. We have previously shown that the initial spike phase of the response mainly requires release of intracellular Ca2+ and is independent of calmodulin, whereas the sustained plateau phase, like the monophasic sustained response elicited by CRF, involves the influx of extracellular Ca2+ via L-type voltage-sensitive Ca2+ channels and activation of calmodulin. We have also shown that the synergism between AVP and CRF does not require extracellular Ca2+ influx. In this study we examined the role of Ca2+/phospholipid-dependent protein kinase-C (PKC) in the two phases of the response to AVP and in the synergism between AVP and CRF. We exploited the observation that prolonged exposure to phorbol esters down-regulates PKC. Dispersed adult male rat anterior pituitary cells were incubated in static suspension culture for 4-5 days, 0.5 microM phorbol 12-myristate 13-acetate (PMA) or 0.0005% dimethylsulfoxide vehicle alone was added, and the incubation was continued for 24 h. The cells were preperifused with PMA-free perifusion medium for 3 h and then perifused with various agents for 10-20 min. Effluent fractions were collected every 30 sec or 1 min and subjected to ACTH RIA. Pretreatment with PMA inhibited the subsequent response to 100 nM PMA and 100 microM dioctanoylglycerol, but not to 5 microM forskolin or to depolarization with 56 mM KCl, demonstrating specific down-regulation of PKC. PMA pretreatment had no effect on the initial spike phase of the response to AVP, but inhibited the sustained plateau phase by 57% (P less than 0.005) and, consequently, the integrated total response by 33% (P less than 0.05). Pretreatment had no effect on the response to CRF. However, pretreatment with PMA completely blocked both phases of the synergistic response to the combination of AVP and CRF. These results indicate that activation of PKC is required for the sustained phase of the response to AVP and both phases of its synergistic interaction with the protein kinase-A pathway, but is not involved in the initial spike phase of the response to AVP, which presumably is mediated by inositol 1,4,5-trisphosphate-stimulated mobilization of intracellular Ca2+, or in the independent activation of the protein kinase-A pathway by CRF.

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)

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.

Increased pro-opiomelanocortin-derived peptide release in myotonic dystrophy.

The response of plasma immunoreactive (IR)-ACTH, IR-beta-endorphin (beta-END) and IR-cortisol to insulin-induced hypoglycaemia, an acute stimulus to the pituitary corticotrophs through the central nervous system, and to synthetic ovine corticotrophin-releasing hormone (CRH), a direct corticotroph stimulator, were studied in normal males and males with myotonic dystrophy. Myotonics had an increased IR-ACTH and IR-beta-END response to hypoglycaemia and an increased IR-ACTH response to CRH compared with normals. Plasma IR-cortisol response were not different in either group of subjects to both stimuli. This neuroendocrine abnormality in myotonic dystrophy may represent a manifestation of the purported specific cell membrane defect underlying the disease. This is the first report of an abnormality in proopiomelanocortin peptide release in myotonic dystrophy.

Pituitary and hypothalamic hormones in normal and neoplastic adrenal medullae: biologically active corticotropin-releasing hormone and corticotropin.

Six normal and 8 neoplastic adrenal medullae were assayed for several immunoreactive (IR) proopiomelanocortin (POMC) and hypothalamic peptides. IR-POMC peptides were found in normal and tumor tissue in concentrations ranging from 0.0003 to 0.1% of those in pituitary. Their molecular sizes resembled those of pituitary intermediate lobe POMC peptides. No intact POMC was found. One pheochromocytoma contained fully bioactive IR-adrenocorticotropic hormone (IR-ACTH; Mr approximately 4,500) and an intermediate-sized (Mr approximately 10,000) IR-ACTH with approximately 69% bioactivity. Normal and tumorous medullae contained IR-corticotropin-releasing hormone (CRH) in concentrations ranging from 0.6 to 4% of those in hypothalamus except for one pheochromocytoma that contained 40 times that amount of IR-CRH, which was chromatographically indistinguishable from hypothalamic CRH and fully bioactive. IR-somatostatin and IR-growth hormone-releasing hormone were found in both tissue types, but IR-gonadotropin-releasing hormone and IR-thyrotropin-releasing hormone (TRH) were not, although IR-histidyl-proline diketopiperazine, a putative TRH metabolite, was found. IR-arginine vasopressin was found in two normal medullae, but not in pheochromocytomas.