Norepinephrine and corticosterone in the medial prefrontal cortex and hippocampus predict PTSD-like symptoms in mice.

This study measured changes in brain extracellular norepinephrine (NE) and free corticosterone (CORT) levels in a mouse model of post-traumatic stress disorder and related them to hyperarousal and fear memory retention. To this end, microdialysis in the medial prefrontal cortex (mPFC) and the hippocampus (HPC) of male C57BL/6NCrl mice was performed during an acoustic startle response (ASR) and following an electric foot shock (FS), as well as during an ASR and recall of contextual fear (CF) 1 day later. Changes in ASR-stimulated NE levels in the mPFC corresponded to ASR 34 days after FS. Changes in basal and ASR-stimulated extracellular NE levels in the HPC, in contrast, were related to expression of early (day 2) and late (day 34) CF after FS. The increase in extracellular NE levels correlated in a U-shape manner with arousal levels and CF, thus suggesting a non-direct relationship. Stress of different modalities/strength (ASR, FS and CF) caused a similar relative increase in free CORT levels both in the mPFC and the HPC. One day after FS, ASR-induced increases in the CORT content in the mPFC tended to correlate with the FS-potentiated ASR in a U-shape manner. Taken together, these data show that the intracerebral increase in free CORT was likely related to an immediate response to stress, whereas NE neurotransmission in the forebrain predicted arousal and CF 1 month after trauma.

Laminin inhibits lactotroph proliferation and is reduced in early prolactinoma development.

Laminin is a component of the extracellular matrix (ECM) that regulates cell proliferation and hormone secretion. Here we describe the effects of laminin on prolactin secretion in normal and tumor cells and analyze laminin expression pattern during prolactinoma development. Prolactin secretion and cell proliferation were inhibited by laminin in GH3 cells. In contrast, no effect was observed in normal pituitary cells. Laminin showed a dynamic expression pattern during prolactinoma development, which was: (a) strong in normal pituitaries from wild type or dopamine D2 receptor deficient mice, (b) lower in pituitary hyperplasia and (c) markedly reduced in prolactinomas from D2R -/- mice. A similar gradual decrease in laminin was found by comparing normal human pituitaries, human pituitary hyperplasia and human prolactinomas. These results show dynamic changes of laminin expression during prolactinoma formation which, due to laminin action on PRL production and cell proliferation, indicate a possible role for laminin in prolactinoma development.

The intrapituitary stimulatory effect of lipopolysaccharide on ACTH secretion is mediated by paracrine-acting IL-6.

During infection/inflammation bacterial lipopolysaccharide (LPS) activates the immune system and thus enhances the level of circulating cytokines. These circulating cytokines induce adaptive processes within the endocrine system and in particular stimulate the HPA axis to increase the level of anti-inflammatory-acting glucocorticoids in the circulation. We have shown recently that LPS stimulates intrapituitary IL-6 production in folliculostellate cells via specific receptors and the p38a mitogen-activated protein kinase/nuclear factor-kappa B pathway. To test the physiological relevance of these findings, we studied whether LPS could enhance ACTH secretion via paracrine-acting intrapituitary IL-6. Lipopolysaccharide stimulated IL-6 secretion both in monolayer and aggregate mouse pituitary cell cultures, but only in aggregates, ACTH secretion was significantly enhanced by LPS. Other hormones, such as GH or PRL, were less stimulated by LPS. My4, an antibody that blocks the interaction of LPS with the LPS receptor CD14, suppressed both LPS-induced IL-6 and ACTH secretion in aggregate cultures. A neutralizing antibody against mouse IL-6 also inhibited LPS-induced ACTH secretion in aggregates. In mouse pituitary fragments, LPS-induced ACTH secretion was blocked by My4 and IL-6 antibodies, identically to re-aggregate cell cultures. LPS-induced ACTH secretion, mediated by intrapituitary IL-6, may represent a pituitary-specific mechanism that stimulates the HPA axis during infection/inflammation.

Retinoic acid prevents experimental Cushing syndrome.

Cushing syndrome is caused by an excess of adrenocorticotropic hormone (ACTH) production by neuroendocrine tumors, which subsequently results in chronic glucocorticoid excess. We found that retinoic acid inhibits the transcriptional activity of AP-1 and the orphan receptors Nur77 and Nurr1 in ACTH-secreting tumor cells. Retinoic acid treatment resulted in reduced pro-opiomelanocortin transcription and ACTH production. ACTH inhibition was also observed in human pituitary ACTH-secreting tumor cells and a small-cell lung cancer cell line, but not in normal cells. This correlated with the expression of the orphan receptor COUP-TFI, which was found in normal corticotrophs but not in pituitary Cushing tumors. COUP-TFI expression in ACTH-secreting tumor cells blocked retinoic acid action. Retinoic acid also inhibited cell proliferation and, after prolonged treatment, increased caspase-3 activity and induced cell death in ACTH-secreting cells. In adrenal cortex cells, retinoic acid inhibited corticosterone production and cell proliferation. The antiproliferative action and the inhibition of ACTH and corticosterone produced by retinoic acid were confirmed in vivo in experimental ACTH-secreting tumors in nude mice. Thus, we conclude that the effects of retinoic acid combine in vivo to reverse the endocrine alterations and symptoms observed in experimental Cushing syndrome.

Normal human pituitary gland and pituitary adenomas express cannabinoid receptor type 1 and synthesize endogenous cannabinoids: first evidence for a direct role of cannabinoids on hormone modulation at the human pituitary level.

Little is known about the expression and function of cannabinoid receptor type 1 (CB1) in the human pituitary gland. The aim of this study was to investigate CB1 expression in human normal and tumoral pituitaries by in situ hybridization and immunohistochemistry using an antibody against CB1. CB1 was found in corticotrophs, mammotrophs, somatotrophs, and folliculostellate cells in the anterior lobe of normal pituitary. After examination of 42 pituitary adenomas, CB1 was detected in acromegaly-associated pituitary adenomas, Cushing's adenomas, and prolactinomas, whereas faint or no expression was found in nonfunctioning pituitary adenomas. Experiments with cultured pituitary adenoma cells showed that the CB1 agonist WIN 55,212--2 inhibited GH secretion in most of acromegaly-associated pituitary adenomas tested and that the CB1 antagonist SR 141716A was generally able to reverse this effect. Moreover, WIN 55,212--2 was able to suppress GHRH-stimulated GH release, and this effect was not blocked by coincubation with SR 141716A, possibly indicating a non-CB1-mediated effect. In contrast, WIN 55,212--2 was ineffective on GH-releasing peptide-stimulated GH release. In four Cushing's adenomas tested, WIN 55,212--2 was not able to modify basal ACTH secretion. However, simultaneous application of CRF and WIN 55,212--2 resulted in a synergistic effect on ACTH secretion, and this effect could be abolished by SR 141716A, demonstrating a CB1-mediated effect. In the single case of prolactinomas tested, WIN 55,212--2 was able to inhibit basal secretion of PRL. Finally, the presence of endocannabinoids (anandamide and 2-arachidonoylglycerol) was investigated in normal and tumoral pituitaries. All tumoral samples had higher contents of anandamide and 2-arachidonoylglycerol compared with the normal hypophysis. Moreover, endocannabinoid content in the different pituitary adenomas correlated with the presence of CB1, being elevated in the tumoral samples positive for CB1 and lower in the samples in which no or low levels of CB1 were found. The results of this study point to a direct role of cannabinoids in the regulation of human pituitary hormone secretion.

Decreased corticotropin-releasing hormone (CRH) concentrations in the cerebrospinal fluid of eucortisolemic suicide attempters.

Several lines of evidence suggest a dysregulation of the adrenocortical (HPA) system with hypersecretion of CRH is associated with suicidal behavior. However, controversial results have emerged from the determination of corticotropin-releasing hormone (CRH) concentrations in the lumbar cerebrospinal fluid (CSF) of suicide attempters probably due to methodological differences. We simultaneously measured CRH concentrations in the CSF and in the plasma of 41 psychiatric in-patients with different diagnoses (affective disorder, schizophrenia, personality disorders, adjustment disorder, substance abuse) and eight neurological control subjects. We also measured plasma cortisol concentrations because data from animal experiments suggest that cortisol may influence CSF CRH concentrations. The major finding was that patients who attempted suicide prior to admission had significantly lower CSF CRH concentrations than psychiatric patients without suicidal behavior. CRH concentrations were significantly higher in the CSF than in plasma in both, psychiatric patients and neurological control subjects. There was no significant difference between suicide attempters and patients with acute suicidal ideations. The latter group showed a trend towards lower CSF CRH concentrations compared with the neurological control subjects. Patients with affective disorder alone as well as patients with multiple diagnoses, but not schizophrenic patients, showed significantly lower CSF CRH concentrations than neurological control subjects. Plasma CRH and plasma cortisol concentrations did not differ among diagnostic groups or between suicide attempters vs. non-attempters. Further studies with more homogeneous samples, drug-free patients and with simultaneous assessment of various parameters of the HPA system are warranted.

Extracellular matrix components regulate ACTH production and proliferation in corticotroph tumor cells.

The extracellular matrix (ECM) conveys signals through membrane receptors called integrins producing changes in cell morphology, proliferation, differentiation and apoptosis. Previous studies suggest that the ECM plays an important role in pituitary physiology and tumorigenesis. In the present work we studied for the first time the effects of fibronectin, laminin, collagen I and collagen IV on hormone secretion and cell proliferation in the corticotroph tumor cell line AtT-20 and in normal pituitary cells, examining the signal transduction mechanisms that mediate these effects. ACTH production in AtT-20 cells was inhibited by fibronectin, laminin and collagen I. A reporter construct with the POMC promoter showed similar results, indicating that the effects of the ECM take place at the level of POMC gene transcription. In contrast, ACTH production was not significantly altered in normal pituitary cells. AtT-20 cell proliferation was stimulated by collagen IV and fibronectin, but inhibited by collagen I and laminin. In parallel, the cell morphology was modified by the ECM. We found that the production of reactive oxygen species mediate the effects of laminin and collagen IV. On the other hand, the effect of fibronectin was mimicked by beta1-integrin and Rho activation. These results show for the first time that the ECM controls ACTH biosynthesis and proliferation in corticotroph tumor cells and suggest a role for the ECM in corticotroph adenoma development.

Characterization of Interleukin-2 (IL-2) receptor expression and action of IL-2 and IL-6 on normal anterior pituitary cell growth.

The pituitary gland is known to express cytokines and their receptors. Interleukin-2 (IL-2) and IL-2 receptor (IL-2R) transcripts and protein products in corticotrophic cells have been previously described. IL-2R were also observed in PRL and GH-producing cells. The synthesis of IL-1 and IL-6 and their receptors by pituitary cells has also been reported. We recently demonstrated that the cytokines in addition to their regulatory effects on anterior pituitary hormone secretion are involved in the autocrine or paracrine regulation of pituitary growth. In the present study we show in normal rat anterior pituitary cells: (a) expression of IL-2Rα chain mRNA, (b) the co-localization of IL-2Rα chain with TSH, FSH and LH-producing cells, (c) the percentage of co-localization of IL-2R with all types of anterior pituitary hormone producing cells: PRL> > > ACTH> > GH> TSH = FSH = LH. (d) that [(3)H]-thymidine is incorporated into the nucleus of all types of hormoneproducing cells without incorporation into other cell types, following IL-2 and IL-6 stimulation. Our results suggest that IL-2 acts on all types of anterior pituitary hormone-producing cells and, through specific functional receptors on the same or other cells, constitutes, as well as IL-6, an inter or intra-cellular factor involved in the coordinate regulation not only of hormone secretion but also of the proliferation of anterior pituitary hormone-producing cells.

Octreotide exerts different effects in vivo and in vitro in Cushing's disease.

The effect of the long-acting somatostatin analog octreotide (SMS 201-995) on adrenocorticotropin (ACTH) secretion was studied in five patients with untreated Cushing's disease in vivo and in six human corticotropic adenoma cell cultures in vitro. For the in vivo study, 100 micrograms of octreotide sc was given 30 and 180 min after cannulation of the cubital vein and 100 micrograms of corticotropin-releasing hormone (CRH) was injected iv at 210 min. Serum ACTH and cortisol levels were measured for 390 min. In vivo, octreotide had no significant effect either on basal or CRH-stimulated ACTH levels and did not influence cortisol levels. The in vitro studies were conducted with corticotropic adenoma cell cultures derived from adenoma tissue obtained from six patients with Cushing's disease. In four of six cell cultures, octreotide (1 nmol/l-1 mumol/l) inhibited basal ACTH secretion in a dose-dependent manner. The inhibition ranged from 70 to 92% for 1 nmol/l octreotide to 14-46% for 1 mumol/l octreotide as compared to controls (100%). In three of three octreotide-responsive adenoma cell cultures investigated. CRH-stimulated ACTH secretion was suppressed by octreotide. Hydrocortisone pretreatment in vitro abolished the inhibitory effect of octreotide on ACTH secretion in one octreotide-responsive corticotropic adenoma cell culture. In conclusion, we showed that octreotide in most cases could inhibit the ACTH release from human corticotropic adenoma cells in vitro but had no suppressive effect on ACTH levels of patients with Cushing's disease in vivo. This discrepancy could be due to a somatostatin receptor down-regulation by cortisol at the hypercortisolemic state in vivo.

Loperamide inhibits corticotrophic cell function by a naloxone-insensitive mechanism in the rat in vitro.

The effect of the antidiarrheal drug loperamide, a mu-opiate agonist, on ACTH secretion and biosynthesis, cAMP generation and phosphoinositide turnover was studied in rat anterior pituitary cell cultures. The cAMP-dependent protein kinase A pathway was stimulated with both corticotropin-releasing hormone (CRH; 2-5 nM) and the membrane-permeable Bu(2)cAMP (0.5-2.5 mM). The protein kinase C pathway was stimulated with 1 microM arginine vasopressin (AVP) and 1-10 nM phorbol 12-myristate 13-acetate (PMA). After 3.5 h, loperamide (10 microM) had no effect on basal ACTH levels but significantly suppressed CRH-induced ACTH release, in a dose-dependent manner, to 60 +/- 4% of control (100%) (p < 0.0001). After 24 h, basal proopiomelanocortin mRNA was significantly decreased to 50% of control by loperamide (p < 0.05). The suppressive effect of loperamide on CRH-induced ACTH secretion was not reversible by naloxone (0.1-1,000 microM). Morphine (0.01-10 microM) had no effect on basal and CRH-induced ACTH secretion. Loperamide did not influence basal and CRH-induced adenylate cyclase activity in anterior pituitary cell membrane preparations, but it significantly blunted Bu(2)cAMP-induced ACTH secretion in cell culture from 100 +/- 4 to 77 +/- 4% (p < 0.05). In Ca(2+)-depleted medium (Ca2+ < 0.1 mM), loperamide had no suppressive effect on CRH-induced ACTH secretion. AVP-induced ACTH secretion was significantly suppressed by loperamide from 100 +/- 5 to 74 +/- 3% (p < 0.0001), while basal and AVP-induced inositol 1-phosphate generation and PMA-induced ACTH secretion were not affected by loperamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of polypeptide growth factors by human nonfunctioning pituitary adenoma cells in culture.

The growth-promoting activities of tumor-conditioned media (TU-CM) obtained from 23 cultured human nonfunctioning pituitary adenomas were studied in vitro. TU-CM obtained from adenoma cell cultures increased both cell counts (range: 108-179%; control = growth in serum-free medium = 100%) and 3H-thymidine incorporation (112-139%) of rat pituitary cell cultures, indicating that TU-CM contains growth-stimulating substances. TU-CM was also able to stimulate the growth of normal fibroblasts (3H-thymidine incorporation: 164-178%; cell counts: 145-157%) and endothelial cells (3H-thymidine incorporation: 131-149%; cell counts: 181-217%), suggesting the presence of - possibly angiogenic-growth factors that act on these cell types. However, the growth of hormone-producing cells was also stimulated, since TU-CM increased 3H-thymidine incorporation into rat pituitary cells in the presence of D-Val-MEM, a medium specifically inhibiting growth of fibroblasts. Addition of neutralizing antibodies against transforming growth factor alpha (TGF-alpha), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), either alone or in different combinations, reduced the growth-promoting activity of TU-CM on rat pituitary cells (range: 96-71%; control = growth effect of TU-CM without antibodies = 100%), strongly indicating the presence of these growth factors in TU-CM. All 4 antibodies together completely inhibited the growth-stimulatory activity of TU-CM, strongly suggesting that these growth factors play the major role among growth-stimulating substances in TU-CM. This is the first study giving evidence that TGF-alpha, EGF, IGF-I and bFGF are secreted by nonfunctioning adenoma cells indicating that the growth factors could be involved in growth regulation of pituitary adenomas by paracrine or autocrine mechanisms.

Interleukin involvement in anterior pituitary cell growth regulation: effects of IL-2 and IL-6.

The pituitary gland plays a central role in the interactions between the immune and neuroendocrine systems. The expression of receptors for interleukin-1 (IL-1), IL-2, and IL-6 and the intrinsic production of these ILs by pituitary cells have been described. Previous studies have focused on the way cytokines influence hormone secretion. We have determined whether, in addition to these effects, ILs could affect pituitary cell proliferation. In GH3 cells, both IL-2 (1-100 U/ml) and IL-6 (10-500 U/ml) significantly stimulated [3H]thymidine incorporation and cell count. In contrast, inhibitory effects of both IL-2 and IL-6 at the same concentrations were observed on normal rat anterior pituitary cell growth. This finding was clearly evident when cells were cultured in Minimum Essential Medium-D-valine medium, a condition that results in cultures virtually free of fibroblasts. Autoradiographic studies confirmed that [3H]thymidine was only incorporated in the nucleus of nonfibroblastic pituitary cells. No direct correlation between the effects of IL-2 and IL-6 on cell growth and hormone secretion was apparent. By immunofluorescence, we observed IL-2 receptor expression on GH3 cells and, for the normal rat cultures, a high percentage of PRL-secreting and a lower percentage of GH-producing cells expressing IL-2 receptors, providing new evidence for a direct site of action of IL-2 on pituitary cells. Considering that uncontrolled division of cells may result from either excessive growth stimulation or deficient growth inhibition, the regulation of pituitary cell growth by IL-2 and IL-6 together with their intrinsic pituitary production could be of potential importance in pituitary adenoma pathogenesis.

Nitroimidazole derivatives inhibit anterior pituitary cell function apparently by a direct effect on the catalytic subunit of the adenylate cyclase holoenzyme.

Nitroimidazole derivatives dose-dependently decreased basal and CRF-stimulated ACTH release, basal and GRF-stimulated rat GH release, and basal rat PRL release in primary cultures of rat anterior pituitary cells. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with the nitroimidazole derivatives. Miconazole, econazole, isoconazole, clotrimazole, and bifonazole had similar or more pronounced effects on anterior pituitary function compared to ketoconazole, whereas metronidazole and etomidate were less effective. The positive correlation between the number of phenylated side-chains or phenolic rings of the imidazole molecule and the efficacy to inhibit activity on pituitary hormone secretion suggests a structure-activity relationship of these compounds. The effects of the nitroimidazole derivatives on anterior pituitary hormone release and biosynthesis were mediated by cAMP. Thus, basal and CRF-, cholera toxin-, and forskolin-stimulated adenylate cyclase activities in rat anterior pituitary cell membranes determined by cAMP formation were suppressed by the nitroimidazole derivatives. Pertussis toxin did not diminish the nitroimidazole derivative effect on cAMP formation. The adenylate cyclase inhibitory effect of these substances was independent of the presence of GTP in the assay system, underlining a direct effect on the catalytic subunit. In addition, basal and forskolin-stimulated cAMP generation in membranes of S49 lymphoma cyc-variants, which lack a functional Gs protein, was efficiently suppressed (by up to 90%) by the nitroimidazole derivatives. In conclusion, ketoconazole and other nitroimidazole derivatives inhibit anterior pituitary hormone synthesis and secretion apparently by a direct effect on the catalytic subunit of the adenylate cyclase system.

Human corticotropin-releasing hormone during pregnancy.

Elevated irCRH levels up to 14 ng/ml were measured in 176 females in the last trimester. The highest maternal CRH levels were found in those females in whom the period from the onset of labour to full dilatation of the cervix and the time span of delivery were shortest. irCRH in amniotic fluid (120 +/- 180 pg/ml; n = 14) was in the same range as in umbilical cord plasma (233 +/- 188 pg/ml; n = 66) and 20-fold lower than in prepartal maternal plasma (5.38 +/- 4.49 ng/ml; n = 66). irCRH in maternal plasma correlated highly to irCRH in umbilical cord plasma (p less than 0.001; n = 66). After delivery irCRH disappeared from maternal plasma with a half-life of 50 minutes (n = 14). One day postpartum irCRH levels (n = 22) were undetectable. The height of the irCRH levels in the various biological fluids did not correlate to the mode or the pathological events of delivery (n = 43). Maternal ACTH levels above the normal range were encountered only in women immediately prepartal and did not correlate to the CRH levels (253 +/- 229 pg/ml; n = 66). Cortisol levels were higher in maternal plasma than in umbilical cord plasma due to elevated CBG (n = 78). Free cortisol levels were higher in the 3rd trimester than in the 1st (2.18 +/- 0.16 vs 1.16 +/- 0.73 ng/ml; n = 42). irCRH in maternal and umbilical cord plasma correlated to the hPl and estriol levels (p less than 0.001 and p less than 0.05; n = 66). We conclude that irCRH is secreted by the placenta into both maternal and fetal circulation. Though placental CRH is undistinguishable from hypothalamic CRH, the biological significance of placental CRH remains open. Our data show that placental CRH might be responsible for the changed function of the adrenal gland during pregnancy, with higher free cortisol levels in the last trimester. The extremely elevated ACTH levels during labour and delivery indicate that CRH is not the only mediator of stress-induced ACTH secretion in the regulation of the maternal hypothalamo-pituitary-adrenal axis.

Ketoconazole inhibits corticotropic cell function in vitro.

The effects of ketoconazole (KC) on secretion and biosynthesis of ACTH and generation of cAMP in rat anterior pituitary cells were investigated in vitro. KC inhibits CRF-stimulated ACTH release from rat anterior pituitary fragments in a dose-dependent fashion between 1.5 and 100 microM. The effect of CRF as a releaser of ACTH was fully restored after KC was removed from the medium. Similar effects were observed in primary cultures of rat anterior pituitary cells. KC dose-dependently decreased basal and CRF-stimulated ACTH release. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with KC. The effects of KC on ACTH release and biosynthesis seem to be mediated by cAMP, since KC inhibits basal and CRF-stimulated cAMP release and content within the same dose range. Since the stimulatory effects of cholera toxin, sodium fluoride, and forskolin were dose-dependently inhibited by KC and since the addition of (Bu)2cAMP abolished the inhibiting effect of KC, it is concluded that KC acts by inhibition of the catalytic component of the adenylate cyclase holoenzyme.

Pharmacological modulation of CRH-stimulated ACTH secretion by ketoconazole.

The effects of ketoconazole (KC) on secretion and biosynthesis of ACTH in rat anterior pituitary cells were investigated in vitro. KC inhibits the corticotropin releasing hormone (CRH) stimulated ACTH release from rat anterior pituitary fragments in a dose dependent fashion between 1.5 and 100 microM. The effect of CRH to release ACTH was fully restored after KC was removed from the medium. Similar effects were observed in primary cultures of rat anterior pituitary cells: KC decreased dose dependently the basal and CRH stimulated ACTH release. In addition, basal and CRH-stimulated mRNA coding for the ACTH precursor was reduced after preincubation with CK. The effects of KC on ACTH release and biosynthesis seems to be mediated by cyclic AMP (cAMP) since KC inhibits basal and CRH stimulated cAMP release and content within the same dose range. Since the stimulatory effect of cholera toxin, sodium fluoride and forskolin were dose dependently inhibited by KC and since the addition of dibutyryl cAMP abolished the inhibiting effect of KC, it is concluded that KC acts by inhibition of the catalytic component of the adenylate cyclase holoenzyme.

Corticotropin-releasing factor in humans. I. CRF stimulation in normals and CRF radioimmunoassay.

The biological activity of ovine (o) and human (h) corticotropin-releasing factor (CRF) in normal volunteers was investigated, using bolus injections with different CRF dosages. There was a significant increase of ACTH, beta-endorphin and cortisol after the injection of all dosages. Repetitive stimulation and continuous infusion of hCRF lead to repetitive release of identical amounts of ACTH or constant elevation of ACTH levels. oCRF and hCRF serum immunoreactivity was measured with specific radioimmunoassays after bolus injection, pulsatile administration and infusion of CRF. The half-time of serum disappearance after acute injection studies was calculated as 9 min for hCRF dand 18 min for oCRF. The 'metabolic clearance' of hCRF calculated using the infusion study was 2.72 ml/min X kg. Endogenous CRF immunoreactivity was detectable in 14 patients during insulin hypoglycemia and in 86 out of 97 pregnant females. Furthermore, CRF could be extracted from human placenta. The chromatographic pattern of extracted placenta CRF, pregnancy serum CRF and CRF standard preparation was identical. Furthermore, CRF immunoreactivity was detectable in some patients with different causes of ACTH hypersecretion.