Stalk-median Eminence Extract Research Articles

The use of inclusion bodies, isolated from Escherichia coli expressing corticotrophin-releasing hormone precursor, to raise specific antibodies against the neuropeptide moiety

We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to beta-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1-41) and CRH(36-41). They did not cross-react with the CRH antagonist CRH(9-41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1-41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human pre-proCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1-41). The results indicate that these antibodies can recognize CRH(1-41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.

Corticotropin-Releasing Hormone (CRH)-Binding Protein: Reduction in the Adrenocorticotropin-Releasing Activity of Placental but not Hypothalamic CRH*

This study is the first to use CRH-binding protein (CRH-BP) purified from human plasma to investigate how the CRH-BP affects the physiological activity of CRH. After incubation at 37 C for 15 min, purified CRH-BP reduced the ACTH-releasing activity of synthetic human (h) CRH in a dose-dependent fashion; using hCRH and CRH-BP at concentrations commonly found in late gestational maternal plasma, (1.5 and 100 ng/mL, respectively) an average 76% reduction in ACTH release was obtained. No effect was observed under the same conditions on ACTH release induced by ovine (o) CRH, which does not bind to CRH-BP. Kinetic estimations are presented to show that the extent of binding of CRH to its BP (and, hence, the reduction in its bioactivity) depends on the time available for binding and the concentration of reactants. Equilibrium between CRH and its BP at the concentrations used in the bioactivity studies take place within 400 s. Since long term secretion of placental CRH into the peripheral circulation occurs during the third trimester of pregnancy, we suggest that the presence of circulating CRH BP may partly explain how markedly elevated plasma levels of CRH coexist with normal ACTH levels at this time. We also propose that stress-induced CRH release will not be similarly quenched by the CRH-BP in the hypothalamic portal system, as the concentration of CRH released will be high, and the exposure time before reaching pituitary corticotropes will be low. Using pituitary cells constantly bathed in BP premixed with hCRH or BP alone (to mimic the situations in pregnancy and nonpregnancy, respectively), we show that short concentrated pulses of synthetic CRH (10 ng in 5 s) or rat stalk median eminence extract (one tenth stalk median eminence in 5 s) retain their ability to induce ACTH secretion despite the presence of CRH-BP. It is, thus, possible that CRH can still exert its role as a stress hormone in late gestation.

NEUROENDOCRINE ALTERATIONS IN NUDE MICE WITH A HUMAN LUNG CARCINOMA PRODUCING PRO‐OPIOMELANOCORTIN, CORTICOTROPHIN‐RELEASING HORMONE AND ARGININE VASOPRESSIN

A mucoepidermoid carcinoma of the lung (ICD classification 8430/3) resected from a patient with no clinical signs of pituitary-adrenal alterations was transplanted into 2-month-old athymic nu/nu nude mice, with the purpose of studying the effects exerted by the human tumour on the host hypothalamo-pituitary-adrenal axis. The tumour produces peptides derived from different regions of pro-opiomelanocortin (POMC: ACTH, 7.6 +/- 0.7; N-terminal POMC, 6.6 +/- 0.6; beta-LPH/endorphin, 7.3 +/- 0.7; and alpha-MSH;3.8 +/- 0.5 pmol/g wet tissue) and the neuropeptides corticotrophin-releasing hormone and arginine vasopressin (CRH: 3.6 +/- 0.4 and AVP: 1.1 +/- 0.2 pmol/g wet tissue). Immunohistochemical staining of consecutive sections of the tumour indicated that staining of tumour cells for the different peptides was not uniform and although some cells co-stained with CRH and AVP, POMC-positive cells appeared to be distinct from CRH and AVP cells. Tumour extracts were chromatographed on Sephadex G-75 and fractions monitored for POMC-derived peptides. A single peak with characteristics of alpha-MSH was detected. The ACTH, N-POMC and beta-LPH/endorphin radioimmunoassays (RIA) detected a peak at large molecular weight, eluting at the position expected for POMC. These RIA systems also revealed an ACTH(1-39) peak and another peak which probably correspond to 13 kDa ACTH, a peak eluting at the position of hN-POMC(1-48), a beta-LPH-like peak, and a smaller sized peak which may represent alpha- or gamma-endorphin. The ACTH, N-POMC and beta-LPH/endorphin contents of anterior lobe (AL) extracts, but not neutrointermediate lobe (NIL) extracts, showed a striking decrease in tumour-bearing (TB) nude mice. However, while no difference was seen in the alpha-MSH content of AL extract between TB and control (C) nude mice, it decreased in NIL extracts of TB animals. The contents of CRH and AVP in stalk-median eminence extracts of TB nude mice was significantly lower than that of C nude mice. Basal plasma corticosteroids were raised in TB nude mice at levels comparable to those in stressed C nude mice, and although adrenal weights did not vary between TB and C nude mice, morphological changes indicating hypertrophy were found in the adrenal glands of the host animals. It was concluded that the tumour dramatically alters the hypothalamo-pituitary-adrenal axis of the host, and that it may be a useful model for studying tumour-host interactions in ectopic hormone-producing tumours.

CAMP-dependent ACTH secretagogues facilitate corticotropin releasing activity of angiotensin II on rat anterior pituitary cells in vitro.

Both arginine vasopressin (AVP) and angiotensin II (AII) potentiate the corticotropin-releasing activity of CRF41 via a potentiation of CRF41-induced cAMP production. In perfused rat anterior pituitary cells, AII (10(-8) mol) showed a transitory 2-fold increase in its ACTH-releasing activity, when tested after application extract of rat stalk median eminence. In order to determine whether this facilitating effect on AII corticotropin-releasing activity occurred through cAMP-dependent mechanisms, the ACTH-releasing activity of AII was tested after stimulation with CRF41, AVP or forskolin, three secretagogues with known effects on cAMP production. When given 16 min after CRF41, 10 micrograms/l, AII (10(-8) mol) showed a significant increase (210%) in its ACTH-releasing activity, which returned to the normal level when AII-stimulation was repeated at 32 min (121%) and 48 min (100%). Similarly, forskolin, 3 X 10(-6) mol, produced a significant transitory increase (208%) in the subsequent AII-induced ACTH release whereas AVP, 10 micrograms/l and 100 micrograms/l, had no effect on the following ACTH response to AII. These results suggest that the AII-induced ACTH secretion--which is cAMP independent--nevertheless may be modulated by previously stimulation of the cAMP pathway.

Mechanism of restoration of ACTH release in rats with long-term lesions of the paraventricular nuclei

The effects of lesions in the paraventricular nucleus (PVN) on the adrenocortical response to ether stress were investigated in neurohypophysectomized and intact rats. During the first 4 days after placement of lesions in the PVN, the corticosterone response to ether stress was almost completely inhibited. It then gradually increased and, within 4-6 weeks of surgery, was restored to about 60% of that in sham-operated rats. Basal plasma concentrations of corticosterone were low in rats after placement of lesions in the PVN and/or after neurointermediate lobectomy (NILX). Corticosterone responses to ether stress were similar in groups submitted to PVN lesions and/or NILX, and lower than those in the appropriate sham-operated groups. In all lesioned groups, plasma ACTH concentrations after a combination of stressors (ether plus laparotomy) were also lower than those in the sham-operated groups. Six weeks after lesioning of the PVN, immunoreactive rat corticotrophin-releasing factor-41 (rCRF-41) concentrations in stalk-median eminence (SME) extract fell to about 5% of that in sham-operated rats, while immunoreactive arginine vasopressin (AVP) concentrations did not change. Immunohistochemistry revealed a substantial decrease in rCRF-41 immunostaining of the median eminence 6 weeks after lesioning of the PVN, though randomly located clusters of stained terminals were still seen in the whole rostro-caudal extent of the median eminence. A mixture containing synthetic rCRF-41 and AVP, in proportions similar to those in SME extracts from sham-operated rats, caused significantly less release of ACTH from anterior pituitary cell cultures than did SME extracts from sham-operated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal by thiols of dopamine-, stalk-median eminence-, and zinc-induced inhibition of prolactin transformation in adenohypophyses of lactating rats.

Depletion-transformation of PRL is a decrease in tissue PRL detectability which precedes and may often be required for increased PRL release. The present studies were designed to determine whether thiol-disulfide interchange mechanisms may be involved in PRL transformation and release by the pituitary of the lactating rat. Thirds of 8-h nonsuckled lactating rat adenohypophyses were incubated with or without thiols (reduced glutathione, the aminothiol cysteamine, or mercaptoethanol), in the presence of known inhibitors of transformation and release such as dopamine (DA), stalk median eminence (SME) extract, or Zn++. PRL concentrations in pre- and postincubated tissues, as well as the amount of released PRL, were determined by polyacrylamide gel electrophoresis and densitometry. In 30-min incubations without additions, 12-22% of the tissue PRL was depleted; however, in the presence of 17-50 microM DA, all doses of SME extracts tested (0.5-2.0 eq), or 0.1 mM Zn++, depletion was partially or totally prevented and PRL release was inhibited 25-60%. On the other hand, when thiols were added in addition to the above agents, a complete, dose-related, restoration of PRL depletion was obtained. In 120-min incubations, thiols similarly reversed the effects of 0.05 mM DA on depletion, but thiols did not reverse the inhibition of PRL release caused by DA, SME, or Zn++. Other data indicate that thiols alone may inhibit rat PRL release and also facilitate or induce PRL depletion; in bovine PRL granules, thiols reverse Zn++ inhibition of PRL release and detectability. These data suggest that thiol-disulfide interchange reactions may be importantly involved in both depletion-transformation and in secretion. The precise thiol sensitivity of the two processes does not appear identical, secretion being more sensitive to DA and less sensitive to thiols than depletion-transformation.

Ovine corticotropin-releasing factor and vasopressin: antibody-quenching studies on hypothalamic extracts of normal and Brattleboro rats.

Stalk median eminence (SME) extracts were preincubated with antibodies to ovine corticotropin-releasing factor (oCRF) and/or vasopressin, and the resulting CRF bioactivity tested with the isolated anterior pituitary cell column bioassay. The ACTH-releasing ability of Wistar rat SME was reduced by 60% with vasopressin antiserum, by 53% with oCRF antiserum, and by 81% after incubation with both antisera simultaneously. SME-stimulated LH release was unaffected by these antisera, which were all used at a dilution of 1:1000. The ACTH-releasing activity of SME could not be completely abolished by increasing the oCRF antibody concentration, or, in the case of ovine SME, by decreasing the tissue concentration preincubated with oCRF antibodies. With Brattleboro SME (which contains no endogenous vasopressin) ACTH-releasing activity was reduced by 37%, 51%, and 57% with anti-oCRF at dilutions of 1:5000, 1:1000, and 1:500, respectively, but could not be reduced further by more concentrated antisera. We conclude, therefore, that the CRF bioactivity of rat SME is probably not due solely to an oCRF-like peptide, but that other substances, one being vasopressin, contribute to its ACTH-releasing ability.

Purification of a high-molecular-weight somatoliberin (growth-hormone-releasing factor) from pig hypothalami.

Preliminary observations [Sykes & Lowry (1980) J. Endocrinol. 85, 42P-43P] had suggested that the major hypothalamic somatoliberin (growth-hormone-releasing factor) was a larger peptide than the other characterized hypothalamic factors, with an elution position on Sephadex G-50 between those of neurophysin and corticotropin. The present paper reports the isolation and preliminary characterization of pig hypothalamic somatoliberin. Acid extracts of pig stalk median eminence were purified by gel filtration and preparative and analytical high-pressure liquid chromatography to yield a preparation that was specific in the release of somatotropin (growth hormone) in vitro, giving a steep dose--response curve at doses in the range 0.20-3.0 ng. Amino acid analysis revealed a non-cysteine-containing peptide with a high number of glutamate (or glutamine) and aspartate (or asparagine) residues. The peptide had about 56-57 amino acid residues and an apparent molecular weight of 6400, in keeping with its elution position on a column of Sephadex G-50.

Responsiveness of adenohypophysial corticotrophs to hypothalamic (SME) extract remains unchanged in rats with hypothalamic lesions inhibiting CRF release.

The corticotrophin-releasing activity of stalk-median eminence (SME) extract was studied using incubated quarters of anterior pituitary glands obtained from rats with long-term anterolateral deafferentiation or complete surgical lesion of the medial basal hypothalamus, as well as with the lesion of the paraventricular nuclei. These hypothalamic interventions are known to cause a complete or partial but significant deficiency in hypothalamic corticotrophin-releasing factor (CRF) release, however, such lesions failed to alter the sensitivity of the ACTH response from incubated pituitary segments towards the SME extract. These results suggest that the specific responsiveness of adenohypophysial corticotrophs is maintained when hypothalamic CRF supply is decreased for a long period of time.

A serine-proteinase inhibitor (Boc-D-Phe-Pro-Arg-H) inhibits the secretion of adrenocorticotropin- and β-endorphin-immunoreactive peptides [formula omitted

The effects of a potent serine protease inhibitor, Boc-D-Phe-Pro-Arg-H, on the secretion and content of ACTH and β-endorphin-like immunoreactivities of cultured rat anterior pituitary cells were studied. Basal release of the hormones was not affected by the drug but secretion in response to stalkmedian eminence extracts was inhibited. The inhibitor did not effect the amount of ACTH or β-endorphin in the medium plus the cells, nor did it change the ratio of the lipotropin and β-endorphin-like immunoreactivities. Thus the compound seems to affect the release of ACTH and β-endorphin, but not the processing of lipotropin to β-endorphin.

Influence of cholecystokinin on hypothalamic-stalk median-eminence-extract stimulation of acth output from isolated pituitary cells

The influence of cholecystokinin (CCK 33) on CRF-like stimulation of ACTH output was tested in vitro using isolated pituitary cells. ACTH was assayed using isolated adrenal cell preparations. The CRF-like material was contained in a crude acetic-acid extract of hypothalamic stalk median eminence (HSME). CCK 33, in doses of 1 U, 10 −3 U, and 10 −6 U/ml cell suspension had no influence on basal or ACTH-stimulated corticosterone output from isolated adrenal cells. Isolated pituitary cells responded in a dose-related fashion of HSME extract, however, the absolute response to a given dose of HSME extract varied according to the basal (non-stimulated) output of a particular cell preparation. CCK 33, in the dose range tested, had no influence on basal ACTH output. In contrast, 10 −3 U/ml of CCK 33, which corresponds to a concentration of 8 × 10 −11 M, significantly inhibited the output of ACTH from isolated pituitary cells stimulated by 0.2 equivalents of HSME. Higher concentrations of CCK 33 had a variable effect. We conclude that cholecystokinin may have a role in the regulation of HSME-stimulated ACTH output from the pituitary.

Growth hormone releasing activity persists in the deafferented medial-basal hypothalamus of the rat.

The possible role of the neural connections of the medial-basal hypothalamus (MBH) in the maintenance of GH releasing activity of the pituitary stalk median eminence (SME) was investigated. Male rats, subjected to sham-operation and to complete and anterolateral cuts around the MBH were used 7-8 days after surgery. Electrical stimulation of neural structures within the MBH caused an increase of plasma GH in pentobarbitone- as well as in urethane-anaesthetized animals. In sham-operated rats the rise of plasma GH levels was apparent only after completion of 10 min of electrical stimulation, while in animals with complete or anterolateral cuts an increase was already evident during electrical stimulation. The results suggest that depolarization of somatostatin secreting fibres in the median eminence may be responsible for the delay in the rise of GH levels in sham-operated rats, while the increment can be attributed to a GH releasing principle in the hypothalamus. Acidic extracts of the SME of rats with complete or anterolateral cuts stimulated the release of GH by primary cultures of rat anterior pituitary cells. Microinjection of 0.05 SME equivalents of SME extract into the anterior pituitary gland of urethane-anaesthetized rats produced a rise in plasma GH levels within 3 min of injection. These data favour the existence of a GH releasing factor, and suggest that the ventromedial and arcuate hypothalamic nuclei are major sites of production of this releasing hormone.

Angiotensin II stimulates the release of ACTH from dispersed rat anterior pituitary cells.

In a rat anterior pituitary dispersed cell system, angiotensin II was found to stimulate the release of ACTH at concentrations ranging from 100 pmol/l to 1 mumol/l, with a maximal response being given by 10 nmol/l. The angiotensin II antagonist, saralasin, was able to block completely at a concentration of 1 mumol/l the stimulation of ACTH induced by 10 nmol/l angiotensin II, but had no effect on the basal release. The log dose-response curve for ACTH release by angiotensin II was shifted to the right in a parallel fashion by saralasin 10 nmol/l, suggesting competitive antagonism. The stimulation of ACTH release by a rat stalk-median eminence extract or by arginine vasopressin was unaffected by saralasin. The data are interpreted as suggesting that rat corticotrophs contain angiotensin II receptors, and that these may be involved in ACTH release in response to hypovolaemic stress.

Ontogenetic development of the inhibition of growth hormone release by somatostatin in the rat: in-vivo and in-vitro (perifusion) study.

An in-vitro study of GH secretion by rat fetal and neonatal pituitary glands was conducted using a perifusion system. After a 2 h period the GH content of the effluent was constant. Theophylline, thyrotrophin releasing hormone (TRH) and rat stalk median emience extract (SME) were effective stimuli of GH release from the pituitary glands of the 19.5-day-old fetuses. Somatostatin, added to the medium (10 microgram/ml), had no inhibitory effect on GH release (basal or stimulated by either theophylline or SME) before day 4 after birth. After postnatal day 5, somatostatin always inhibited GH secretion. These findings were consistent with the results of experiments in vivo. In rats tested within 4 days of birth, sodium pentobarbitone-stimulated plasma GH levels were not reduced by somatostatin; on day 4 and thereafter somatostatin depressed the response to pentobarbitone injection. These results indicate a postnatal maturation of the regulation of GH release by the hypothalamo-hypophysial system in the rat.

Corticoliberin activity of rat neurohypophysis is distinct from vasopressin.

Electrical stimulation of the neural lobe of the pituitary resulted in an increase of corticosterone secretion in both normal and Brattleboro rats. Bioassaying the corticoliberin (CRF) activity of stalk-median eminence and neural lobe extracts obtained from normal and Brattleboro rats revealed that the endogenous vasopressin was not a prerequisite of ACTH-releasing potency. Arginine-8-vasopressin failed to potentiate the CRF activity of the different extracts. These data suggest that a nonvasopressin substance(s) with CRF activity can be released from the neurohypophysis of the rat, and it may contribute to activating the pituitary-adrenal axis under certain experimental conditions.

Effects of paraventricular lesions on stimulated ACTH release and CRF in stalk-median eminence of the rat.

The effects of destroying the paraventricular nucleus (PVN) of the rat hypothalamus on pituitary-adrenal function were studied. Four days after PVN lesions were placed with a rotating knife, the basal plasma corticosterone level was normal, but the corticosterone response to electrical stimulation of the medial basal hypothalamus, surgical trauma, and ether-venesection stress was significantly inhibited. Four and 8 days after PVN lesioning and adrenalectomy, the basal plasma ACTH level was lower, and the rise of plasma ACTH level elicited by a 3-min ether inhalation was significantly smaller than in the adrenalectomized controls. Corticotropin-releasing factor (CRF) activity in the stalk-median eminence extracts from PVN-lesioned rats was significantly less than in the control extracts. The weight of the adrenals was decreased by both 2 and 4 wk after PVN destruction, and 2 wk after hemiadrenalectomy, the compensatory adrenal hypertrophy was inhibited. The plasma corticosterone response to ether-venesection stress was inhibited only temporarily because it returned to normal by the end of the 4th postoperative week. The results are consistent with the hypothesis that a substantial portion of CRF-containing fibers in the stalk-median eminence region either originate from or run though the PVN or its immediate vicinity.

Effects of treatment with adrenocorticotrophin on the hypothalamo-pituitary-adrenal system.

Long-term treatment with adrenocorticotrophin (ACTH) inhibited the stress-induced response of the hypophysial-adrenocortical system 24 h after the final ACTH injection. The mechanism of this phenomenon was studied in both normal and adrenalectomized rats, the latter receiving corticosterone at various doses. The effect of electrical stimulation of the medial basal hypothalamus on the concentration of corticosterone in plasma (an indicator of ACTH secretion), the corticotrophin releasing factor (CRF) content of the stalk median eminence (SME), the ACTH content of the pituitary gland and the in-vitro release of ACTH by the pituitary gland incubated with or without addition of SME extract were investigated. Electrical stimulation of the medial basal hypothalamus failed to induce a rise in concentrations of corticosterone in plasma of normal rats treated with ACTH; moreover the levels of hypothalamic CRF and hypophysial ACTH were significantly decreased. Hemipituitary glands of ACTH-treated rats released markedly less ACTH in vitro in response to SME extract than did the control glands. This indicated that long-term hormone administration caused a serious impairment of the responsiveness of the corticotrophic cells toward CRF. Pituitary ACTH content and in-vitro responsiveness of pituitary glands obtained from ACTH-treated, adrenalectomized rats receiving corticosterone replacement seemed to be dependent on the amount of exogenous corticosteroid, but not on that of exogenous ACTH. Our previous and present findings suggest that long-term treatment with ACTH elicits repeatedly increased secretion of endogenous corticosterone, impairing the stress-induced CRF-ACTH release at both the hypothalamic and hypophysial levels. Our data challenge the view that ACTH itself is able to inhibit its own secretion.

Secretion of ACTH, LPH and beta-endophin from human pituitary tumours in vitro.

Basal and stimulated secretion of immunoreactive ACTH, LPH and beta-endorphin from four human pituitary tumours has been studied in vitro using a superfused, isolated cell system. Chromatography of cell secretions under acid-dissociating conditions demonstrated that the human tumor cells released immunoreactive peptides with the elution profiles of alpha h (1-39) ACTH, beta h-LPH, gamma h-LPH and beta h-endorphin confirming that beta h-endorphin is secreted by human pituitary tumour cells and is not formed by enzymic cleavage from beta h-LPH in blood. No alpha- or beta h-MSH, nor any higher molecular weight forms of ACTH or LPH were detected. The cells from all four tumours responded to stimulation with rat stalk-median eminence extract (SME) and synthetic AVP with a concomitant release of ACTH, beta-LPH, gamma-LPH and beta-endorphin. In contrast to the isolated rat anterior pituitary cells, the pattern of responses to SME and AVP were indistinguishable and the release provoked by rat SME could be accounted for virtually entirely by its vasopressin content. No stimulation of release was observed when the cells were exposed to a variety of biogenic amines. Addition of hydrocortisone to the perfusion buffer of two tumours resulted in a slow inhibition of both basal and stimulated ACTH and LPH release. These data demonstrate that human pituitary tumour tissue from patients with Cushing's disease and Nelson's syndrome can be studied in vitro and that such studies may contribute to a greater understanding of the aetiology of these diseases.

Adrenocorticotropin and lipotropin secretion by dispersed cell cultures of a human corticotropic adenoma: effect of hypothalamic extract, arginine vasopressin, hydrocortisone, and serotonin.

Basal and modulated secretion of ACTH and lipotropin (LPH) by cultures of trypsin-dispersed cells of a biopsy of a human corticotropic adenoma have been examined. ACTH secretion was detectable throughout the period of culture (13 days) but declined steadily from an initial production rate of 238 +/- 124 ng/3 X 10(5) cells/12 h. The time course of secretion showed a slower phase over the first 4 h, with increases up to 12 h. An extract of rat stalk median eminence caused a significant (P less than 0.005) dose-dependent increase in both ACTH and LPH secretion during 30 min. The patterns of response for ACTH and LPH were very similar; both exhibited a decline in the basal release of peptide subsequent to the period of stimulation. The addition of hydrocortisone (0.2 micrograms/ml) did not suppress basal ACTH secretion during 30 min but significantly (P less than 0.05) inhibited stimulation produced by rat stalk median eminence extract. Arginine vasopressin (dose range, 1-9 ng/ml) significantly (P less than 0.025) stimulated both ACTH and LPH secretion during 30 min. The patterns of response were again very similar. Serotonin (dose range, 0.01-10 micrograms/ml) did not affect ACTH secretion during incubations of 30 min to 4 h. The results obtained with the cell cultures of a human corticotropic cell adenoma concur with in vivo findings of incomplete autonomy of secretion, parallel secretion of ACTH and LPH in response to provocative stimuli, and suppression by corticosteroids. The technique has potential for exploring the cellular mechanisms controlling secretion by human corticotropic adenomas as well as the nature of the hormones produced.

Evidence that the dopaminergic prolactin-inhibiting factor mechanism regulates only the depletion-transformation phase and not the release phase of prolactin secretion during suckling in the rat.

We have compared the effectiveness of stalk-median eminence (SME) extracts, dopamine, and bromocriptine (CB-154) in inhibiting the release of PRL into the plasma during suckling when injected before depletion with that when the injections were given after the pituitary stores of PRL had been depleted and transformed by 10 min of suckling. We found that a single injection of either neutralized acidic extracts of SME at a dose of one or two SME equivalents per rat or 625 ng dopamine effectively prevented both the depletion of PRL within the pituitary and the subsequent rise in plasma PRL when given 1-3 min before the onset of suckling. Likewise, a single injection of bromocriptine (0.5 mg) given 20-30 min before the onset of suckling totally blocked pituitary depletion and the subsequent release of PRL into the circulation. However, the PRL concentration rose normally in the plasma in response to suckling when either SME extract or bromocriptine was given after PRL depletion had first been effected by prior short term (10 min) suckling. These data indicate that the depletion-transformation phase but not the release phase (into the circulation) of PRL secretion is under inhibitory control by a dopaminergic PRL-inhibiting factor system.