Hypothalamic Stalk Median Eminence Research Articles

Effect of Photoperiod and Morphine on Plasma Prolactin Concentrations and Thyrotropin-Releasing Hormone Secretion in the Ewe

This study investigated the effects of daily photoperiod length and morphine on thyrotropin-releasing hormone (TRH) and prolactin secretion in sheep. Two groups of adult ewes were kept under either 16 h L:8 h D or 8 h L:16 h D photoperiods for approximately 60 days. Then the photoperiods were reversed and approximately 60 days later push-pull cannulae were implanted into the hypothalamic stalk-median eminence. After a 7-day recovery period, the ewes were subjected to hypothalamic perfusion and blood was collected at 15-min intervals from the jugular vein. Perfusates also were collected into 15-min fractions. The first 3 h of perfusion served as an equilibrium period. During the next 2 h, saline was infused into one jugular vein. This was followed by a 2-hour morphine infusion (1 mg.kg-1.h-1). Data from 13 ewes were analyzed for effect of photoperiod and drug on TRH concentrations in the perfusates and prolactin in the serum. Prolactin was significantly (p less than 0.01) higher under 16 h L:8 h D than 8 h L:16 h D and was greatly increased (p less than 0.001) by morphine infusion. TRH only tended to be higher (p = 0.05) under 16 h L:8 h D than under 8 h L:16 h D, but morphine infusion induced a rapid and highly significant (p less than 0.01) increase in TRH secretion. There were no photoperiod and drug interactions for either TRH or prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The effect of cholecystokinin octapeptide on pituitary-adrenal hormone secretion

The purpose of this investigation was to determine the influence of cholecystokinin octapeptide (CCK-OP) on pituitary-adrenal hormone secretion. CCK-OP at a dose of 5 μg/kg (i.p.) elevated plasma corticosterone from 27 to 43 μg/100 ml in one experiment and from 12 to 50 μg/100 ml in a second experiment: Lower doses of CCK-OP (0.5 μg/kg) elevated corticosterone from 12 μg/100 ml to 20 μg/100 ml. CCK-OP (1, 10, and 100 ng/ml) had no effect on ACTH-induced corticosterone released by isolated adrenal cells in vitro when tested in the presence of 50 pg of ACTH 1−24. 100 and 500 ng of CCK-OP resulted in an increased pituitary ACTH release equal to 123% (n.s.) and a 206% ( P < 0.05) of control, respectively. In comparison, a 3 5 hypothalamic stalk median eminence equivalent increased ACTH release to 313% of control ( P < 0.05). The exact mechanism of this CCK effect on pituitary ACTH release is unknown. Although it is likely that the direct effects on the pituitary in vitro represent a pharmacologic and not a physiologic effect of this peptide, in vivo doses are between doses used for pancreatic effects and satiety effects suggesting that there may be a physiologic stimulating action of this peptide on the hypothalamic-pituitary-adrenal axis but at a level above the adrenal and pituitary.

&lt;i&gt;In vitro &lt;/i&gt;Release of ACTH from Dispersed Rat Pars intermedia Cells. III. Multiple Forms of ACTH Biological Activity

We have reported that both an extract of rat hypothalamus-stalk-median eminence (HE) and 5-hydroxytryptamine (5-HT) will stimulate the release of ACTH biological activity from acutely, non-enzymically dispersed rat pars intermedia (PI) cells. We have also found that the ACTH activity within the PI cells is composed of 4 chromatographically separable entities, but only a small proportion of the total ACTH activity co-elutes with ACTH1-39. In the present study we subjected the media, after incubation of PI cells with either HE or 5-HT, to sequential chromatography on Sephadex G-50 F and G-100 SF. We found the same 4 ACTH-like moieties as in the PI cell extracts. Only the release of the 2 earliest eluting components, presumably of much larger molecular dimensions than ACTH1-39, was enhanced by either HE or 5-HT. There was no augmented release of the ACTH component having molecular dimensions similar to ACTH1-39. Thus, in our in vitro system, the ACTH biological activity released from PI cells is not the size of ACTH1-39, but is composed of at least 2 much larger molecules which possess ACTH (as well as some MSH) biological activity.

Control of Bioactive Corticotropin Release from the Neuro-Intermediate Lobe of the Rat Pituitaryin Vitro

The purpose of this study was to identify agents capable of regulating the release of biologically active ACTH from the isolated neuro-intermediate lobe of the rat pituitary. Agents found to be potent secretagogues included acetylcholine (100 mug/ml), hypothalamic stalk-median eminence extract (0.33 eq), arginine antidiuretic hormone (100 mU/ml) and serotonin (100 mug/ml). Lower doses of arginine antidiuretic hormone (5.5 mU/ml) and serotonin (2 mug/ml) were ineffective. Dopamine 2 and 5 mug/ml) inhibited the release of biologically active ACTH whereas norepinephrine (5 mug/ml) did not. Dexamethasone (0.25 mug/ml) did not alter the basal or stimulated release of ACTH from the isolated neuro-intermediate lobe in contrast to its effect on ACTH release from the isolated anterior pituitary. Similarly, the tripeptide, prolyl-leucyl-glycinamide, which has been reported by some to inhibit MSH release, had no effect on either the basal or stimulated release of ACTH. The data suggest that regulation of ACTH release from the neuro-intermediate lobe in vivo may involve both stimulatory (acetylcholine) and inhibitory (dopamine) inputs.

Involvement of intracellular calcium in hormone secretion from rat pituitary cells

Cells were dissociated from normal rat pituitaries by a combination of mechanical agitation and enzymatic action, seeded into culture flasks and grown in monolayer culture. When such cells were exposed to an extract of the hypothalamic stalk-median eminence area (HSME) a dosedependent secretion of ACTH was observed. A 2-h exposure to Ca-free media significantly reduced the HSME-stimulated release of ACTH but the measured levels were still greater than the unstimulated controls. When the 45Ca 2+ uptake into the cultured cells was measured both control and HSME-stimulated cells yielded identical results (60–80 nmol Ca/mg cell protein). Upon removal of the calcium associated with the surface coat it was found that HSME actually decreased the cellular uptake of calcium. Since variations in uptake can result from changes in influx or efflux as well as from variations in pool size or turnover times of calcium exchange with intracellular compartments, a series of isotope washout experiments were performed. Neither HSME nor theophylline affected the rate constant of calcium efflux from what is believed to be the cytosol pool to the extracellular media. Both agents, however, prompted a shift of intracellular calcium into a more tightly bound compartment. The data suggest that the calcium required for pituitary hormone secretion is derived primarily from an intracellular rather than extracellular origin. It may be that, via the action of cyclic AMP, such calcium can be mobilized from intracellular stores and shifted to a more tightly bound compartment where it can participate in the intracellular processes associated with secretion.

Effects of Hypothalamic Extract and Corticosterone on Incorporation of &lt;sup&gt;14&lt;/sup&gt;C-Phenylalanine into Newly Synthesized ACTH-Like Protein &lt;i&gt;in vitr&lt;/i&gt;&lt;i&gt;o&lt;/i&gt;

The purpose of the present experiments was firstly, to examine the efficacy of the oxycellulose-Amberlite CG-50 extraction procedure for the isolation of rat pituitary ACTH and secondly, to study the effects of both hypothalamic extract and corticosterone on the synthesis of rat pituitary ACTH. Briefly, 'cold' rat pituitary ACTH was monitored by bioassay in each step of the isolation procedure as a function of corticosterone stimulation in incubated adrenal slices. Labeled ACTH was isolated from rat pituitaries previously incubated in vitro and obtained from adrenalectomized animals and cortisone acetate treated animals to provide further data to substantiate that the labeled isolated protein contained ACTH. Cold ACTH was detected in two purified fractions (oxycellulose and Amberlite CG-50). Adrenalectomy stimulated and cortisone acetate suppressed the incorporation of 14C-phenylalaine into the ACTH-like protein in these two fractions. A continuously increasing uptake of the label into ACTH-like protein in the isolated fractions occurred with time providing evidence that de novo synthesis had taken place. Corticosterone inhibited ACTH synthesis at both 10(-4) and 10(-6) M, whereas, hypothalamic stalk median eminence (HSME) caused a significant, but not dose-related, increase in ACTH synthesis with 3/5, 1, and 2 HSME equivalents added.

Physical characteristics of the Ca++ compartments associated with in vitro ACTH release.

Two separate Ca++ compartments necessary for in vitro release of ACTH from the rat adenohypophysis are revealed by respective mild or vigorous washing procedures with Ca-free media. Vasopressin (500 mU/ml), like elevated [K+], requires the presence of a loosely bound compartment for ACTH release. A crude acid extract of hypothalamus-stalk-median eminence (HSME) or theophylline (10 mM) require a tightly bound compartment, since their potency as ACTH secretagogues is not diminished when the loosely bound compartment is removed. Estimations of the intracellular concentrations of Ca+ + show that incubation in Krebs-Ringer bicarbonate media increases the Ca+ + level as much as threefold from the normal in vivo value of 2.70 mM/kg wet wt. The mild wash procedure produces only a slight reduction in this elevated value. The vigorous wash procedure reduces the internal concentration to about 65% of the in vivo level. We conclude that Ca+ + need not play a critical role in the release process of adenohypophysial ho...

In vitro effect of hypothalamic extract and other stimulating agents on glucose oxidation and luteinizing hormone (LH) release from rat anterior pituitary glands.

Rat hemi-pituitaries were incubated with an ovine hypothalamic stalk-median eminence extract (HE) or its ultrafiltrate (HEUF). LH release was measured by radioimmunoassay (RIA) and glucose oxidation was estimated by measuring 14CO2 derived from 14C-glucose. HEUF produced a dose-dependent LH-releasing effect, but did not alter the total amount of LH in the incubation system. The effect of HE on LH release could not be studied because of the presence of crossreactive contaminants which interfered with the RIA. Both HE and HEUF increased glucose oxidation by anterior pituitary tissue, an effect observed during the first 10 min of incubation. Iodoacetate which severely suppressed glucose oxidation increased basal release of LH and prevented any further stimulation of LH release by HEUF. 2,4-Dinitrophenol which abolished the promotion of glucose oxidation by HEUF caused an increase in the basal LH release and also blocked the LHreleasing effect of HEUF. A high potassium medium (60 HIM) which stimulated LH release also promoted glucose oxidation. Calcium-free medium abolished the effect of high potassium on LH release even though glucose oxidation was increased. Although theophylline enhanced the effect of HEUF and dibutyryl cyclic AMP on LH release, it decreased the stimulating effect of dibutyryl cAMP on glucose oxidation. The LHreleasing effect of HEUF appears to be independent from its effect on glucose oxidation. (Endocrinology90: 690, 1972)

Suppression by corticosterone of adrenocorticotropin release from adenohypophyses incubated in Krebs-Ringer bicarbonate ultrafiltered plasma.

Adenohypophyses were incubated in plasma obtained from adrenalectomized rats and pressure dialyzed against Krebs–Ringer bicarbonate. Corticosterone, in concentrations between 20 and 40 μg/100 ml of ultrafiltered plasma protein, suppressed the augmented release of adrenocorticotropin induced by a crude acid extract of rat hypothalamus–stalk–median eminence. Thus corticosterone, in concentrations and conditions comparable to those found in the circulation, will act directly on the adenohypophysis to decrease its response to corticosterone-releasing factor activity.

Effect of colchicine on in vitro ACTH release induced by HIGH K+ and by hypothalamus-stalk-median eminance extract.

The addition of colchicine (10-5M) to adenohypophyses incubated in Krebs-Ringer bicarbonate suppressed the augmented release of ACTH induced by a 5-fold increase in K+ concentration. However, this concentration of colchicine was without effect on the augmented release induced by the addition of a crude acid extract of rat hypothalamus-stalk-median eminence (HSME). Since colchicine binds to and blocks the contractile microtubular system which might be involved in the translocation of hormone-containing granules within the cell, these findings suggest (a) that this system does not mediate the augmented release of ACTH induced by releasing factor activity and (b) that the mechanism of augmented release of ACTH induced by high K+ differs from that governing the release induced by the HSME extract. (Endocrinology 89:408,1971)

Suppression of ACTH release from adenophypophysis by corticosterone: an in vitro study.

The addition of corticosterone (1 µg/ml) to rat adenohypophyses incubated in KRB depressed spontaneous ACTH release and suppressed the augmented release of ACTH induced by a crude acid extract of rat hypothalamus stalk-median eminence (HSME). Concentrations of corticosterone within the physiological range (0.50 and 0.25 µg/ml) did not depress spontaneous release, but did suppress the HSMEinduced release of ACTH. We conclude that corticosterone, the circulating adrenal glucocorticoid of the rat, will, in physiological concentrations, act directly on the cells of the adenohypophysis to decrease their responsivity to CRF activity. This will allow variation in the rate of secretion of ACTH even with a constant rate of secretion of CRF. (Endocrinology 87: 371, 1970)

Assay of corticotropin-releasing factors by injection into the anterior pituitary of intact rats.

Injection of small volumes (0.5 μl) of test materials were made into the median eminence (ME) or into one lobe of the adenohypophysis (AP) of adult female rats pretreated with dexamethasone + Nembutal + morphine. Test materials included: vehicle solutions; substances likely to affect cell membrane stability or permeability; histamine, epinephrine; vasopressin compounds; crude and partially purified corticotropin-releasing factor from ovine hypothalamic stalk median eminence tissue; and nonspecific control materials. Neither histamine nor epinephrine caused ACTH release after AP or ME injections. In doses that were ineffective intravenously, arginine vasopressin, lysine vasopressin or a modified vasopressin all caused slight ACTH release after AP or ME injections, but the responses from the 2 sites were either equal, or the one in the ME was larger than that in the AP. In contrast, both crude and purified corticotropin-releasing factor (CRF) provoked more ACTH release following AP injection than was observed after ME injections. The slight response to corticotropin-releasing factor following ME injections may have been attributable to spread of the material to the pituitary, since the positive responses to CRF were obtained only in the limited region in which the portal vessels were most concentrated. Although direct pituitary assay of corticotropin-releasing factor activity appears to be feasible and useful, we observed some nonspecific ACTH release following AP injection of large doses of bovine plasma albumin, crude cerebral cortex extract, and crude extract of skeletal muscle. The present assay for CRF defines a substance as having corticotropinreleasing properties if it meets the following criteria: 1) It is more effective in provoking ACTH release (or adrenocortical response) when given into the anterior pituitary than when given into the median eminence of animals treated with dexamethasone, Nembutal or morphine; 2) it is ineffective intravenously at the dose used in the above test; 3) the effective intrapituitary dose is not large (i.e., less than 25 μg). If these criteria are met, the assay is much more specific and about 6 times more sensitive than are intravenous assays for CRF. (Endocrinology84: 544, 1969)

Pituitary growth hormone content and hypothalamic growth hormone releasing activity in neonatal rats after stress.

A remarkable growth hormone releasing (GRF) activity was found in hypothalamic stalk median eminence (SME) extracts of neonatal rats. The application of electric shock to rats of 1, 5, and 10 days of age induced a clear-cut decrease in pituitary growth hormone content. In the shocked animals GRF activity in the hypothalamic SME extracts was significantly reduced. These results indicate that in the very early stages of development all mechanism(s) which bring about growth hormone release are available, and that a response to stressful stimuli may be expected in infant rats as well as in adult rats.