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Readers are referred to a recent symposium (1) and reviews (2–4) for a comprehensive and broad introduction to somatostatin (SS) and its actions. This chapter will be restricted to the mechanisms (primarily ionic) by which SS inhibits growth hormone (GH) release. The abundant literature on the actions of SS on other cell types will not be discussed unless directly relevant to ionic mechanisms in somatotrophs. We will begin by summarizing the ionic mechanisms by which growth hormone releasing factor (GRF) stimulates the acute release of GH via cyclic AMP (cAMP) and cytosolic free Ca++ ([Ca++]i) as second messengers (Fig. 2.1). This is discussed in more detail elsewhere in this volume.

Acid extracts of a) acutely dispersed rat pars intermedia (PI) cells, b) media after incubation of PI cells, c) whole nervosa-intermedia, and d) whole pars distalis, were chromatographed on Sephadex G-50 Fine in 1% acetic acid. Three peaks of ACTH biological activity were resolved in all four extracts. Peak I eluted in the void volume of the column, peak III co-eluted with synthetic ACTH1–39, and peak II eluted in an intermediate position. The predominant ACTH activity derived from the PI tissue was peak I, amounting to over 70% of the total ACTH activity present in that lobe. The positions of PI peaks I and II remained unaltered after rechromatography as well as after treatment with and chromatography in 8 M urea. However, peak I of PI ACTH was further resolved into two separate peaks by chromatography on Sephadex G-100 SF. Thus pars intermedia ACTH activity appears to be composed of four separate entities, with the predominant forms being larger than ACTH1–39.

Studies were carried out to further define the sites of interaction of Ca++, cAMP, and somatostatin (SRIF) in the mechanisms governing GH release from acutely dispersed purified somatotrophs obtained from rat adenohypophyses. Both high [K+] and the Ca++ ionophore A23187 stimulated the release of GH. The release induced by high [K+] was accompanied by a small but significant transient increase in intracellular cAMP, while A23187 did not alter basal cAMP levels. The augmented release of GH induced by both secretagogues was blocked by SRIF (1 ng/ml) and by removing Ca++ from the incubation medium (85 microM). The transient increase in cAMP induced by high [K+] was not blocked by SRIF or by low Ca++ incubation. These results are consistent with a model whereby an increase in free cytosol Ca++ will result in GH release by exocytosis. This increase in free cytosol Ca++ can come from either intracellular bound Ca++ or from the extracellular compartment. Secretagogues which act via cAMP increase intracellular cAMP levels. The increase in cAMP would then stimulate the translocation of Ca++ from a bound to a free cytosol form. Secretagogues may also stimulate GH release by bypassing cAMP to increase free cytosol Ca++ by directly increasing the influx of Ca++ into the cells or by increasing the intracellular movement of Ca++ to the free cytosol compartment. SRIF would block GH release by acting at the level of the plasma membrane to block Ca++ influx and by inhibiting a step(s) subsequent to the increase in cytosol Ca++.

The exposure of rat adenohypophysial tissue to iced media for periods of 30–60 min causes accumulation of Ca2+ by the tissue and an increased "basal" release of growth hormone into the media. The Ca2+ permeability following the chill, estimated from the uptake of 45Ca2+ by the tissue at 37 °C, is unchanged by the exposure to iced media. This suggests that the accumulation of Ca2+, which occurs during the chill, may be due simply to decreased efflux of Ca2+. The insensitivity of 45Ca2+ influx to the increased cellular content is readily explained if Ca2+ is rapidly sequestered after it has entered the cell. Our previous investigations snowed an increase in 45Ca2+ uptake (permeability) associated with hormone release which had been induced by elevated K+ or by partially purified releasing factor. We used tissue that had been chilled during collection. Our studies here indicate that our previous observations of increased uptake are qualitatively correct despite the cellular accumulation of Ca2+ that must have occurred before exposure to the secretagogues. The release of hormone seems to be related to the absolute cytoplasmic concentration of Ca2+ and release will occur in any circumstance that increases this concentration. The source of Ca2+ is not critical.

The presence of immunoreactive α melanocyte stimulating hormone (IαMSH) was investigated in both mucosal and muscular layers of the various areas of the gastrointestinal tract. IαMSH was present in both layers in all areas of the gastrointestinal tract but the esophageal mucosa and muscularis in saline extracts. The highest concentrations were found in the duodenum. Hypophysectomized males tended to have higher content than intact males. There was no difference between intact estrogen-primed females and hypophysectomized females up to 1 month post hypophysectomy in any area. The tract of 3 month hypophysectomized females showed lower levels than the intact estrogen-primed females in 5 areas; however, in similar groups of 3 month hypophysectomized females which were estrogen primed, 8 of the 10 areas contained more IαMSH than the intact estrogen-primed females. Acid extracts from female rats during the estrous cycle showed no cycle-dependent differences. Comparison of acid and saline extracts showed an absence of IαMSH in gastric tissues and a decrease in the duodenal muscularis in acid extracts but no consistent differences were found in other areas. These results suggest that the IαMSH found in the gastrointestinal tract is not of pituitary origin but may be produced in the gastrointestinal tract. The induction of increased content by estrogen priming in hypophysectomized rats suggests that estrogen priming may induce production. The absence of IαMSH in acid extracts of the stomach suggests that a difference in distribution of pro-opio-cortin products may exist in the gastrointestinal tract.

We previously reported methods for the preparation of enriched and purified dispersed somatotrophs from the rat adenohypophysis. We now demonstrate that the somatotrophs retain (a) their GH content during the purification proceduresand (b) their responsiveness to dibutyryl cyclic AMP and to a partially purified hypothalamic extract, rich in GHRF (GH-releasing factor) activity. This dispersed somatotroph preparation may provide a novel and useful tool in the study of the mechanisms governing the synthesis and release of GH. (Endocrinology 94: 1525, 1974)

cAMP is thought to be an essential intracellular mediator in the release of GH from somatotrophs. Ca++ is required in the incubation medium to elicit GH release iin vitro. We have carried out studies using a purified preparation of rat somatotrophs to see whether the Ca++ requirement precedes or follows the accumulation of cAMP induced by prostaglandin E2 (which increases adenylate cyclase activity) and 3-isobutyl-lmethylxanthine (a phosphodiesterase inhibitor). Incubation of somatotrophs in low Ca++ medium (

Gonadotrophic and ACTH activity were assayed in a segment of the pituitary rich in peripheral gonadotrophs (delta cells) and in an area almost devoid of this type of cell. The region rich in delta cells was found to contain three times the gonadotrophic activity and one-third the ACTH activity of the region poor in delta cells. Thus the delta cells are the site of gonadotrophin and not of ACTH production.

Adenohypophyses were incubated in Krebs-Ringer bicarbonate and the release of ACTH was measured under various conditions. A 5-fold increase in K+ augmented ACTH release. This augmented release was significantly greater from adenohypophyses of adrenalectomized rats than from those of intact animals and was prevented when Ca++ was removed from, or corticosterone was added to, the incubating medium. The increased release of ACTH in the high K+ medium and its prevention when Ca++ was removed were reversible and repeatable. These findings are consistent with the model of “excitation- secretion coupling” which has been proposed to describe the process of hormone release. These findings also suggest that corticosterone may operate directly on releasing mechanisms, possibly in the adenohypophysial cell membrane, to inhibit ACTH release. (Endocrinology 85: 1144, 1969)

Changes in adenohypophyseal DNA and RNA content were determined as a function of time following adrenalectomy, sham adrenalectomy, and the chronic injection of cortisol. The stess of operation resulted in a transient increase in adenohypophyseal nucleic acid content, whereas the increase in secretion of ACTH induced by feedback control did not. An attempt is made to correlate the changes in nucleic acid content with the concurrent changes in the intensity of biosynthesis of ACTH.

Control, adrenalectomized, and cortisol-treated rats were maintained under rigidly controlled conditions, and the adenohypophyses were examined histologically with two staining procedures which differentiate six distinct cell types. Only one cell type demonstrated cytological evidence of increased synthetic activity 32 days after adrenalectomy (the changes were, however, minimal) and decreased synthetic activity following the chronic injection of cortisol. This cell type, which we designate as the corticotroph, would be classed as a chromophobe (no stainable granules) with use of standard histological techniques, but is, in fact, as Herlant's Tetrachrome demonstrates, a distinct acidophilic cell type different from the prolactin cell and the somatotroph. The determination of adenohypophyseal DNA and RNA revealed no evidence of increased protein synthetic activity following bilateral adrenalectomy, but did reveal evidence of decreased protein synthetic activity following the chronic injection of cortisol.

Measurements of transmembrane potential (Vm) were obtained in 117 cells from the adenohypophyses of 14 rats in vitro when the glands were superfused with Krebs—Ringer bicarbonate (KRB). The mean Vm was -12.0 mV ±.4 SE, in agreement with previous in vitro findings. No positive Vm's were observed. Vm and R (resistance) of individual cells were recorded while the superfusion fluid was changed. A depolarization of the cells of the adenohypophysis was demonstrated in an elevated [K+]o medium (5x[K+]0 KRB). This depolarization appeared to be dependent upon the calcium ion concentration of the medium. The percent depolarization averaged 37.3% ± 3.0 SE for 23 cells in 5x[K+]0 –normal [Ca++]0 KRB, but was significantly smaller (p less than 0.05) in 5x[K+]o– [Ca++]0 free KRB, averaging 28%±2.3 SE for 19 cells. A small but significant depolarization (24.5% ± 3.5 SE, 20 cells) was demonstrated when the normal medium (KRB) was replaced with a calcium—free medium (0[Ca++]o KRB). No change in membrane resistance from co...

6α-meth}d-17α-hydroxyprogesterone acetate (6α-MAP) was injected daily to male and female rats from the first postnatal days to maturity. The degree of atrophy and the histological change in the adrenal glands and in the gonads were similar to those reported late after hypophysectomy. The morphological and functional recovery of these glands was extremely slow after the cessation of the injections. The underdeveloped glands, however, reacted promptly to their trophic hormones. The characteristic change in the pituitary was the complete absence of delta cells or gonadotrophs.

A sensitive and specific radioimmunoassay for LH-releasing hormone (LHRH) was developed. Using a specific antibody we have attempted to define or dissociate a separate FSHRH, antigenically distinct from LHRH. In an in vitro system, LH release by hypothalamic extract was inhibited by a certain dose of LHRH antiserum but FSH release was not affected. Diurnal patterns of LHRH, FSH and prolactin were studied but no clear cyclic changes were shown. LHRH and LH levels in the serum were completely dissociated. We suggest that negative feedback systems play a more critical role than hypothalamic LHRH in the release of LH.

Elevation of [K+] in the incubation media to 24.9 mM increased the in vitro uptake of Ca++ by the rat adenohypophysis as measured by 45Ca. There was no change in the extracellular space as measured with tritiated mannitol. The addition to the incubation media of other materials which also provoke the release of the various adenohypophysial hormones had no effect upon Ca++ uptake. The materials added were a crude acid extract of rat hypothalamus-stalk-median eminence, synthetic lysine vasopressin, dibutyryl cyclic AMP, or theophylline. In each case the concentration employed was sufficient to cause release of at least 2 of the adenohypophysial hormones. Thus, in this tissue, only the hormone release produced by elevated K+ is consistent with the “stimulussecretion coupling” model. Perhaps only the presence of Ca++ and not an increased uptake of Ca++ is required for the release of preformed hormone from the adenohypophysis. (Endocrinology 89: 766, 1971)

The part played by adrenaline and noradrenaline in the adrenal cortical response to insulin-induced hypoglycaemia was investigated. Both exogenously administered adrenaline and noradrenaline produced an increase in plasma corticosterone. This response was suppressed by pretreatment with the adrenergic blocking agent, Dibenzyline. The increase in plasma corticosterone produced by Protamine Zinc Insulin-induced hypoglycaemia was unchanged by previous adrenal demedullation or pretreatment with Dibenzyline. Thus neither medullary nor extra medullary adrenaline and/or noradrenaline play an essential mediating role in the adrenal cortical response to insulin-induced hypoglycaemia.

A purified extract of sheep hypothalamus possessing growth hormone releasing factor activity caused an increased uptake of 45Ca from the incubation medium into the rat adenohypophysis concurrent with an increased release of growth hormone. The data are consistent with the "stimulussecretion coupling" model proposed for the release of preformed hormone from the adenohypophysis.

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.

1. The in vitro uptake of (22)Na and (42)K was measured simultaneously in rat adenohypophyses during hormone release produced by several secretagogues and during inhibition of hormone release in Ca-free media.2. Intracellular adenohypophysial [Na(+)] and [K(+)] changed only slightly when the uptake changed. This would indicate that relative permeability changes were the primary effect of the treatments.3. The uptake of (42)K was increased by elevated external [K(+)], but was unaffected by the presence or absence of Ca(2+). Acid extracts of hypothalamus-stalk-median eminence or cerebellum also increased the (42)K uptake.4. The uptake of (22)Na or (24)Na was decreased by elevated [K(+)]. Uptake was increased in Ca-free Krebs-Ringer bicarbonate; but was unaltered when [K(+)] was concurrently increased.5. Neither purified growth hormone releasing hormone, synthetic lysine-vasopressin, dibutyryl cyclic AMP nor theophylline had an effect on the uptake of either K(+) or Na(+).6. The rapid uptake of (22)Na and its smaller volume of distribution compared to absolute measurements of intracellular [Na(+)] suggest that the plasma membrane of adenohypophysial cells is relatively impermeable to Na(+).7. We conclude that changes in the uptake of Na(+) and K(+) associated with hormone release are incidental to the release process.8. Hormone release produced by elevated external [K(+)] is most likely due to a non-specific increase in permeability of the cell membranes, facilitating Ca(2+) entry into the cytoplasm.9. The results suggest that the low resting transmembrane potentials of adenohypophysial cells may be due to their conjoint relatively high permeability to both K(+) and Ca(2+), rather than K(+) and Na(+).

The changes in pituitary beta and delta cells (basophiles) and/the state of thyroid activity and the potentiality to secrete TSH after treatment with 6 alpha-methyl-17-alpha-hydroxy-progesterone acetate (6 alpha-MAP) were studied in albino rats. Thyroid weight cell height and weight per 100 mg of body weight were not consistently or markedly altered after 110 days of 6 alpha-MAP treatment. A propothiouracil diet elicited increased thyroid activity in both the control and 6 alpha-MAP treated animals. Pituitary beta cells (thyrotrophs) were unaffected by treatment.

In 28 6-h experiments on 10 conscious resting trained male dogs, plasma growth hormone (GH) was determined at 5-min intervals by radioimmunoassay. For all experiments, the basal GH concentration in plasma was 0.80 ± 0.06 ng mL−1. In each experiment, 1–3 secretory bursts of GH occurred, raising plasma GH 2.4 to 15.3 times basal concentrations (for all 43 bursts, 6.6 ± 0.4 times the basal value). Metabolic clearance rates (MCR) and apparent distribution volumes (V) were determined, using stepwise infusions of canine GH. The MCR (3.99 ± 0.30 mL kg−1 min−1) and V (57.9 ± 5.5 mL kg−1) were used to transform the GH concentration versus time data into GH secretion rates, using a single compartment approach. Basal GH secretion rates for all 28 experiments were 3.12 ± 0.24 ng kg−1 min−1. The secretory bursts yield peak GH secretion rates of 9.4 ± 0.8 times basal secretion and these steep-sloped bursts last 25.1 ± 1.2 min. Six-hour infusions of 0.15 μg kg−1 min−1 of somatostatin (SRIF) abolished all secretory bursts but did not lower basal secretion rates. In five of seven SRIF infusion experiments in which samples were taken after the infusion ceased a secretory burst was seen in the hour following cessation of infusion (in four cases within 10 min). These secretory bursts lasted 23.0 ± 2.9 min and were similar to those seen in control experiments. Infusions of SRIF at 0.05 μg kg−1 min−1 had no effect. These results imply that during basal GH secretion, a surfeit of SRIF impinges on the somatotrophs, as extra SRIF does not further lower basal secretion. However, during secretory bursts, very little SRIF must be present, as exogenous SRIF blocks these bursts. The bursts are similar in duration to overshoots provoked in perifused dispersed rat somatotrophs by removal of an SRIF signal. It seems likely that their cause in vivo is similar. (All values are means ± SEM.)

Sheep hypothalamic extracts, chromatographed on Sephadex G-25, revealed 3 peaks with CRF biological activity. The first eluting peak co-chromatographed with ACTH. After separation from ACTH by CMC, this CRF peak contained vasopressin immunoactivity and pressor activity. The second eluting CRF peak also contained vasopressin immunoactivity and pressor activity and was found to represent the presumed authentic vasopressin. The third eluting CRF peak contained vasopressin immunoactivity but no pressor activity. Further processing of this putative CRF on Sephadex G-10 resulted in a 500-fold purification. The material is inactivated by proteolytic enzymes. We tentatively conclude that this CRF is a peptide with a molecular weight of 700–1000 and that it shares common antigenic determinants with vasopressin.

The problems inherent in the study of the control of adenohypophyseal hormone secretion in vitro using intact tissue or tissue fragments are fourfold: variability of response, lack of viability of tissue in the gland core, relative lack of sensitivity, and the heterogeneity of cell types within the gland, which precludes interpretation of intracellular metabolic events within a specific cell type. Several investigators have used acutely dispersed or cultured adenohypophyseal cells to examine the effects of hypothalamic regulatory hormones as described in recent reviews (Labrie et al., 1976b; Vale et al., 1976). These preparations overcome the problems of variability, viability, and sensitivity associated with the classic whole or hemipituitary studies. However, the difficulties arising from the heterogeneity of the cell type remain. It is not possible, using presently available preparations, save for the use of autoradiographic techniques, to localize alterations of intracellular metabolite involved in the release of one hormone to a specific adenohypophyseal cell type. In light of much evidence showing a lack of specificity of several of the hypothalamic hypophysiotropic hormones (Labrie et al., 1976a,b; Vale et al., 1976), it has become imperative to study a uniform cell population. Cloned tumor cell lines have been used in an attempt to overcome this problem (Tashjian et al., 1968; Hertelendy and Keay, 1974; Dannies et al., 1976), but there is no assurance that intracellular events are not grossly altered in tumor cells.

The parenchymal cells of the pars intermedia (PI) and corticotrophs of the pars distalis (PD) synthesize pro-opiomelanocortin (POMC), which, through posttranslational processing, gives rise to a group of structurally related peptides, including MSHs, ACTH, CLIP, LPHs and endorphins. We investigated the control of release of these peptides using an in vitro system. We perifused either intact neurointermediate lobes (NI) or PD halves obtained from rats. Perifusion medium and tissue extracts were subjected to a battery of bioassays (BA) and radioimmunoassays (RIA) (including MSH-BA, alpha-MSH-RIA, ACTH-BA, ACTH-RIA, LPH-RIA) and a receptor-binding assay for morphine-like activity (MLA). The relative amounts of released peptide activities were examined under basal conditions and after challenging with synthetic ovine corticotrophin-releasing factor (CRF) and somatostatin. CRF stimulated the release of all assayed peptides from both the PD and PI in a dose-related manner. Stimulated release was immediate (within 3 min), constant, reversible and repeatable. Somatostatin (up to 100 ng/ml) did not alter basal release from either PD or PI. Somatostatin did block CRF-induced release from the PI but not from the PD. These observations support an action of both CRF and somatostatin in the control of secretion of POMC-derived peptides from the PI.

In order to define the roles of intracellular Ca++ and cAMP in the mechanisms governing GH release, we have carried out perifusion studies using a purified preparation of acutely dispersed somatotrophs obtained from rat adenohypophyses. Two groups of secretagogues were used: those that act by increasing intracellular cAMP [(Bu)2cAMP], the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, and prostaglandin E2; and those thought to act primarily by increasing Ca++ influx into the cells (high extracellular K+ and the Ca++ ionophore A23187). The release of GH induced by the three secretagogues that increase cAMP levels is instantaneous and maintained during perifusion, whereas the release induced by high K+ and A23187, unlike the cAMP-induced release, reaches a peak within 2 min and then falls rapidly back to baseline levels, even though the secretagogues are still present. After GH release has returned to baseline levels but while high K+ is maintained, the somatotrophs respond briskly to (Bu)2cAMP, 3...

The purpose of this study was to ascertain whether the pars intermedia of the rat adenohypophysis, isolated from direct innervation via the infundibular stem, could maintain adrenal cortical weight and plasma corticosterone levels. We compared the adrenal cortical response of rats 40 days after either complete hypophysectomy, hypophysectomy with reinsertion of only the pars distalis, or hypophysectomy with reinsertion of only the nervosa-intermedia. Adrenal weight and plasma corticosterone levels were partially maintained in the group with reinserted pars distalis. These parameters were not different from the complete hypophysectomy group in the animals with reinserted nervosa-intermedia. Thus, the pars intermedia, with its nerve supply disrupted, cannot maintain adrenal cortical function.

Studies were carried out to simultaneously measure cAMP and cGMP accumulation and GH release from acutely dispersed purified somatotrophs obtained from rat adenohypophyses. cAMP accumulation was dramatically increased by both prostaglandin E2 (10(-6) M) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor, 0.5 mM) within 1 min of their addition, while there was a delay of 8--16 min before a significant increase in GH release was seen. SRIF (100, 10, or 1 ng/ml) completely blocked the stimulated release of GH. SRIF also consistently decreased the elevation of cAMP induced by the two secretagogues, but this decrease was small and not always significant. cGMP was unmeasurable (less than 0.02 fmol/1000 cells) in all of our experiments, while basal cAMP levels were about 1 fmol/1000 cells. We conclude that cAMP plays a role in the intracellular mechanisms governing GH release and that SRIF primarily acts subsequent to cAMP elevation, with a possible secondard or minor action on cAMP formation.

An acutely dispersed and purified preparation of somatotrophs obtained from rat adenohypophyses was used to study the mechanism of action of GH-releasing factor (GRF). Synthetic GRF [human pancreatic, hpGRF-(1–40)-OH] stimulated the immediate (within 4 min) release of GH in a dose-related manner, with a preceding or concurrent increase in cAMP in the somatotrophs. Somatostatin, at concentrations as low as 1.0 ng/ml, completely blocked the GRF-induced increase i n GH release, with only a partial reduction in the GRF-induced accumulation of cAMP in the somatotrophs. 3-Isobutyl-l-meth-ylxanthine, a phosphodiesterase inhibitor, potentiated the action of GRF in increasing cAMP in the somatotrophs, with subsequent GH release. These results along with those of previous studies suggest that cAMP is an intracellular mediator in the action of GRF and that somatostatin has a major effect on blocking GH release at a step subsequent to cAMP accumulation. (Endocrinology 117: 2364–2370, 1985)

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...

We compared adrenals from rats obtained from six suppliers in the in vitro ACTH assay. The slope of the response of one strain (Canadian Breeding Laboratories, Sprague–Dawley) was consistently greater and the adrenals were consistently more sensitive than those from the other strains tested.

Incubation of rat adenohypophyses in potassium ion of sufficient concentration to provoke the release of several of the adenohypophysial trophic hormones produces a reversed, positive transmembrane potential in more than half the cells. This finding is consistent with a process of "stimulus-secretion coupling" in which hypothalamic releasing factors act by selective depolarization of their "target" cells. The positive potentials may be due to a prolonged preferential permeability to calcium ions triggered by an initial depolarization of the cell membrane to a threshold value by increased external potassium ion.

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)

Incubation of rat adenohypophyses in a high concentration of potassium increases adrenocorticotropic hormone release. This increased release is suppressed by the addition of corticosterone to the incubating medium. Our findings are consistent with a process of "stimulus-secretion coupling" proposed for other glands and suggest that corticosterone may operate directly on the adenohypophysial cell membrane to inhibit releasing mechanisms.

In order to identify the cell type responsible for ACTH production in the rat adenohypophysis, the incorporation of thymidine- 3H into the various cell types of the adenohypophysis has been studied using autoradiography. Sections were examined 1 month following adrenalectomy, when ACTH secretion is greatly increased, and 1 month following the daily administration of cortisol, when ACTH secretion is suppressed. Only 1 cell type, restricted to the pars intermedia, responded to altered levels of ACTH secretion with both an increase in label index following adrenalectomy and a decrease in label index following cortisol administration. Histological changes were restricted to the pars intermedia, where numerous dark cells were observed only after adrenalectomy. Following adrenalectomy, increases in weight were restricted to the colloid and the pars intermedia. Since no changes in plasma or hypophysial MSH content were found, the histological and autoradiographic changes do not simply reflect alterations in MSH ...

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)

A single subcutaneous insertion will stimulate the secretion of corticosterone. Rats pretreated by repeated daily subcutaneous needle insertions can be subsequently injected without the injection itself causing an increase in plasma corticosterone. This conditioning procedure will allow the study of the effect of parenterally administered materials in animals without the injection per se imposing an acute release of adenohypophyseal ACTH and of adrenal corticosteroids.