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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.

Dispersed rat lactotrophs were treated with bromocriptine (10−10 M) for either 30 min or 3 h to investigate its effect on cell morphology using light and electron microscopy and ultrastructural morphometry. After 30 min, lactotrophs treated with bromocriptine exhibited an increase in storage granule size (p < 0.05). Crinophagy was also evident and a significant increase in lysosome volume density was observed (p < 0.001). However, no significant change in prolactin content in the media was detected until 3 h of incubation in bromocriptine (p < 0.05). At this time a nonsignificant decrease in Golgi region volume density was also observed.

The exposure of rat adenohypophysial tissue to iced media for brief periods of 2–7 min provokes a biphasic release of growth hormone (GH). An increased release of adrenocorticotropin (ACTH) and prolactin (PRL) is also detected in the "instantaneous" first phase which takes place while the tissue is still cold. No detectable movement of Ca2+ into or out of the cells occurs during this phase and the magnitude of release is unaffected when Ca-free media (45Ca2+ uptake or Ca2+ content. This delayed release is unaffected by Ca-free media. The release of the cytoplasmic marker enzyme, lactic dehydrogenase (LDH), was also measured after a brief chill. LDH release was an order of magnitude less than the release of hormone measured in the same experiment indicating that exocytosis was reponsible for hormone release rather than nonspecific damage to the cells. I conclude that the hormone release following a brief chill does not employ a "stimulus–secretion coupling" mechanism. However, as the release is via exocytosis, some common aspect of the release mechanism beyond the "coupling" stage is activated to provoke the release of at least three adenohypophysial hormones, GH, ACTH, and PRL.

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)

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)

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(+).

Serum and hypothalamic luteinizing hormone releasing hormone (LH-RH) was lowered in young mature male rats after castration. Testosterone injections raised the hypothalamic LH-RH content significantly. The mean value of serum LH level was elevated by testosterone, but not significantly. Hypothalamic LH-RH content was also lowered by hypophysectomy. In this circumstance, testosterone injections significantly increased LH-RH content. These results suggest that there may be a positive feedback of testosterone upon the hypothalamic LH-RH release and synthesis mechanisms.

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.

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...

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.

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)