Monte A. Greer

Regulation of Hormone Secretion by Acute Cell Volume Changes: Ca2+-Independent Hormone Secretion

Exocytosis of intravesicular material should help a cell meet a relative extracellular hyposmotic challenge by expanding the plasmalemma through fusion with vesicular membrane. Cell swelling evokes an immediate secretory burst of hormones stored in secretory vesicles with dynamics indistinguishable from those induced by specific secretagogues. Hormone secretion induced by cell swelling is not associated with a rise in cAMP, IP3, or prostaglandins, and it is not depressed by inhibition of stretch mechano-receptors or aquaporin channels. In contrast to most types of regulated secretion, that induced by cell swelling in normal cells does not require a rise in intracellular Ca2+ through opening L-type Ca2+ channels. However, such Ca2+ influx is essential for cell-swelling induced secretion in tumor-derived pituitary cells. Cell swelling induces universal secretion of exocytotic material. The response of cells specialized in osmoregulation is, however, different. Possible physiological significance: Consistent stimulation of secretion occurs with a 4% hyposmolar challenge. It is likely that fluctuations in osmotic pressure with resultant cell volume changes have a significant regulatory role in hormone secretion. Released hormones could also play an important role in the pathophysiology of ischemia. Exocytosis itself does not have an essential role in volume regulation.

Cell swelling induced by the permeant molecules urea or glycerol induces immediate high amplitude thyrotropin and prolactin secretion by perifused adenohypophyseal cells

The permeant molecules, urea and glycerol, evoked a prompt secretory burst of TSH and PRL when added to the extracellular medium of acutely dispersed anterior pituitary cells. Secretion of both hormones was proportional to the concentration of urea or glycerol between 26 and 104 mM (r greater than 0.89, P less than 0.001). Equivalent concentrations of the impermeant molecule, mannitol, did not induce secretion. The acute TSH and PRL secretory responses to TRH, hyposmolarity, and permeant molecules were qualitatively indistinguishable. These data support our hypothesis that cell swelling and resultant plasmalemma expansion is a potent inducer of hormone secretion. Since the secretory response to permeant molecules was not reduced in a Ca2+-free medium containing 0.1 mM EGTA, an increase in Ca2+ transport across the plasmalemma to raise cytosol Ca2+ concentration does not appear involved.

Evidence that the Major Physiological Role of TRH in the Hypothalamic Paraventricular Nuclei May Be to Regulate the Set-Point for Thyroid Hormone Negative Feedback on the Pituitary Thyrotroph

If a primary physiologic action of TRH is to regulate the set-point for negative feedback, a sudden drop in plasma thyroid hormone concentration should stimulate the same rate of in vivo increase in TSH secretion from normal and TRH-deprived thyrotrophs. To test this hypothesis, 3 experiments were performed in which young adult female rats were divided into 3 groups of 6-10 rats each: intact controls, hypothalamic paraventricular nuclei ablation (PVN) and sham-ablated (Sham). Sham and PVN rats were thyroparathyroidectomized 2-4 weeks after brain lesions and serial blood samples taken in all groups at frequent intervals from 0 to 58 days post-thyroidectomy. Plasma TSH was significantly higher than in intact controls by 3 days post-thyroidectomy in both the Sham and PVN groups (p < 0.05). At 14 days PVN plasma TSH was 4 x higher and at 30 days 8 x higher than in intact controls and remained consistently at 50% of that of the Sham group. There was no statistical difference between PVN and Sham in the rate of increase in TSH. Plasma T4 was 40% lower in PVN than in Sham at the time of thyroidectomy and became undetectable in both by day 3. The prompt parallel rate of rise of plasma TSH in Sham and PVN groups following thyroidectomy indicates that a primary physiologic action of TRH in the thyrotroph is to control the set-point for thyroid hormone negative feedback on TSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The permeant molecule urea stimulates prolactin secretion in GH4C1 cells by inducing Ca2+ influx through dihydropyridine-sensitive Ca2+ channels

Isotonic urea in medium with a normal 1.2 mM Ca2+ concentration induced a striking rise in both cytosolic Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion, each of whose peaks were proportional to the concentration of urea between 5 and 120 mM. There was a significant linear relationship between the peaks of induced [Ca2+]i and PRL secretion (r = 0.99, P less than 0.001). The increase in both [Ca2+]i and PRL secretion was completely abolished by removal of medium Ca2+ or by 2 microM nifedipine. Hypertonic urea was ineffective in inducing either an increase in [Ca2+]i or PRL secretion. These data support the hypothesis that plasma membrane expansion is a potent non-toxic inducer of hormone secretion and that in GH4C1 cells an increase in [Ca2+]i produced by enhanced influx of extracellular Ca2+ through dihydropyridine-sensitive Ca2+ channels plays an important role in this phenomenon.

Dynamics of thyrotropin-releasing hormone-induced thyrotropin and prolactin secretion by acutely dispersed rat adenohypophyseal cells. Evidence for 'all-or-none' secretion by heterogeneous secretory units, each with a specific response threshold.

We have examined the dynamics of thyrotropin-releasing hormone (TRH)-stimulated secretion of prolactin (PRL) and thyrotropin-stimulating hormone (TSH) using enzymatically dispersed rat adenohypophyseal cells suspended in a perfusion chamber with a volume of 0.2 ml to minimize mixing and dilution. One-min exposure to 3-300 nM TRH, the effective dose range, elicited immediate pulses of PRL and TSH secretion with dose-dependent amplitudes. At all TRH concentrations, following a brief burst of secretion lasting less than 1 min, release of both hormones declined precipitously. Increasing the duration of stimulation up to 30 min with half-maximal TRH concentrations did not alter the dynamics of the initial response and was ineffective in maintaining the initial amplitude of secretion. This phenomenon could not be attributed to exhaustion of readily releasable intracellular PRL and TSH, since an increment in TRH concentration elicited a second pulse of hormone secretion with temporal response characteristics identical to the first. The amplitude of the second pulse was dependent on both the initial concentration of TRH and the magnitude of the increment in TRH concentration. With a stepwise increase in TRH concentration during continuous perfusion, the sum of PRL or TSH secreted from all bursts of secretory activity approximated that achieved with a single exposure to the highest concentration of TRH employed. The high-amplitude secretory response to a given concentration of TRH was restored after an 8-min perfusion with medium alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyposmolar stimulation of secretion of thyrotropin, prolactin, and luteinizing hormone does not require extracellular calcium and is not inhibited by colchicine, cytochalasin B, ouabain, or tetrodotoxin.

Abstract Hyposmolar stimulation of thyroid-stimulating hormone, prolactin, and luteinizing hormone secretion by dispersed perifused rat pituitary cells was not depressed by removal of Ca2+ from the perifusion medium or by 0.1 mM colchicine, 20 μM cytochalasin B, 0.1 mM ouabain, or 3 μM tetrodotoxin. The secretory response induced by medium hyposmolarity or by thyrotropin-releasing hormone was not appreciably different at 23, 37, or 43°C, but was markedly reduced or abolished when the experiments were performed at 1°C. These data indicate that microtubules or microfilaments, transport of extracellular Ca2+ into the cytoplasm, and plasmalemma ion transport mechanisms sensitive to ouabain or tetrodotoxin are not essential components of the mechanism by which extracellular hyposmolarity induces secretion.

HYPOSMOLAR MEDIUM AND ETHANOL IN ISOSMOTIC SOLUTION INDUCE THE RELEASE OF THYROTROPIN-RELEASING HORMONE (TRH) BY ISOLATED RAT PANCREATIC ISLETS

Cell swelling induced by hypotonic medium or small isotonic permeant molecules results in an immediate secretory response in various types of cells. We have expanded exploration of this phenomenon by examining the effect of either isotonic ethanol or hyposmotic medium on the release of TRH by freshly isolated islets of Langerhans in static incubation and perifusion. Ethanol (40, 80 or 160 mM in isotonic solution) dose-dependently evoked the release of TRH by statically incubated islets. The dynamics of TRH release induced by 80 mM isotonic ethanol or 30% hypotonic medium were similar to those induced by 50 mM KCl, with the highest secretion rate during the first 5 min of incubation irrespective of the duration of stimulation. Ca2+ depletion of the incubation medium abolished the response to 50 mM KCl but did not diminish the response to 80 mM isotonic ethanol. We conclude that osmotic stimuli known to induce cell swelling also induce release of TRH by isolated pancreatic islets.

Evidence that Ca2+-activated K+ channels participate in the regulation of pituitary prolactin secretion

We evaluated the role of Ca(2+)-activated K+ channels in the regulation of prolactin (PRL) secretion with a perifusion system using acutely dispersed rat anterior pituitary cells. Apamin, which blocks Ca(2+)-activated K+ channels, induced PRL secretion in a dose-dependent fashion between 1 and 300 nM (r = 0.99, P < 0.01). Charybdotoxin, another Ca(2+)-activated K+ channel-blocker, also induced PRL secretion at 20 nM concentration. These were not non-specific toxic effects, since stimulation of PRL secretion by 10 nM thyrotropin-releasing hormone (TRH) was not different before and after applying the channel-blockers. Both 10 microM dopamine and 2 microM nifedipine significantly, but incompletely, depressed PRL secretion induced by 100 nM apamin; 10 microM dopamine completely blocked PRL secretion induced by 20 nM charybdotoxin. Our data indicate that Ca(2+)-activated K+ channels may play an important role in the regulation of PRL secretion.

Comparison of hyposmolar and hyperosmolar effects on in vitro luteinizing hormone secretion by anterior pituitary cells.

Abstract It has previously been described that perifusion of acutely dispersed adenohypophyseal cells with hypotonic medium causes an immediate high-amplitude “on” burst of luteinizing hormone (LH) secretion. In the present report the converse study with hyperosmolar solutions has been made. Perifusion with hypertonic medium depressed LH secretion; return to isotonicity caused an immediate high-amplitude “off” burst of LH secretion closely resembling that induced by hypotonic perifusion. The data give further support to the theory that exocytotic secretion may involve expansion of the outer cell membrane, thus drawing secretory granules to the cell surface where their contents are extruded.

Cell volume induced hormone secretion: studies on signal transduction and specificity.

Cell swelling causes an immediate secretory response in various cell types. Induced secretion possesses some unique features suggesting the involvement of a specific signal transduction pathway. The effect of 10-20 µM GdCl3, 100 µM HgCl2, 1-100 µM indomethacin and 1-20 µM nordihydroguaiaretic acid (NDGA) on cell swelling-induced hormone secretion (isosmotic 80 mM ethanol or 15-30% hyposmotic medium) from incubated rat hypothalamic paraventricular nucleus (PVN) and posterior pituitary (oxytocin and TRH), isolated pancreatic islets (TRH) and perifused anterior pituitary cells (prolactin) were examined. To determine how general the effect of cell swelling is on exocytotic secretion, the release of two different neurohormones (thyrotropin releasing hormone -TRH and oxytocin) from the same tissue explant were studied. Both hyposmotic medium or isosmotic ethanol containing medium induced immediate TRH and prolactin release from the tested tissues.The effect of GdCl3, HgCl2, NDGA or indomethacin showed no inhibition of cell swelling induced secretion. In contrast to TRH, oxytocin release was not induced by isosmotic ethanol containing medium from the PVN or posterior pituitary. CONCLUSION: These data indicate that signal transduction leading to exocytosis after cell swelling does not involve GdCl3 sensitive stretch activated channels, mercury sensitive aquaporins, or indomethacin and NDGA sensitive mediators including prostaglandins and leukotriens. Cell swelling-induced exocytosis possesses limited selectivity; cells specifically involved in water and salt regulation retain their specific response to osmotic stimuli.