Regulation of hormone secretion by acute cell volume changes: Ca(2+)-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, IP(3), 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 Ca(2+) through opening L-type Ca(2+) channels. However, such Ca(2+) 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.

Hypo-osmolarity stimulates and high sodium concentration inhibits thyrotropin-releasing hormone secretion from rat hypothalamus.

The hypothalamic paraventricular nucleus, representing cell bodies in which thyrotropin-releasing hormone is synthesized, and the median eminence, representing nerve terminals, were incubated in vitro. Various hypo- and hyperosmotic solutions were tested to determine osmotic sensitivity of thyrotropin-releasing hormone secretion. High KCl (56 mM) causing membrane depolarization was used as a non-specific control stimulus to induce thyrotropin-releasing hormone secretion. A 30% decrease of medium osmolarity (from 288 to 202 mOsmol/l) increased thyrotropin-releasing hormone secretion from both the paraventricular nucleus and median eminence. A 30% decrease of medium NaCl content by its replacement with choline chloride did not affect basal thyrotropin-releasing hormone secretion. Increasing medium osmolarity with biologically inactive L-glucose did not affect basal or KCl-induced thyrotropin-releasing hormone secretion from either structure. Medium made hyperosmotic (350-450 mOsmol/l) by increasing the NaCl concentration resulted in a dose-dependent decrease of basal thyrotropin-releasing hormone secretion and abolished KCl-induced thyrotropin-releasing hormone secretion. If an osmotically equivalent amount of choline chloride was substituted for NaCl, there was no effect on thyrotropin-releasing hormone secretion, indicating a specific action of Na+. This study indicates a specific sensitivity to high concentrations of Na+ ions of both thyrotropin-releasing hormone-producing parvocellular paraventricular neurons and thyrotropin-releasing hormone-containing nerve terminals in the median eminence.

Both iso- and hyperosmotic ethanol stimulate release of hypothalamic thyrotropin-releasing hormone despite opposite effect on neuron volume.

Previous studies have indicated that isosmolar, but not hyperosmolar, ethanol induces in vitro gonadotropin-releasing hormone secretion from the basal hypothalamus, presumably by causing cell swelling. Moreover, ethanol reduces secretion of another hypothalamic neuropeptide vasopressin. We have studied the acute effect of ethanol on specific hypophysiotropic basal and K+-stimulated thyrotropin-releasing hormone secretion in vitro especially in relation to cell swelling. Isosmotic 40-160 mM ethanol increased thyrotropin-releasing hormone release from the hypothalamic paraventricular nucleus and median eminence in a dose-dependent manner. Both a 30% decrease of osmolarity and isosmotic 80 mM ethanol induced 12% swelling of hypothalamic neurons. Hyperosmotic 80 mM or 160 mM ethanol induced release of thyrotropin-releasing hormone from both hypothalamic structures but did not cause cell swelling (80 mM) or even induced cell shrinkage (160 mM). Depletion of medium Ca2+ did not affect thyrotropin-releasing hormone secretion caused by either isosmotic or hyperosmotic ethanol. Our data indicate that both iso- and hyperosmotic ethanol stimulated release of hypophysiotropic thyrotropin-releasing hormone despite opposite effects on neuron volume. The mechanism of ethanol action appears complex and variable depending on the type of cell and neuropeptide affected.

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.

Pituitary PRL secretion induced by tetraethylammonium is inhibited by dopamine through D2 receptors.

We have evaluated the inhibitory effect of dopamine on PRL secretion induced by blocking K+ channels. Tumor-derived GH4C1 cells and collagenase-dispersed normal anterior pituitary (AP) cells from young adult male rats were perifused with Krebs-Ringer Hepes medium. In both cell types blocking K+ channels with tetraethylammonium (TEA) induced PRL secretion but did not stimulate cyclic AMP generation. Blocking Na+ channels with 1 microM tetrodotoxin had no effect on basal or TEA-induced PRL secretion. Dopamine inhibited the TEA-induced rise in [Ca2+]i in GH4C1 cells expressing dopamine D2 short receptors. In normal AP cells, 1-100 nM dopamine blocked PRL secretion induced by 20 mM TEA in a log-linear concentration-dependent fashion, with a plateau at > 100 nM dopamine (IC50 30 nM). The D2 dopaminergic receptor agonist, quinpirole, at 100 nM completely blocked PRL secretion induced by 20 mM TEA. The D2 dopaminergic receptor antagonist, sulpiride, at 10 microM reversed the inhibitory effect of 10 microM dopamine on PRL secretion induced by 20 mM TEA. Pretreatment of cells with 100 ng/ml pertussis toxin (PTX) for 24 h prevented 100 nM dopamine inhibition of PRL secretion induced by 20 mM TEA. The data indicate that in both normal lactotroph cells and in tumor-derived cells expressing D2 receptors, PRL secretion stimulated by blocking K+ channels is inhibited by dopamine binding to D2 receptors on the plasma membrane. This inhibition involves interaction with PTX-sensitive Gi protein.

Blocking K+ channels with TEA induces plasmalemma depolarization, increased [Ca2+]i, and ACTH secretion in AtT-20 cells.

Blocking K+ channels induces hormone secretion in various pituitary cell lines by a mechanism which is not completely delineated. In the present study, we employed the mouse pituitary tumor-derived AtT-20 cell as a model to evaluate this phenomenon. We correlated the effect of the K+ channel-blocker, tetraethylammonium (TEA), on K+ current and membrane potential utilizing whole cell recording, on cytosol Ca2+ ([Ca2+]i) concentration utilizing fura-2, and on ACTH secretion utilizing a perifusion system. TEA inhibited voltage-dependent K+ current and initiated membrane depolarization in a dose-dependent fashion. Divergences in the sensitivity to TEA between voltage-dependent K+ currents and membrane depolarization indicate that voltage-dependent K+ channels are not responsible for TEA-induced depolarization TEA (1-30 mM) also induced a concentration-dependent rise in [Ca2+]i concentration and ACTH secretion, both of which were inhibited by removing medium Ca2+. Our data indicate that TEA inhibits K+ currents and induces membrane depolarization; this opens Ca2+ channels in the plasmalemma, causing a rise in [Ca2+]i which initiates ACTH secretion. Alteration of K+ channel permeability by hormones or neurotransmitters may thus play an important regulatory role in controlling pituitary hormone secretion.

Evidence that Ca(2+)-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 ramp and square-wave exposure to thyrotrophin-releasing hormone or depolarizing K+ on cytosolic Ca2+ concentration and prolactin secretion in GH4C1 cells.

The standard method of studying hormone secretion in vitro is to make instantaneous changes in the concentration of stimulators in the medium. However, in vivo the extracellular concentration of such substances changes more gradually; secretion does not occur in square-wave bursts and agonists or antagonists transmitted through the bloodstream are diluted and diffused by plasma or tissue fluid to further decelerate the rate of change in concentration at the cell surface. We have therefore compared in GH4C1 cells the dynamics of changes in cytosolic Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion in response to two very different secretagogues, thyrotrophin-releasing hormone (TRH) and depolarizing K+, using a square-wave or ramp exposure for 5 min. The dynamics of hormone secretion were analysed by column perifusion (2 x 10(6) cells/column). Ca2+ dynamics were monitored by dual excitation microfluorimetry from 20-30 optically isolated cells using the Ca2+ indicator, fura-2. With square-wave exposure, both TRH (0.1-100 nM) and K+ (10-50 mM) induced dose-dependent increases in [Ca2+]i and PRL secretion. Concentrations of TRH > 1 nM caused a two-phase increase in [Ca2+]i with an initial high-amplitude first phase and a low-amplitude second phase. Depolarizing K+ induced a sharp increase in [Ca2+]i which peaked within 15 seconds, then declined gradually on a sloping plateau. Both TRH and K+ induced an acute dose-dependent PRL secretory burst peaking within 2.5 min with a subsequent rapid decline.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that tolbutamide induces prolactin secretion by a mechanism which does not involve blocking ATP-sensitive potassium channels.

Tolbutamide is an important member of the sulfonylureas, drugs which stimulate secretion of several hormones, including insulin and prolactin (PRL), through a mechanism postulated to involve blocking ATP-sensitive K+ channels. In the present study, we have evaluated the hypothesis relating the induced secretion to an effect on K+ channels by examining the effect of tolbutamide and the ATP-sensitive K+ channel-opener, diazoxide, on PRL secretion by acutely dispersed perifused adenohypophyseal cells from young adult male rats. Five-min perifusion of 0.1-50 microM tolbutamide induced a concentration-correlated secretion of PRL with a minimum effective concentration of 0.1 microM. Both basal and 50 microM tolbutamide-induced PRL secretion were significantly suppressed by 10 microM dopamine or 2 microM nifedipine, which block Ca2+ influx through L-type channels, but not by 100 microM diazoxide, indicating that tolbutamide induces PRL secretion by a mechanism involving Ca2+ influx through L-type Ca2+ channels which is not related to its ability to block ATP-sensitive K+ channels.

Rat Prl and TSH secretion are regulated differently by K(+)-channel blockers.

All four different K(+)-channel blockers [tetraethylammonium (TEA), a nonselective K(+)-channel blocker; tolbutamide, an ATP-sensitive K(+)-channel blocker; quinine and 4-aminopyridine, both primarily voltage-dependent K(+)-channel blockers] stimulated prolactin (Prl) secretion by acutely dispersed anterior pituitary cells but had no effect on thyroid-stimulating hormone (TSH) secretion. TEA stimulated Prl secretion in a dose-dependent manner between 1 microM and 20 mM, but even as high as 20 mM, TEA did not induce TSH secretion. Valinomycin (2 microM), a K+ ionophore, inhibited both basal and TEA-induced Prl secretion. TEA-stimulated Prl secretion was abolished by using a Ca(2+)-depleted medium or adding 10 microM dopamine. TEA did not reverse the inhibitory effect of dopamine on thyrotropin-releasing hormone-induced Prl secretion. Our data indicate that K+ channels may play a role in the secretion of adenohypophysial hormones that is idiosyncratic for each hormone. Differences in the role of K+ channels may reflect differences between the various pituitary cell types in plasma membrane ion channel composition, membrane potential, or the mechanism of exocytosis.

Tetraethylammonium blockade of K+ channels in GH4C1 cells regulates prolactin secretion by inducing Ca2+ influx.

Tetraethylammonium (TEA), a K+ channel blocker, induced PRL secretion and an increase in cytosol Ca2+ concentration [Ca2+]i in a dose-dependent manner between 5-20 mM in GH4C1 rat pituitary tumor-derived cells. Removal of medium Ca2+ or the addition of 1 microM nifedipine abolished both the induced [Ca2+]i increment and PRL secretion. TEA augmented the TRH-induced rise in [Ca2+]i and inhibited the rise in [Ca2+]i induced by 30 mM K+. The dynamics of TEA-, TRH- and K(+)-induced PRL secretion were different, with the TEA-induced secretory peak occurring at about 10 min compared to 2-3 min for TRH and K+. Tolbutamide, which blocks ATP-sensitive K+ channels, induced PRL secretion without causing a rise in [Ca2+]i. The results suggest that: (a) K+ channels have a complex interaction with the PRL secretory process in GH4C1 cells; (b) TEA induces PRL secretion by causing Ca2+ influx through dihydropyridine-sensitive Ca2+ channels; and (c) K+ channels play a different role in the [Ca2+]i rise induced by TRH than in that induced by depolarizing K+.

Isotonic but not hypertonic ethanol stimulates LHRH secretion from perifused rat median eminence.

We utilized luteinizing hormone-releasing hormone (LHRH) secretion by perifused minced rat hypothalamic median eminence (ME) tissue to evaluate whether isotonic ethanol would stimulate neurosecretion as it does secretion from pituitary cells. Isotonic ethanol induced a dose-dependent burst of LHRH secretion which was maximal at 2-3 min and returned to near baseline by 10 min. In Ca(2+)-depleted media (< 2 microM Ca2+), stimulation of secretion by isotonic ethanol was enhanced, but secretion induced by depolarizing 30 mM K+ was abolished. Hypertonic ethanol was ineffective in stimulating LHRH secretion in either normal or Ca(2+)-free media. The secretory response of hypothalamic LHRH-secreting cells to ethanol and its negative modulation by medium Ca2+ is thus identical to that of normal anterior pituitary cells and presumably is caused by cell swelling resulting from influx of permeant ethanol molecules across the plasmalemma.

TSH secretory responses to prolonged infusion of TRH in hypothyroid rats.

Continuous infusion of 0.1-10 micrograms/ml of TRH was performed through chronic i.v. cannulas in unrestrained, unanesthetized hypothyroid rats. Infusion of a constant concentration of TRH induced a peak in the plasma TSH concentration within 5-15 min which declined to the baseline within 1 h. The refractory period lasted 20-40 min after stopping the continuous TRH infusion. A second burst of TSH secretion was induced by increasing the TRH concentration during the refractory period while TRH was being continuously infused. These data indicate that in hypothyroid rats TSH secretion rapidly becomes refractory to continuous exposure to the same concentration of TRH but is stimulated by a higher TRH concentration. This suggests there is heterogeneity in TSH secretory units, which may consist of a constellation of thyrotrophs or of discrete intracellular secretory units, each with a threshold of specific response to TRH.

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)

Cyclic adenosine monophosphate is not part of the transduction chain by which cell-swelling induces secretion in either normal or tumor-derived GH4C1 pituitary cells.

We have evaluated whether cyclic adenosine monophosphate (cAMP) generation plays a role in the burst of hormone secretion caused by osmotic cell-swelling in tumor-derived and normal pituitary cells. Up to 45% hyposmolarity induced a dose-dependent increase in prolactin (PRL) secretion, but had no effect on cAMP generation. However, hyposmolarity inhibited forskolin-induced cAMP accumulation in a dose-dependent manner, but had no effect on 3-isobutyl-1-methyl-xanthine (IBMX)-induced cAMP accumulation. Ca2+ depletion of the medium partially blocked the inhibitory effect of hyposmolarity on forskolin-induced cAMP generation and completely blocked stimulation of PRL secretion by hyposmolarity. High levels of K+ in medium inhibited forskolin-induced cAMP generation, while thyrotropin-releasing hormone (TRH) enhanced it. Our results indicate that in both GH4C1 and normal pituitary cells, cAMP is not a regulatory factor for hormone secretion induced by cell-swelling.

Adenosine 3',5'-cyclic monophosphate-mediated prolactin secretion in GH4C1 cells involves Ca2+ influx through L-type Ca2+ channels.

In normal anterior pituitary cells adenosine 3',5'-cyclic monophosphate (cAMP)-mediated prolactin (PRL) secretion requires Ca2+ influx. However, the role of Ca2+ in cAMP-induced secretion in the clonal rat pituitary tumor-derived GH4C1 cells remains uncertain. We examined in GH4C1 cells the effects of forskolin (FSK), an adenylate cyclase activator, and dibutyryl cAMP (DB-cAMP) on PRL secretion and intracellular Ca2+ ([Ca2+]i) dynamics. Ca2+ depletion of the medium inhibited FSK and DB-cAMP stimulated PRL secretion approximately 50%. Both FSK and DB-cAMP increased [Ca2+]i in a dose-dependent fashion. The peak amplitude in [Ca2+]i in response to each concentration of these stimuli was achieved in 3 min, corresponding to the peak PRL response to the same stimuli. Either Ca2+ depletion of the medium or addition of 2 microM nifedipine (NF) abolished the increase in [Ca2+]i caused by FSK or DB-cAMP. Our data indicate that an increase in intracellular c-AMP in GH4C1 cells produces an elevation of [Ca2+]i by opening L-type Ca2+ channels and that c-AMP-mediated PRL secretion is augmented by Ca2+ influx in GH4C1 cells as in normal pituitary cells.

Cell swelling induced by medium hyposmolarity or isosmolar urea stimulates gonadotropin-releasing hormone secretion from perifused rat median eminence.

Medium hyposmolarity between 10 and 50% and isotonic urea between 22.5 and 90 mM induced a dose-dependent burst of gonadotropin-releasing hormone (GnRH) secretion from perifused median eminence tissue which was maximal at 2-3 min and returned to near baseline by 5 min in spite of continued exposure to the stimulus. If Ca(2+)-free medium was used, osmotic stimulation of secretion was increased or unchanged, but secretion induced by 30 mM K+ was markedly reduced. Our data indicate that cell swelling induced by medium hyposmolarity or permeant molecules stimulates GnRH secretion from median eminence cells or cell processes as it does secretion from normal endocrine cells containing hormone stored in intracellular vesicles. In both, Ca2+ influx is not required or has a negative modulating influence on cell swelling-induced secretion.

Lidocaine inhibits prolactin secretion in GH4C1 cells by blocking calcium influx.

The mechanism of the inhibitory effect of local anesthetics on hormone secretion was studied in the GH4C1 line of rat pituitary tumor-derived cells. Lidocaine between 0.1 and 5 mM exerted significant dose-dependent inhibition on the increment in cytosol Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion induced by 30 mM K+. For both effects the IC50 was 0.25 mM and maximal inhibition occurred at 5 mM. A normal response returned within 20 min after removal of lidocaine from the incubation medium. 1 microM tetrodotoxin had no effect on the 30 mM K+ induced [Ca2+]i transient or PRL secretion, indicating that Na+ channels are not involved in the inhibitory effect of lidocaine. Lidocaine similarly inhibited the [Ca2+]i increment and PRL secretion induced by 30% medium hyposmolarity and 1 microM Bay K 8644. Lidocaine was much less effective in inhibiting secretion induced by 1 microM phorbol 12-myristate 13-acetate (TPA) or 5 microM forskolin. 5 mM procaine produced effects similar to those of lidocaine. Our data suggest that in GH4C1 cells local anesthetics depress secretagogue-induced PRL secretion primarily by blocking Ca2+ influx, probably through L-type Ca2+ channels.

Alpha-adrenergic inhibition of thyrotropin-releasing hormone-induced prolactin secretion in GH4C1 cells is associated with a depressed rise in intracellular Ca2+.

alpha-Adrenergic receptors are present on the plasma membrane of normal anterior pituitary cells and alpha-adrenergic agonists may play a role in the secretion of corticotropin (ACTH) and thyrotropin (TSH). However, alpha-adrenergic involvement in prolactin (PRL) secretion is uncertain. We have therefore examined this question in the PRL-secreting clonal rat pituitary tumor-derived GH4C1 cells. Norepinephrine (NE), an alpha-adrenergic agonist, had no effect on basal PRL secretion but abolished thyrotropin-releasing hormone (TRH)-induced PRL secretion in a dose-dependent manner (EC50 100 nM). NE also significantly suppressed the TRH-stimulated rise in [Ca2+]i. Phentolamine (PA), a non-selective alpha-adrenergic antagonist, reversed the inhibitory effect of NE on both the TRH-stimulated PRL secretion and [Ca2+]i rise. NE did not inhibit the rise in PRL secretion or [Ca2+]i induced by depolarizing 30 mM K+, 30% hyposmolarity or BAY K-8644, a specific L-type Ca2+ channel agonist. The inhibitory effect of NE on TRH-induced PRL and [Ca2+]i changes was also present when Ca2+ influx was prevented by removing medium Ca2+ or by blocking L-type Ca2+ channels with 2 microM nifedipine. The TRH-stimulated first-phase rise in [Ca2+]i in GH4C1 cells is believed to result primarily from release of sequestered Ca2+ from an intracellular pool through the activation of inositol 1,4,5-trisphosphate (IP3) and this [Ca2+]i spike stimulates PRL secretion. Our data thus suggest that GH4C1 cells have alpha-adrenergic receptors and that alpha-adrenergic agonists either suppress IP3 generation or block IP3 release of sequestered intracellular Ca2+.