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.     

Effects of trifluoperazine on prolactin release and cyclic AMP formation and degradation in GH4C1 pituitary cells

In GH4C1 cells, the calmodulin antagonist trifluoperazine (TFP) showed a dose-dependent, biphasic effect on the basal release of PRL. An inhibition of PRL release was observed with 15-50 mumol/l TFP, whereas a concentration of 100 mumol/l and above had a stimulatory effect. The increase in basal hormone release evoked by TRH (1 mumol/l) and high extracellular concentration of K+ (50 mmol/l) was eliminated by 30 mumol/l TFP. The stimulatory effect of 100 mumol/l TFP on basal hormone release was not affected by addition of TRH (1 mumol/l) or K+ (50 mmol/l). The Ca2+ antagonists Co2+ (5 mmol/l) and verapamil (100 mumol/l), and the Ca2+ chelator EgTA (4 mmol/l) abolished the stimulatory effect of TRH (1 mumol/l) and of K+ (50 mmol/l) on PRL release, whereas only Co2+ inhibited the stimulation caused by 100 mumol/l TFP. TFP (75 mumol/l) caused a transient increase in the concentration of cellular cAMP. Incubation of intact GH4C1 cells with TFP (75 mumol/l), had an inhibitory effect on both the low and the high affinity form of cAMP phosphodiesterase. Basal as well as TRH-stimulated adenyl cyclase activity were inhibited by TFP, and this effect was counteracted by addition of calmodulin.     

Effects of metabolic inhibitors on hormone release, cyclic AMP levels, and oxygen consumption in rat pituitary cells in culture

The metabolic inhibitors antimycin A (2 mumol/l), dinitrophenol (0.5 mmol/l), and iodoacetate (6 mmol/l) were tested for their effects on hormone release, cAMP levels, and oxygen consumption in clonal strains of rat pituitary cells (GH3 cells). Basal release of growth hormone (GH) and prolactin (PRL) was reduced by all three inhibitors, and thyrotropin-releasing hormone (TRH) (1 mumol/l) and K+ (50 mmol/l) stimulated hormone release were blocked. Trifluoperazine, a calmodulin antagonist, inhibited basal GH and PRL release at concentrations up to 30 mumol/l and stimulated above 50 mumol/l. The stimulatory effect of 80 mumol/l trifluoperazine on basal hormone release was eliminated by antimycin A, dinitrophenol, and iodoacetate, whereas the inhibitory effect of antimycin A, dinitrophenol and iodoacetate on basal hormone was not affected by 30 mumol/l trifluoperazine. None of the inhibitors had any effect on the level of cellular cAMP (i.e. intracellular plus extracellular). Oxygen consumption of GH3 cells was blocked by antimycin A, reduced by 25% by iodoacetate and increased by about 100% by dinitrophenol. In contrast, hormone secretion stimulated by TRH and K+ was not accompanied by any measurable alteration in oxygen consumption. Trifluoperazine (greater than or equal to 80 mumol/l) reduced the basal oxygen consumption and blocked the stimulatory effect of dinitrophenol on oxygen consumption. In conclusion, inhibition of the energy generation of GH and PRL-producing cells severely affects the action of secretagogues, although stimulated hormone secretion may not be accompanied by any measurable increase in oxygen consumption. The cellular energy supporting hormone secretion is mostly generated via oxidative phosphorylation.     

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+.     

Role of calcium in thyrotrophin-releasing hormone-stimulated release of melanocyte-stimulating hormone from frog neurointermediate lobe

The effect of modifications of extracellular calcium concentrations on alpha-MSH release has been studied using perifused frog neurointermediate lobes. Increasing concentrations of calcium (from 2 to 10 mmol/l) gave rise to a dose-related stimulation of alpha-MSH secretion, whereas reduction of Ca2+ from 2 to 1.5 mmol/l partially inhibited alpha-MSH release. The direct effect of extracellular Ca2+ on alpha-MSH secretion was confirmed by the dose-dependent stimulation of alpha-MSH release induced by the calcium ionophore A23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mmol Co2+/l both resulted in an inhibition of spontaneous and TRH-induced alpha-MSH release. Conversely, administration of verapamil or methoxyverapamil (10 mumol/l each) did not alter basal secretion and had no effect on the response of the glands to TRH. Nifedipine (10 mumol/l), which was able to block KCl (20 mmol/l)-evoked alpha-MSH release, induced a slight inhibition of basal alpha-MSH secretion, indicating that extracellular Ca2+ levels may regulate alpha-MSH release in part by Ca2+ influx through voltage-dependent Ca2+ channels. In contrast TRH-induced alpha-MSH release was not affected by nifedipine or dantrolene (10 mumol/l), and BAY-K-8644 (1 mumol/l) did not significantly modify the response of neurointermediate lobes to TRH. Taken together, these results suggest that TRH-induced alpha-MSH secretion is associated with calcium influx across the plasma membrane and that calcium entry caused by TRH may occur through nifedipine/verapamil-insensitive Ca2+ channels.     

Dopamine inhibits cell swelling-induced prolactin secretion in MMQ cells by blocking Ca2+ influx.

To evaluate the role of Ca2+ influx on hormone secretion induced by cell swelling, we have utilized a prolactin (PRL)-secreting rat tumor cell line, MMQ, which has plasmalemma dopamine receptors. Medium hyposmolarity or osmotically equivalent isotonic urea caused prompt cell swelling and a rise in both [Ca2+]i and PRL secretion in a dose-dependent manner. Dopamine inhibited the induced increase in both [Ca2+]i and PRL secretion in a dose-dependent manner but the maximum inhibition was only 50%. This effect of dopamine was prevented by haloperidol. Depletion of medium Ca2+ or blocking Ca2+ influx with nifedipine completely abolished the osmotically induced rise in both [Ca2+]i and PRL secretion. These data indicate that Ca2+ influx through nifedipine-sensitive Ca2+ channels is an essential component of PRL secretion induced by osmotic cell swelling in MMQ cells and that a dopaminergic receptor-linked mechanism influences the opening of these channels.     

Protein kinase C modulates cell swelling-induced Ca2+ influx and prolactin secretion in GH4C1 cells.

In GH4C1 rat pituitary cells, cell swelling stimulates prolactin (PRL) secretion by increasing Ca2+ influx through nifedipine-sensitive Ca2+ channels; however, the mechanism by which cell swelling opens Ca2+ channels is still unclear. To evaluate the role of protein kinase C (PKC) in this phenomenon, we studied the effect of down-regulating PKC by 12-h pretreatment with phorbol ester or by treatment with H-7, a protein kinase C inhibitor. Cell swelling induced by either 27% medium hyposmolarity or 80 mM isotonic urea caused a prompt rise in both [Ca2+]i and PRL secretion in otherwise untreated control GH4C1 cells. Removal of medium Ca2+ enhanced the osmotically induced cell swelling but prevented the increase in [Ca2+]i and PRL secretion. Both PKC down-regulation and H-7 suppressed the cell swelling-induced increases in [Ca2+]i concentration and PRL secretion, although they enhanced the induced cell volume expansion. Our data indicate that in GH4C1 cells PKC plays an important positive modulating role in the osmotic opening of plasmalemma Ca2+ channels, a critical component of the early transduction chain by which cell swelling causes PRL secretion in tumor-derived clonal pituitary cells.     

The role of calcium and calmodulin in mediating release of thyrotrophin-releasing hormone by cultured hypothalamic cells

We have developed a fetal rat hypothalamic cell culture system for the study of factors controlling the acute release of TRH. Release of TRH by the cells has been characterized by reversed-phase high pressure liquid chromatography and about 86% of the total immunoreactivity in the medium co-eluted with synthetic TRH. Release of TRH by the cells in response to 56 mmol K+/l increased between days 5 and 9 of culture but reached a plateau thereafter. Cell contents of TRH did not change significantly between days 5 and 14 of culture. Release of TRH from the cells was stimulated by K+ (56 mmol/l), veratridine (100 mumol/l) and ouabain (100 mumol/l) to 550, 480 and 335% of basal release respectively over a 1-h period. Release of TRH was dependent upon calcium in that it was absent when calcium-free medium was used and could be blocked by verapamil (20 mumol/l); however it could not be blocked by nifedipine (50 mumol/l). The calcium ionophore blocked by nifedipine (50 mumol/l). The calcium ionophore A23187 (1 mumol/l) stimulated TRH release to 340% of basal release. Tetrodotoxin (1 mumol/l) completely abolished the release in response to veratridine but had no effect on the release stimulated by K+ (56 mmol/l). The calmodulin antagonists trifluoperazine and triflupromazine (50 mumol/l) inhibited veratridine-stimulated TRH release. This was at a site after calcium influx as they also inhibited A23187-stimulated TRH release. The highly specific calmodulin antagonist W7 (10 mumol/l) also inhibited both veratridine and A23187-stimulated TRH release whereas, at the same concentration, its inactive analogue W5 did not significantly inhibit TRH release in response to either stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that ethanol induces prolactin secretion in GH4C1 cells by producing cell swelling with resultant calcium influx

Although acute exposure to ethanol has been reported to affect hormone secretion, the data are sometimes conflicting, and the mechanism of action of ethanol is unclear. We have examined in GH4C1 cells the effect of isotonic ethanol on cell volume measured with a Coulter counter, the cytosolic Ca2+ concentration ([Ca2+]i) monitored with fura-2, and PRL secretion analyzed in a perifusion system. Isotonic ethanol caused prompt cell swelling and an explosive rise in both [Ca2+]i and PRL secretion proportional to the concentration of ethanol between 5-120 mM. The increases in both [Ca2+]i and PRL secretion induced by 80 mM isotonic ethanol were essentially abolished by removal of medium Ca2+ or by nifedipine; the nifedipine IC50 was approximately 20 nM. Cell swelling induced by hyposmolarity or isotonic urea similarly increased both [Ca2+]i and PRL secretion. Hypertonic ethanol did not cause cell swelling and was ineffective in inducing an increase in either [Ca2+]i or PRL secretion. These data suggest that in GH4C1 cells a major mechanism by which ethanol stimulates PRL secretion is to induce cell swelling, thus producing enhanced Ca2+ influx through dihydropyridine-sensitive Ca2+ channels.     

Lidocaine and procaine inhibit the increase in cytosol Ca2+ induced by thyrotropin-releasing hormone or K+ depolarization in GH4C1 cells

Lidocaine at greater than or equal to 1 mM and procaine at greater than or equal to 2.5 mM exerted dose-dependent inhibition of the increment in [Ca2+]i induced by 100 nM thyrotropin-releasing hormone (TRH) or 30 mM K+ in GH4C1 cells. The rise in [Ca2+]i induced by K+ was more sensitive to this inhibition than that induced by TRH. Lidocaine was more potent than procaine in inhibiting the [Ca2+]i increment induced by secretagogues. Maximal lidocaine inhibition of the TRH-induced [Ca2+]i increment occurred within 15-20 min and a normal response to secretagogues returned within 20 min after removal of lidocaine from the incubation medium. Our data suggest that in GH4C1 cells local anesthetics depress secretagogue-induced intracellular Ca2+ mobilization, depolarization of the cell membrane, and the opening of voltage-dependent Ca2+ channels. This may explain the depression of secretagogue-stimulated hormone secretion induced by these agents.     

Is calcium or cyclic AMP involved in the inhibitory effect on pituitary hormone secretion of the tripeptide aldehyde proteinase inhibitors?

The mechanism by which tripeptide aldehyde proteinase inhibitors decrease prolactin (PRL) and growth hormone (GH) secretion was studied. Agents known to modify the intracellular levels of cyclic adenosine monophosphate (cAMP) or cytosolic free calcium were used in monolayer cultures of the rat anterior pituitary gland. The phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), 8-bromo-cAMP and forskolin all stimulated PRL release. Boc-D-Phe-Pro-arginal (Boc-DPPA) at 1 mmol/l concentration was a potent inhibitor of basal PRL release and significantly decreased the effect of 8-Br-cAMP, forskolin or IBMX (0.5 mmol/l). Forskolin (1 mumol/l) stimulated ACTH, PRL and GH release and all these effects were decreased by 100 mumol/l of Boc-D-Phe-Phe-lysinal (Boc-DPPL). Neither tripeptide aldehyde affected the forskolin-induced rise in intracellular cAMP. Growth hormone releasing factor (hpGRF, 1 nmol/l) stimulated both GH release and intracellular cAMP generation; Boc-DPPL (100 mumol/l) significantly decreased stimulated GH release without affecting cAMP accumulation. Increasing medium K+ to 10 times normal level stimulated PRL release presumably by enhancing Ca2+ entry into the cells and 1 mmol/l Boc-DPPA decreased high potassium-stimulated PRL release. The ionophore A-23187 stimulated PRL release at 10 mumol/l but not at 1 mumol/l. At 1 mumol/l A-23187 prevented the Boc-DPPA-induced inhibition of PRL release. These findings suggest that the tripeptide aldehyde proteinase inhibitors inhibit PRL and GH release at a site beyond cAMP formation.     

Mechanism of suppressed prolactin secretion due to medium hyperosmolarity--the importance of Ca2+ influx]

It is still unknown how extracellular hyperosmolarity suppresses exocytosis. To evaluate the possibility that extracellular hyperosmolarity affects one of the most important second messenger system, Ca2+ signal, we evaluated the effect of hyperosmolarity on the thyrotropin releasing hormone (TRH)-induced changes in both intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion in GH4C1 cells. TRH caused two phases of [Ca2+]i: an initial high-amplitude phase (first phase), which was not inhibited by Ca2+ free medium, and a sustained low-amplitude phase (second phase), which was abolished by Ca2+ free medium. Medium hyperosmolarity (isotonic = 300mOsm, hypertonic = 338, 375, 450, and 600mOsm) suppressed both TRH-induced phases of [Ca2+]i in a dose dependent manner, however, the suppressive effect was clearly stronger in the second phase of [Ca2+]i than in the first phase of [Ca2+]i. Low doses of medium hyperosmolarity (338 and 375mOsm) suppressed PRL secretion, which was dependent on Ca2+ influx. However, high doses of medium hyperosmolarity (450 and 600mOsm) also blocked PRL secretion, which was dependent on Ca2+ mobilized from cytosolic Ca2+ pools. These data indicate that in GH4C1 cells medium hyperosmolarity may inhibit PRL secretion by both blocking Ca2+ influx and a mechanism unrelated to Ca2+.     

Thyrotropin-releasing hormone stimulation of prolactin secretion is coordinately but not synergistically regulated by an elevation of cytoplasmic calcium and 1,2-diacylglycerol

The precise roles of the calcium and lipid pathways in TRH-stimulated PRL secretion from rat pituitary (GH3) cells are controversial. In particular, it is debated whether elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i) is sufficient to cause burst secretion (0-2 min) or whether an increase in 1,2-diacylglycerol must accompany the Ca2+ elevation. In this study, the effects of TRH, which elevates 1,2-diacylglycerol, on [Ca2+]i and stimulation of burst secretion were compared with those of depolarization by high extracellular K+, which does not increase 1,2-diacylglycerol. A maximal concentration of TRH (1 microM) and depolarization by 17.5 mM K+ caused elevation of [Ca2+]i from the resting level of 140 +/- 20 nM to 470 +/- 70 nM and 514 +/- 60 nM, respectively, and stimulated burst secretion from 0.6 +/- 0.2 ng/10(6) cells/min to 3.3 +/- 0.8 and 3.1 +/- 0.4 ng/10(6) cells/min, respectively, when a small component of TRH-stimulated secretion that is independent of elevation of [Ca2+]i was subtracted. A detailed comparison of multiple levels to which [Ca2+]i was elevated (up to 600 nM) and the degree of stimulation of burst phase secretion demonstrated the same positive linear correlation (correlation coefficient = 0.96) for TRH and K+ depolarization. Hence, elevation of [Ca2+]i is sufficient to cause burst secretion irrespective of elevation of 1,2-diacylglycerol. Optimal stimulation by TRH of sustained secretion of PRL did not depend on elevation of [Ca2+]i; sustained PRL secretion stimulated by 10 nM TRH was 2.6 +/- 0.4 and 2.7 +/- 0.2 ng/10(6) cells/min in control cells and arachidonic acid-pretreated cells in which [Ca2+]i was not elevated, respectively. The data from this and previous studies demonstrate that elevation of [Ca2+]i and 1,2-diacylglycerol may act coordinately, but not synergistically, to mediate TRH stimulation of PRL secretion from GH3 cells.     

Medium hyperosmolarity inhibits prolactin secretion induced by depolarizing K+ in GH4C1 cells by blocking Ca2+ influx.

Medium hyperosmolarity between 300 (normal medium osmolarity) and 600 mOsm inhibited in a concentration-correlated fashion (r greater than 0.97, p less than 0.001) the rise in intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion induced in GH4C1 cells by depolarizing 30 mM K+. [Ca2+]i concentration and PRL secretion were tightly related between 300 and 600 mOsm (r = 0.976, p less than 0.001); 50% inhibition of both occurred at 450 mOsm. Medium hyperosmolarity slowed the rate of Ca2+ influx. At 600 mOsm the rise in both [Ca2+]i and PRL secretion was abolished but PRL secretion induced by 1 microM phorbol 12-myristate 13-acetate was not significantly reduced. Our data suggest that inhibition of Ca2+ influx may be the primary mechanism by which extracellular hyperosmolarity inhibits PRL secretion induced by high medium K+ in GH4C1 cells. Depression of the Ca2+ intracellular transduction system may play a pathophysiological role in vivo in conditions such as dehydration and hypertonic coma.     

Medium hyperosmolarity depresses thyrotropin-releasing hormone-induced Ca2+ influx and prolactin secretion in GH4C1 cells.

We studied the influence of graded degrees of hyperosmolarity on the dynamics of the thyrotropin-releasing hormone (TRH)-induced rise in cytosol Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion in GH4C1 cells. TRH caused two phases of increase in [Ca2+]i that were differentially altered by hyperosmolarity: 100% hyperosmolarity (600 mOsm) depressed only 20% of an initial high-amplitude [Ca2+]i burst (first phase) dependent on Ca2+ mobilized from intracellular pools, but it abolished a sustained low-amplitude second phase dependent on extracellular Ca2+ influx. Low degrees of hyperosmolarity suppressed PRL secretion due to Ca2+ influx while high degrees suppressed secretion due to mobilized Ca2+. These data suggest that in GH4C1 cells hypertonic inhibition of secretion may result from both blocking Ca2+ influx and mechanisms unrelated to [Ca2+]i.     

The effects of ions and hypothalamic factors on the in vitro activity of rainbow trout prolactin cells.

The influence of various ions and of dopamine and somatostatin on the in vitro activity of rainbow trout prolactin (PRL) cells was investigated. There was a positive correlation between medium Ca2+ concentration and both PRL synthesis and release up to 1.8 mM Ca2+, above which no further increase occurred. Even with no Ca2+ in the medium, there was still PRL secretion during the incubation. Replacement of Ca2+ with Ba2+ in the medium did not elevate either total PRL levels or PRL release above that in Ca2 +)-free medium. Neither elevated Mg2+ nor increased medium K+ had any effect on PRL synthesis or release. Dopamine inhibited PRL release but not synthesis, as did the D2 receptor agonist, apomorphine. However, the D2 receptor antagonist, (+)-butaclamol was unable to prevent the action of dopamine on PRL release. Somatostatin inhibited both PRL synthesis and release in normal Ca2+ medium, but release only in reduced Ca2+ medium. Thus, Ca2+, dopamine, and somatostatin may all have roles in regulating prolactin secretion in this fish. In addition, oPRL reduced trout PRL release, indicating a possible negative feedback mechanism for trout PRL secretion.     

Thyrotropin-releasing hormone rapidly stimulates a biphasic secretion of prolactin and growth hormone in GH4C1 rat pituitary tumor cells

The characteristics of TRH-induced acute PRL and GH secretion were studied in GH4C1 cells, a clonal rat anterior pituitary tumor cell line which secretes PRL and GH. The experiments were carried out both in a flow system in which microcarrier (Cytodex)-attached cells were perifused at a constant rate and in a conventional static culture system. In both systems, cells responded to TRH in a qualitatively similar manner. TRH significantly stimulated PRL and GH secretion within 5 sec without a detectable lag period. The secretion rate was highest during the initial 1 min, declined sharply thereafter despite the continuous presence of TRH, and plateaued at a lower level. The maximum dose of TRH caused 250-700% of basal secretion during the early period (approximately 8 min; first phase) and about 150% of basal secretion thereafter (second phase). The sustained lower secretion (second phase) was maintained as long as cells were exposed to TRH (up to 2.5 h), and the secretion rate returned to the basal level within 30 min of removal of TRH from the medium. The half-maximal doses for the first and second phase secretion were 2-3 and 0.5-1 nM, respectively, in both the perifusion and static culture systems. Over a 2-day period, TRH stimulated PRL synthesis and inhibited GH synthesis. The dose-response curves for these long term effects on hormone synthesis were similar to the dose-response curves for the first phase of release. [N3-methyl-His2]TRH gave similar results, but was more potent than TRH. [N3-methyl-His2]TRH stimulated first phase release with an ED50 of 0.4-0.8 nM, second phase release with an ED50 of 0.1-0.2 nM, and hormone synthesis with an ED50 of 0.7-0.8 nM. Preincubation of the cells with Ca+2-free medium significantly depressed both first and second phase secretion. Preexposure of the cells to cycloheximide (10 micrograms/ml) had little effect on the first phase of secretion, but reduced second phase secretion. The acute effects of TRH on GH and PRL were identical, except that the secretory response tended to be greater for PRL. We conclude that 1) TRH causes hormone secretion very rapidly in a biphasic manner; 2) the first phase of secretion consists primarily of the release of stored hormone, whereas the second phase includes the release of newly synthesized hormone; 3) the dose-response curve of second phase secretion is shifted to the left compared with that of first phase secretion; and 4) both phases of secretion are at least partially dependent on extracellular Ca+2.     

Role of calcium and sodium ions in the inhibitory control of baseline and stimulated prolactin release

The mechanism of dopamine (DA) inhibition of pituitary PRL release is still unclear. To study it, we utilized enzymatically dispersed anterior pituitary cells obtained from adult female Sprague-Dawley rats. The cells were incubated in media with or without Na+ and in the presence or the absence of various drugs for 30 min for evaluating the secretion of PRL under baseline and experimental conditions. In some experiments, 45Ca2+ (1 microCi/ml) was added after 30 min of incubation and the latter prolonged for an additional minute to determine Ca2+ uptake. DA inhibited baseline PRL release and 45Ca2+ uptake in a dose-dependent manner only in the presence of Na+ and was totally inactive in its absence. The inhibitory effects of Nifedipine and Nicardipine, two Ca2+ channel antagonists, on PRL release were also found to be Na+ dependent. BAY K 8644, a Ca2+ channel agonist, stimulated PRL release and Ca2+ uptake in a dose-dependent manner, and these effects were enhanced by Na+-free media. DA antagonized the stimulatory actions of BAY K 8644 on PRL release in a similar dose-dependent manner both in the presence and the absence of Na+. However, on stimulated 45Ca2+ uptake DA was less effective in the absence of Na+. The stimulatory action of TRH on PRL release was enhanced by the absence of Na+. DA antagonized the effect of TRH in a dose-dependent manner both in the presence and in the absence of Na+ but appeared more effective in the absence of the ion. The PRL-releasing effects of phorbol ester and of the Ca2+ ionophore A23187 were antagonized by DA in a Na+- independent manner. These results suggest the existence of two mechanisms of DA inhibitory action: one exerted on baseline PRL release which is Na+ dependent, receptor linked, and probably implicates potential operated Ca2+ channels; the other is exerted on stimulated PRL release, is Na+ independent, and appears to be a postreceptorial intracellular event probably involving protein kinase C and/or cytosolic Ca2+ levels.     

Effects of hypophysiotropic factors on growth hormone and prolactin secretion from somatotroph adenomas in culture

In an attempt to characterize GH and PRL secretion in acromegaly, the effects of various stimuli on GH and PRL release by cultured pituitary adenoma cells derived from acromegalic patients were studied. In addition, the PRL responses of somatotroph adenoma cells were compared to those of prolactinoma cells. GH-releasing hormone-(1-44) (GHRH) consistently stimulated GH secretion in all 14 somatotroph adenomas studied in a dose-dependent manner. The sensitivity as well as the magnitude of the GH responses to GHRH were highly variable in individual tissues. Somatotroph adenomas that did not respond to dopamine were more sensitive and had greater GH responses to GHRH. In 8 of 9 somatotroph adenomas that concomitantly secreted PRL, the addition of GHRH likewise increased PRL release. Omission of extracellular Ca2+ blocked the stimulatory effect of GHRH on GH and PRL secretion. When cells were coincubated with 0.1 nM somatostatin, GH and PRL secretion induced by 10 nM GHRH were completely blocked in most adenomas. Similarly, coincubation of dopamine resulted in inhibition of GHRH-induced hormone secretion in some adenomas. Addition of TRH to the incubation medium, on the other hand, significantly stimulated GH secretion in 8 of 14 adenomas, while TRH stimulated PRL release in all of the adenomas. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) produced an increase in GH and PRL secretion in other adenomas. In prolactinoma cells, somatostatin and dopamine unequivocally suppressed PRL secretion; however, other stimuli including GHRH, VIP, and CRF were ineffective. TRH induced a significant increase in PRL secretion in only one prolactinoma. These results suggest that responsiveness to GHRH and somatostatin is preserved in somatotroph adenomas; the responsiveness to GHRH is inversely correlated to that to dopamine; and PRL cells associated with somatotroph adenomas possess characteristics similar to those of GH cells. Further, the GH stimulatory actions of TRH and VIP are different.