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Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.

We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.

We have developed a system in which the fusion of pancreatic zymogen granules with plasma membranes can be studied in vitro. Here we show that this membrane fusion event is stimulated specifically by peptides of the effector domain of rab3, a small, monomeric GTP-binding protein. In addition, we demonstrate that the stimulatory effect of the peptides involves their binding to a target on the plasma membrane, and is both qualitatively and quantitatively different from the effect of GTPγS, which also enhances membrane fusion. We suggest that regulated exocytosis in the pancreatic acinar cell may be under the control of more than one type of GTP-binding protein.

The dihydropyridine Ca2+ channel activator BAY K 8644 (1 microM) stimulated basal prolactin secretion from perifused primary cultures of anterior pituitary cells and potentiated the stimulation of prolactin secretion by 1 microM thyrotropin-releasing hormone (TRH) 5-fold over 30 min. This potentiation was mimicked by other dihydropyridine agonists CGP 28392 and (+)-SDZ 202-791 and by (-)-BAY K 8644 (1 microM), but not by (+)-BAY K 8644. The Ca2+ channel antagonist nimodipine, at a concentration sufficient to block BAY K 8644-stimulated 45Ca2+ uptake in GH4C1 anterior pituitary tumor cells, decreased basal prolactin secretion and blocked the enhancement of basal and TRH-stimulated secretion by BAY K 8644. These results suggest that dihydropyridine agonists potentiate TRH-induced secretion through interaction with known stereospecific sites on Ca2+ channels. In GH4C1 cells, BAY K 8644 alone did not affect inositol polyphosphate accumulation, but potentiated TRH-stimulated accumulation of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Accumulation of the Ca(2+)-mobilizing isomer inositol 1,4,5-trisphosphate was not potentiated, suggesting that potentiation of TRH-stimulated hormone secretion by BAY K 8644 does not result from synergistic stimulation of phospholipase C, but may correlate with enhanced inositol trisphosphate-3-kinase activity.

To investigate the relationship of changes in cytosolic free calcium concentrations [( Ca2+]c) caused by TRH to changes in PRL secretion, we simultaneously monitored PRL release and [Ca2+]c, using the fluorescent Ca2+ indicator indo-1, in freshly isolated perifused cells from rat anterior pituitary glands. We found that a 30-sec pulse of 100 nM TRH triggered a transient spike of [Ca2+]c, but prolonged PRL release for up to 30 min; continuous administration of TRH caused a sustained elevation in [Ca2+]c, but the same pattern and amount of PRL release as that caused by the pulse of TRH. PRL secretion was refractory to further pulses of TRH given at 10-min intervals for 40 min, but did respond to a second pulse of TRH given 40 min after the first pulse with no intervening pulses. Pulses of TRH given every 10 min still triggered spikes of [Ca2+]c of the same magnitude as the first pulse, indicating that the cause of the refractory state must occur at a post-receptor step that is after the mobilization of [Ca2+]c. A 30-sec pulse of a high concentration of KCl caused a transient spike of [Ca2+]c and transient, not prolonged, release. Additional pulses of KCl cause progressively less PRL release, although the magnitude of the spikes in [Ca2+]c did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate whether dopaminergic reduction of PRL secretion is caused by an inhibition of release of Ca2+ from intracellular stores, we used a perifusion system to monitor simultaneously changes in intracellular Ca2+ concentrations ([Ca2+]c) and changes in PRL secretion from rat anterior pituitary cells, and from enriched populations of lactotrophs. We eliminated influx of extracellular Ca2+ by using medium with no added Ca2+ and 0.2 mM EGTA, conditions which abolished the increase in [Ca2+]c caused by 56 mM KCl. In this low Ca2+-containing medium, 100 nM TRH induced a burst of [Ca2+]c; the magnitude of the peak was the same whether the cells were perifused with low Ca2+-containing medium for 2 or 20 min before adding TRH, indicating the source of intracellular Ca2+ was stable under these conditions. After 2 min in this medium, TRH was still able to stimulate almost as much PRL release as in medium containing 1.8 mM Ca2+, and 1 microM dopamine inhibited this release, but did not affect the magnitude of the TRH-induced increase in [Ca2+]c. Preincubation for 5 min with dopamine did not affect the ability of a 30-sec incubation with TRH to stimulate the accumulation of inositol phosphate, inositol bisphosphate, and inositol trisphosphate, either in medium containing 1.8 mM Ca2+, or in low Ca2+-containing medium. Preincubation with dopamine for 5 min had no effect on TRH-induced mobilization of intracellular calcium. A source of Ca2+ is needed to refill internal Ca2+-stores discharged by TRH, and as dopamine lowered [Ca2+]c which might fill these stores, we tested to see if dopamine prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin-releasing hormone (TRH) (1 microM) and the Ca2+-channel agonist BAY K 8644 (1 microM) each induced transient increases in prolactin secretion from primary cultures of rat anterior pituitary cells in perifusion. When BAY K 8644 was added after a TRH-induced secretory peak, the additional effect of BAY K 8644 on prolactin release was approximately twofold greater over a 30-min period than the effect of BAY K 8644 on previously untreated cells. TRH and BAY K 8644 were also synergistic when added in the opposite order or simultaneously. Substitution of other agents for BAY K 8644 revealed that only high K+ (40 mM) was at least additive with TRH in stimulating prolactin secretion; treatment with TRH inhibited, rather than facilitated, subsequent stimulation of prolactin secretion by angiotensin II (100 nM) or the ionophore A23187 (20 microM). The cooperative effect was not specific for TRH because BAY K 8644 also acted synergistically with angiotensin II or 40 mM K+. In GH4C1 cells, in which TRH and BAY K 8644 were also synergistic in releasing prolactin, measurements with the fluorescent indicator indo-1 showed that TRH and BAY K 8644 could each elevate cytosolic Ca2+ above the level stimulated by the other. Unexpectedly, TRH was found to inhibit BAY K 8644-stimulated 45Ca2+ uptake in both GH4C1 and primary cultured cells. These results indicate that BAY K 8644 and TRH synergistically stimulate prolactin secretion by a mechanism other than a cooperative effect on the activity of dihydropyridine-sensitive Ca2+ channels.

The hormones bombesin and thyrotropin-releasing hormone (TRH) stimulated formation of inositol-monophosphate, bisphosphate, trisphosphate and tetrakisphosphate with parallel time courses in GH 4 C 1 cells, while a more polar inositol polyphosphate peak, consisting of inositol-pentakisphsophate and perhaps also inositol-hexakisphosphate, was unaffected by either hormone. Although bombesin and TRH had similar potencies in stimulating inositol trisphosphate production (K m = 30 nM and 40 nM, respectively), TRH was significantly more efficacious than bombesin. Maximal stimulation of inositol-1,4,5-trisphosphate formation by TRH was not further increased by addition of a maximally effective dose of bombesin, suggesting that the two hormones act through stimulation of a common pool of phospholipase C, and this enzyme pool can be fully stimulated by TRH, alone.

Measurements of concentrations of cytosolic free Ca2+ ([Ca2+]c) in individual cells has frequently demonstrated periodic transients in [Ca2+]c rather than sustained elevated levels. To determine in anterior pituitary cells if such short and repetitive [Ca2+]c transients stimulated prolactin release, we used a perifusion system with cells loaded with the fluorescent Ca2+-indicator, indo-1. A one second pulse of 100 mM KCl caused an increase in [Ca2+]c with a half peak width of about 18 seconds and an almost coincident increase in prolactin secretion. Subsequent pulses of KCl each caused increases in [Ca2+]c and prolactin release that were the same as the first, up to a pulse frequency of one every two minutes. Increasing the frequency to 1 pulse every minute or 1 pulse every 30 seconds, however, resulted in a serial decline in the amount of prolactin released by each pulse even though each pulse caused a similar peak Ca2+ response. These findings demonstrate that cells become adapted to transient increases in [Ca2+]c of the same magnitude so that they no longer release prolactin if the increases in [Ca2+]c occur frequently enough. Cells may use frequency-encoded Ca2+ signals to stimulate release of prolactin at low frequency to prevent the adaptation that occurs at higher frequencies.