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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.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.

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.

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.

Human pancreatic growth hormone-releasing factor (GRF-44-NH2) stimulated growth hormone (GH) secretion and intracellular cyclic AMP levels in cultured pituitary cells from both sheep and rat. Somatostatin (SRIF), over a wide range of doses and time, showed no significant effect on the elevated cyclic AMP levels in sheep cells, but did block the GH release in a dose-dependent manner. In rat cells, however, SRIF inhibited GRF-stimulated cyclic AMP levels by 75% maximum (still 8-fold greater than the basal levels) and GH release to almost half the basal value. We conclude that somatostatin inhibits GRF-elevated cyclic AMP levels in rat pituitary cells but not in sheep cells.

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.

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.