No abstract

We have investigated the effects of histamine on the intracellular calcium concentration ([Ca2+]i) of cultured rat cerebellar astrocytes using fura‐2‐based Ca2+ imaging microscopy. Most of the cells responded to the application of histamine with an increase in [Ca2+]i which was antagonized by the H1 receptor blocker mepyramine. When histamine was applied for several minutes, the majority of the cells displayed a biphasic Ca2+ response consisting of an initial transient peak and a sustained component. In contrast to the initial transient [Ca2+]i response, the sustained, receptor‐activated increase in [Ca2+]i was rapidly abolished by chelation of extracellular Ca2+ or addition of Ni2+, Mn2+, Co2+ and Zn2+, but was unaffected by nifedipine, an antagonist of L‐type voltage‐activated Ca2+ channels. These data indicate that the sustained increase in [Ca2+]i was dependent on Ca2+ influx. When intracellular Ca2+ stores were emptied by prolonged application of histamine in Ca2+‐free conditions, Ca2+ re‐addition after removal of the agonist did not lead to an ‘overshoot’ of [Ca2+]i indicative of store‐operated Ca2+ influx. However, Ca2+ stores were refilled despite the absence of any substantial change in the fura‐2 signal. Depletion of intracellular Ca2+ stores using cyclopiazonic acid in Ca2+‐free saline and subsequent re‐addition of Ca2+ to the saline resulted in an increase in [Ca2+]i that was significantly enhanced in the presence of histamine. The results suggest that besides capacitative mechanisms, a non‐capacitative, voltage‐independent pathway is involved in histamine‐induced Ca2+ entry into cultured rat cerebellar astrocytes.

We have investigated control mechanisms involved in the propagation of agonist-induced Ca2+ waves in isolated mouse pancreatic acinar cells. Using a confocal laser-scanning microscope, we were able to show that maximal stimulation of cells with acetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initial Ca2+ release of comparable amounts with both agonists at the luminal cell pole. Subsequent Ca2+ spreading to the basolateral membrane was faster with ACh (17.3 ± 5.4 μm/s) than with bombesin (8.0 ± 2.2 μm/s). The speed of bombesin-induced Ca2+ waves could be increased up to the speed of ACh-induced Ca2+waves by inhibition of protein kinase C (PKC). Activation of PKC significantly decreased the speed of ACh-induced Ca2+ waves but had only little effect on bombesin-evoked Ca2+waves. Within 3 s after stimulation, production of inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3] was higher in the presence of ACh compared with bombesin, whereas bombesin induced higher levels of diacylglycerol (DAG) than ACh. These data suggest that the slower propagation speed of bombesin-induced Ca2+ waves is due to higher activation of PKC in the presence of bombesin compared with ACh. The higher increase in bombesin- compared with ACh-induced DAG production is probably due to activation of phospholipase D (PLD). Inhibition of the PLD-dependent DAG production by preincubation with 0.3% butanol led to an acceleration of the bombesin-induced Ca2+ wave. In further experiments, we could show that ruthenium red (100 μM), an inhibitor of Ca2+-induced Ca2+ release in skeletal muscle, also decreased the speed of ACh-induced Ca2+ waves. The effect of ruthenium red was not additive to the effect of PKC activation. From the data, we conclude that, following Ins(1,4,5) P 3-induced Ca2+ release in the luminal cell pole, secondary Ca2+ release from stores, which are located in series between the luminal and the basal plasma membrane, modifies Ca2+spreading toward the basolateral cell side by Ca2+-induced Ca2+ release. Activation of PKC leads to a reduction in Ca2+release from these stores and therefore could explain the slower propagation of Ca2+ waves in the presence of bombesin compared with ACh.

We have investigated the effect of genistein on the hormone-stimulated Ca2+ influx and on a 28pS nonselective cation channel in mouse pancreatic acinar cells using the Ca2+ indicator fluo3 and the patch-clamp technique. The identity of the Ca2+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca2+-dependent nonselective cation channel as a potential pathway for Ca2+ influx. Capacitative Ca2+ entry was induced by depletion of intracellular Ca2+ stores with 500nM acetylcholine or with the Ca2+ ATPase inhibitor 2,5di-tert- butylhydroquinone. In the presence of 100microM genistein, Ca2+ release was unimpaired, whereas Ca2+ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca2+-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28pS nonselective cation channel does not contribute to Ca2+ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca2+ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca2+ entry mechanism in mouse pancreatic acinar cells is unclear.

We have used fluo 3-loaded mouse pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). The Ca2+-mobilizing agonist ACh (500 nM) induced a Ca2+ release in the luminal cell pole followed by spreading of the Ca2+ signal toward the basolateral side with a mean speed of 16.1 ± 0.3 μm/s. In the presence of an acidic pHi, achieved by blockade of the Na+/H+exchanger or by incubation of the cells in a Na+-free buffer, a slower spreading of ACh-evoked Ca2+ waves was observed (7.2 ± 0.6 μm/s and 7.5 ± 0.3 μm/s, respectively). The effects of cytosolic acidification on the propagation rate of ACh-evoked Ca2+ waves were largely reversible and were not dependent on the presence of extracellular Ca2+. A reduction in the spreading speed of Ca2+ waves could also be observed by inhibition of the vacuolar H+-ATPase with bafilomycin A1 (11.1 ± 0.6 μm/s), which did not lead to cytosolic acidification. In contrast, inhibition of the endoplasmic reticulum Ca2+-ATPase by 2,5-di- tert-butylhydroquinone led to faster spreading of the ACh-evoked Ca2+ signals (25.6 ± 1.8 μm/s), which was also reduced by cytosolic acidification or treatment of the cells with bafilomycin A1. Cytosolic alkalinization had no effect on the spreading speed of the Ca2+ signals. The data suggest that the propagation rate of ACh-induced Ca2+ waves is decreased by inhibition of Ca2+ release from intracellular stores due to cytosolic acidification or to Ca2+ pool alkalinization and/or to a decrease in the proton gradient directed from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool to the cytosol.