Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 104/cm2 showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 104/cm2 largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.

Evidence increasingly shows that astrocytes play a pivotal role in brain physiology and pathology via calcium dependent processes, thus the characterization of the calcium dynamics in astrocytes is of growing importance. We have previously reported that the epidermal growth factor and basic fibroblast growth factor up-regulate the oscillation of the calcium releases that are induced by stimuli, including glutamate in cultured astrocytes. This calcium oscillation is assumed to involve protein kinase C (PKC), which is activated together with the calcium releases as a consequence of inositol phospholipid hydrolysis. In the present study, this issue has been investigated pharmacologically by using astrocytes cultured with and without the growth factors. The pharmacological activation of PKC largely reduced the glutamate-induced oscillatory and non-oscillatory calcium increases. Meanwhile, PKC inhibitors increased the total amounts of both calcium increases without affecting the peak amplitudes and converted the calcium oscillations to non-oscillatory sustained calcium increases by abolishing the falling phases of the repetitive calcium increases. Furthermore, the pharmacological effects were consistent between both glutamate- and histamine-induced calcium oscillations. These results suggest that PKC up-regulates the removal of cytosolic calcium in astrocytes, and this up-regulation is essential for calcium oscillation in astrocytes cultured with growth factors.

Abstract. Phosphoinositide-3 kinase (PI3K) and phospholipase C (PLC) utilize the same phosphoinositides as substrates to produce different signaling molecules. These enzymes are activated by a similar set of cell signaling mechanisms, i.e., tyrosine kinases and G proteins, and affect common cell functions, including proliferation, motility, and intracellular trafficking. Despite these similarities, the interplay between these enzymes is not well understood. To address this issue, the effects of the PI3K inhibitor LY294002 on carbachol-induced calcium increase in PC12h cells were examined. As carbachol stimulates both Gq-and Gi-coupled muscarinic acetylcholine receptors (mAChRs), PI3K and PLC are activated simultaneously in this protocol. LY294002 was found to reduce the carbachol-induced calcium increase, and the reduction was attributed to suppression of calcium entry. As LY294002 did not affect either carbachol-induced calcium release or calcium entry induced by calcium store depletion, this agent was found to suppress calcium entry directly activated by mAChRs. Although PI3K was supposed to compete for substrates with PLC, the PI3K inhibitor did not enhance PLC-dependent cellular responses. As LY294002 was still effective by treating cells after carbachol stimulation, it is likely that this agent blocks the calcium entry channels directly.

Abstract. We investigated the effects of bifemelane, a nootropic drug, on the intracellular calcium concentration ([Ca 2+ ] i ) in rat cerebral astrocytes using a Ca 2+ imaging device. At concentrations of 10 -30 µM, bifemelane induced a slow onset and small increase in the [Ca 2+] i , while at higher concentrations (100 -300 µM), it induced a rapid transient increase in the [Ca 2+] i during administration and a second large increase was seen during drug washout. The first peak was observed in Ca 2+ -free medium, but its onset was significantly delayed, and no second peak was seen. Neither of these effects was seen in cells treated with thapsigargin, a specific inhibitor of endoplasmic reticulum Ca 2+-ATPase, in Ca 2+ -free medium. When thapsigargintreated astrocytes were returned to normal medium containing Ca 2+ (1.8 mM), the [Ca 2+ ] i increased significantly, and this effect was reversely inhibited by bifemelane. We conclude that bifemelane causes the first peak by stimulating release from intracellular Ca 2+ stores and the second by capacitive entry through store-operated Ca 2+ channels. Although the detail mechanisms of action of the drug are still unknown, bifemelane will be provided as a pharmacological tool for basic studies on astrocytes.

Phosphatidylinositol hydrolysis and subsequent increases in intracellular calcium, activated by G-protein-coupled receptors or receptor tyrosine kinases, are important regulators of various cellular functions [1,2].The initial step in receptor-mediated phosphatidylinositol (PtdIns) metabolism involves the activation of phospholipase C (PLC), which in turn hydrolyzes PtdIns. When the substrate is phosphatidylinositol The production and further metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] require several calcium-dependent enzymes, but little is known about subsequent calcium-dependent changes in cellular Ins(1,4,5)P 3 . To study the calcium dependence of muscarinic acetylcholine receptor-induced Ins(1,4,5)P 3 increases in PC12h cells, we utilized an Ins(1,4,5)P 3 imaging system based on fluorescence resonance energy transfer and using green fluorescent protein variants fused with the pleckstrin homology domain of phospholipase C-d1. The intracellular calcium concentration, monitored by calcium imaging, was adjusted by thapsigargin pretreatment or alterations in extracellular calcium concentration, enabling rapid receptor-independent changes in calcium concentration via storeoperated calcium influx. We found that Ins(1,4,5)P 3 production was increased by a combination of receptor-and calcium-dependent components, rather than by calcium alone. The level of Ins(1,4,5)P 3 induced by the receptor was found to be half that induced by the combined receptor and calcium components. Increases in calcium levels prior to receptor activation did not affect the subsequent receptor-induced Ins(1,4,5)P 3 increase, indicating that calcium does not influence Ins(1,4,5)P 3 production without receptor activation. Removal of both the receptor agonists and calcium rapidly restored calcium and Ins(1,4,5)P 3 levels, whereas removal of calcium alone restored calcium to its basal concentration. Similar calciumdependent increases in Ins(1,4,5)P 3 were also observed in Chinese hamster ovary cells expressing m1 muscarinic acetylcholine receptor, indicating that the observed calcium dependence is common to Ins(1,4,5)P 3 production. To our knowledge, our results are the first showing receptor-and calciumdependent components within cellular Ins(1,4,5)P 3 .