Possible mechanisms regulating ATP- and thimerosal-induced Ca2+ oscillations in the HSY salivary duct cell line

The ATP-induced oscillatory changes in cytosolic Ca2+ concentration ([Ca2+] (i)) were analysed in HSY cells, a salivary ductal cell line from human par otid, using a fluorescence ratio imaging system. At concentrations higher t han 1 muM, ATP caused sinusoidal [Ca2+](i) oscillations due to the periodic release and reuptake of Ca2+ by intracellular Ca2+ stores. The phorbol eat er 4 beta -phorbol 12,13-dibutyrate (PDBu) changed the [Ca2+](i) oscillatio ns to a single spike. The inhibitory effect of PDBu on the [Ca2+](i) signal s was reversed by protein kinase C (PKC) inhibitors such as staurosporine a nd chelerythrine chloride. However, preincubation of the cells with the PKC inhibitors did not affect the pattern of the ATP-induced [Ca2+](i) oscilla tions. The desensitization of the [Ca2+](i) response observed during prolon ged stimulation with ATP was also not prevented by the PKC inhibitors. Incu bation of HSY cells with the sulphydryl reagent thimerosal, which enhances the sensitivity of inositol 1,4.5-trisphosphate (IP3) receptors. caused rep etitive Ca2+ release from intracellular Ca2+ stores resulting in baseline s pikes of [Ca2+](i). The thimerosal-induced [Ca2+](i) oscillations did not c hange in the presence of PDBu and the phospholipase C inhibitor U73122. Thu s, we could not provide evidence that negative feedback by PKC plays a cent ral role in the regulation of ATP-induced [Ca2+](i) oscillations. These res ults suggest that the [Ca2+](i) oscillations, at least the baseline spikes, in HSY cells can be generated without stimulating the formation of IP3. (C ) 2001 Elsevier Science B.V. All rights reserved.