Possible mechanisms regulating ATP- and thimerosal-induced Ca 2+ oscillations in the HSY salivary duct cell line

Abstract

The ATP-induced oscillatory changes in cytosolic Ca 2+ concentration ([Ca 2+] i) were analysed in HSY cells, a salivary ductal cell line from human parotid, using a fluorescence ratio imaging system. At concentrations higher than 1 μM, ATP caused sinusoidal [Ca 2+] i oscillations due to the periodic release and reuptake of Ca 2+ by intracellular Ca 2+ stores. The phorbol ester 4β-phorbol 12,13-dibutyrate (PDBu) changed the [Ca 2+] i oscillations to a single spike. The inhibitory effect of PDBu on the [Ca 2+] i signals was reversed by protein kinase C (PKC) inhibitors such as staurosporine and chelerythrine chloride. However, preincubation of the cells with the PKC inhibitors did not affect the pattern of the ATP-induced [Ca 2+] i oscillations. The desensitization of the [Ca 2+] i response observed during prolonged stimulation with ATP was also not prevented by the PKC inhibitors. Incubation of HSY cells with the sulphydryl reagent thimerosal, which enhances the sensitivity of inositol 1,4,5-trisphosphate (IP 3) receptors, caused repetitive Ca 2+ release from intracellular Ca 2+ stores resulting in baseline spikes of [Ca 2+] i. The thimerosal-induced [Ca 2+] i oscillations did not change in the presence of PDBu and the phospholipase C inhibitor U73122. Thus, we could not provide evidence that negative feedback by PKC plays a central role in the regulation of ATP-induced [Ca 2+] i oscillations. These results suggest that the [Ca 2+] i oscillations, at least the baseline spikes, in HSY cells can be generated without stimulating the formation of IP 3.