Sequential activation of different Ca2+ entry pathways upon cholinergic stimulation in mouse pancreatic acinar cells.

1. We have studied capacitative calcium entry (CCE) under different experimental conditions in fura-2-loaded mouse pancreatic acinar cells by digital microscopic fluorimetry. CCE was investigated during [Ca2+]i decay after cell stimulation with a supramaximal concentration of ACh (10 microM) or during Ca2+ readmission in Ca2+-depleted cells (pretreated with thapsigargin or ACh). 2. La3+ and Zn2+ (100 microM) inhibited CCE during Ca2+ readmission but had negligible effects during ACh decay. In contrast flufenamic acid (100 microM), an inhibitor of non-selective cation channels, genistein (10 microM), a broad-range tyrosine kinase inhibitor, and piceatannol (10 microM), an inhibitor specific for non-receptor Syk tyrosine kinase, inhibited CCE during ACh decay but not during Ca2+ reintroduction. 3. Simultaneous detection of Mn2+ entry and [Ca2+]i measurement showed that, in the presence of extracellular calcium, application of 100 microM Mn2+ during ACh decay resulted in manganese influx without alteration of calcium influx, whilst when applied during Ca2+ readmission, Mn2+ entry was significantly smaller and induced a clear inhibition of CCE. 4. Application of the specific protein kinase C inhibitor GF109293X (3 microM) reduced CCE in Ca2+-depleted cells, whereas the activator phorbol 12-myristate, 13-acetate (3 microM) increased Ca2+ entry. 5. Based on these results we propose that cholinergic stimulation of mouse pancreatic acinar cells induces Ca2+ influx with an initial phase operated by a non-specific cation channel, sensitive to flufenamic acid and tyrosine kinase inhibitors but insensitive to lanthanum and divalent cations, followed by a moderately Ca2+-selective conductance inhibited by lanthanum and divalent cations.