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

1We 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+]idecay after cell stimulation with a supramaximal concentration of ACh (10 μM) or during Ca2+readmission in Ca2+‐depleted cells (pretreated with thapsigargin or ACh).2La3+and Zn2+(100 μM) inhibited CCE during Ca2+readmission but had negligible effects during ACh decay. In contrast flufenamic acid (100 μM), an inhibitor of non‐selective cation channels, genistein (10 μM), a broad‐range tyrosine kinase inhibitor, and piceatannol (10 μM), an inhibitor specific for non‐receptor Syk tyrosine kinase, inhibited CCE during ACh decay but not during Ca2+reintroduction.3Simultaneous detection of Mn2+entry and [Ca2+]imeasurement showed that, in the presence of extracellular calcium, application of 100 μM 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.4Application of the specific protein kinase C inhibitor GF109293X (3 μM) reduced CCE in Ca2+‐depleted cells, whereas the activator phorbol 12‐myristate, 13‐acetate (3 μM) increased Ca2+entry.5Based 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.