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

C. Camello, J. A. Pariente, G. M. Salido and P. J. Camello

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 µM) or during Ca²⁺ readmission in Ca²⁺-depleted cells (pretreated with thapsigargin or ACh).

La³⁺ 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.

Simultaneous detection of Mn²⁺ entry and [Ca²⁺]_i measurement 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.

Application 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.

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.

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