Short pulses of acetylcholine stimulation induce cytosolic Ca2+ signals that are excluded from the nuclear region in pancreatic acinar cells

We have investigated the spreading of cytosolic Ca2+ signals generated by acetylcholine stimulation (using either microionophoresis or pressure application) of isolated pancreatic acinar cells (or small cell clusters) using confocal microscopy of Ca2+-sensitive fluorescence (fura red). We have been particularly interested in the effects of short vigorous pulses of acetylcholine (ACh) stimulation since, in the pancreas, ACh secreted from nerve endings is quickly eliminated by the action of ACh esterase. We focused on three regions: the secretory pole (secretory granule area), the nucleus and the basal area outside the nucleus, The nuclei were visualized by using the specific nuclear stain Hoechst 33342, With ion ophoretic application, a long-lasting stimulation with ACh (10 s and longer) induces large Ca2+ transients of similar amplitude in all the three selected regions of the cell, Short applications (about 3 s) of ACh result in a Ca2+ rise in the secretory pole, whereas no changes in cytoplasmic Ca2+ were detected in the basal, nonnuclear region or in the nucleus. We found that at the peak of such localised Ca2+ responses, evoked either by ACh ionophoresis or pressure application, significant Ca2+ concentration gradients (up to 400 nM/mu m) can be established along the line connecting the secretory pole with the nucleus, In some experiments slightly longer applications (about 5 s) of ACh produce Ca2+ transients in both the secretory region and in the basal, nonnuclear regions of the cells, whereas the nuclear [Ca2+] remained largely unaffected. Estimation of the ACh concentration in the vicinity of the cell under investigation indicated that values of about 1 mu M were attained in the pressure application experiments. These results show directly that the nucleus of pancreatic acinar cells can be effectively protected from relatively large Ca2+ transients generated in the secretory pole of pancreatic acinar cells by short pulses of near-maximal ACh concentrations. This indicates that calcium-dependent secretion (both fluid and digestive enzymes) can occur without changes of the intranuclear [Ca2+] and consequently without activation of numerous calcium dependent nuclear processes.