SHORT PULSES OF ACETYLCHOLINE STIMULATION INDUCE CYTOSOLIC CA2-CELLS(SIGNALS THAT ARE EXCLUDED FROM THE NUCLEAR REGION IN PANCREATIC ACINAR)

We have investigated the spreading of cytosolic Ca2+ signals generated by acetylcholine stimulation (using either microionophoresis or press ure application) of isolated pancreatic acinar cells (or small cell cl usters) using confocal microscopy of Ca2+-sensitive fluorescence (fura red). We have been particularly interested in the effects of short vi gorous pulses of acetylcholine (ACh) stimulation since, in the pancrea s, ACh secreted from nerve endings is quickly eliminated by the action of ACh esterase. We focused on three regions: the secretory pole (sec retory granule area), the nucleus and the basal area outside the nucle us, The nuclei were visualized by using the specific nuclear stain Hoe chst 33342, With ion ophoretic application, a long-lasting stimulation with ACh (10 s and longer) induces large Ca2+ transients of similar a mplitude in all the three selected regions of the cell, Short applicat ions (about 3 s) of ACh result in a Ca2+ rise in the secretory pole, w hereas no changes in cytoplasmic Ca2+ were detected in the basal, nonn uclear region or in the nucleus. We found that at the peak of such loc alised Ca2+ responses, evoked either by ACh ionophoresis or pressure a pplication, significant Ca2+ concentration gradients (up to 400 nM/mu m) can be established along the line connecting the secretory pole wit h 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 [Ca 2+] remained largely unaffected. Estimation of the ACh concentration i n the vicinity of the cell under investigation indicated that values o f about 1 mu M were attained in the pressure application experiments. These results show directly that the nucleus of pancreatic acinar cell s can be effectively protected from relatively large Ca2+ transients g enerated in the secretory pole of pancreatic acinar cells by short pul ses of near-maximal ACh concentrations. This indicates that calcium-de pendent secretion (both fluid and digestive enzymes) can occur without changes of the intranuclear [Ca2+] and consequently without activatio n of numerous calcium dependent nuclear processes.