CYTOPLASMIC CA-2+ OSCILLATIONS EVOKED BY RECEPTOR STIMULATION, G-PROTEIN ACTIVATION, INTERNAL APPLICATION OF INOSITOL TRISPHOSPHATE OR CA-2+ SIMULTANEOUS MICROFLUORIMETRY AND CA-2+ DEPENDENT CL- CURRENT RECORDING IN SINGLE PANCREATIC ACINAR-CELLS

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-γ-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration ([Ca2+](i)) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2+-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 μM) evoked an oscillating increase in [Ca2+](i) measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2+-dependent Cl- current reporting [Ca2+](i) close to the cell membrane. In the same cells a lower ACh concentration (0.05 μM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 μM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+](i) oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+](i) oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 μM GTP-γ-S evoked [Ca2+](i) oscillations with a similar pattern. Ins (1,4,5) P3 (10 μM) evoked repetitive shortlasting spikes in [Ca2+](i) that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Cafeine (1 mM) broadened these Ca2+ pulses. [Ca2+](i) was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette. The relatively smooth rise and fall in [Ca2+](i) seen in the microfluorimetric recordings in response to a 3 min Ca2+ infusion were accompanied by a similar rise and fall in the Cl- current. Before the peak and in the following falling phase repetitive short-lasting Cl- current spikes occurred. We conclude that receptor stimulation generates cytoplasmic Ca2+ spikes by Ins (1,4,5) P3-evoked Ca2+ release causing pulses of Ca2+-induced Ca2+ release primarily from pools close to the plasma membrane.