No abstract

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP‐gamma‐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 microM) 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 microM) 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 microM 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 microM GTP‐gamma‐S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) 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. Caffeine (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.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol 1,4,5-trisphosphate (Ins P3) is a second messenger releasing intracellular Ca2+ into the cytosol. It has recently been proposed that inositol 1,3,4,5-tetrakisphosphate (Ins P4), which is formed from Ins P3 by Ins P3-3-kinase, acts with Ins P3 as a second messenger by promoting extracellular Ca2+ entry. It has been suggested that Ins P3 itself can act to stimulate Ca2+ uptake from the extracellular fluid, although a physiological function for Ins P4 was not excluded. Transmembrane currents can now be measured in single cells by voltage clamping under conditions where the intracellular perfusion fluid can be changed several times during individual experiments. We have used this method to test the effects of Ins P3 and Ins P4 on the Ca2+-activated K+ current, and now show that neither Ins P3 alone nor Ins P4 alone can activate a sustained current, whereas Ins P3 and Ins P4 in combination evoke a sustained increase in Ca2+-activated K+ current which is dependent on external Ca2+.

Nervous or hormonal stimulation of many exocrine glands evokes release of cellular K+ (ref. 1), as originally demonstrated in mammalian salivary glands2,3, and is associated with a marked increase in membrane conductance1,4,5. We now demonstrate directly, by using the patch-clamp technique6, the existence of a K+ channel with a large conductance localized in the baso-lateral plasma membranes of mouse and rat salivary gland acinar cells. The K+ channel has a conductance of approximately 250 pS in the presence of high K+ solutions on both sides of the membrane. Although mammalian exocrine glands are believed not to possess voltage-activated channels1,7, the probability of opening the salivary gland K+ channel was increased by membrane depolarization. The frequency of channel opening, particularly at higher membrane potentials, was increased markedly by elevating the internal ionized Ca2+ concentration, as previously shown for high-conductance K+ channels from cells of neural origin8-10. The Ca2+ and voltage-activated K+ channel explains the marked cellular K+ release that is characteristically observed when salivary glands are stimulated to secrete.