Membrane potential regulates Ca2+ uptake and inositol phosphate generation in rat sublingual mucous acini.

In salivary acinar cells, muscarinic-induced fluid secretion is associated with a 1,4,5-IP3 induced increase in the cytosolic free Ca2+ concentration ([Ca2+]i), which in turn activates Ca(2+)-dependent K+ and Cl- channels that modulate the membrane potential. In the present study the influence of the membrane potential on [Ca2+]i and inositol phosphates was monitored in rat sublingual mucous acini. Depolarization induced by switching from 5.8 mM extracellular K+ ([K+]e) to 116 mM [K+]e resulted in a transient increase in the [Ca2+]i measured using the Ca2+ sensitive fluorescent indicator Fura-2. This initial rapid (t1/2 approximately 5 s) increase (approximately 3-fold) in [Ca2+]i was dependent on extracellular Ca2+, insensitive to nifedipine, and followed by establishment of a 'new' resting [Ca2+]i, approximately 35% higher than the level in physiological [K+]e. Depolarization also induced a significant rise in the resting cellular inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) contents, but not 1,4,5-IP3 content. Stimulation with 10 microM carbachol (CCh, a muscarinic agonist) produced a biphasic increase in [Ca2+]i, the initial transient phase due to mobilization of Ca2+ from an intracellular pool, and a sustained phase mediated by an influx of Ca2+. Membrane depolarization had no effect on the initial phase, while, the sustained increase in [Ca2+]i was eliminated. The CCh-enhanced quench of the Fura-2 signal by Mn2+ (an index for divalent cation entry) was reversibly inhibited by depolarization. The enhanced Mn2+ uptake induced by inhibiting microsomal Ca(2+)-ATPase with thapsigargin was similarly inhibited by membrane depolarization, consistent with the effect of depolarization primarily acting on the Ca2+ entry pathway and not on receptor coupling. Depolarization did not alter the initial CCh-induced increases in IP3, IP4 or 1,4,5-IP3 content, or the sustained increase in 1,4,5-IP3, whereas, depolarization significantly blunted (> 70%) the sustained, CCh-induced generation of IP3 and IP4. The membrane potential, therefore, appears to modulate Ca2+ activated fluid secretion by controlling the driving force for Ca2+ entry via a depletion-activated Ca2+ entry pathway. Inositol phosphate metabolism is also influenced by the membrane potential, but this effect apparently plays a minor role in regulating [Ca2+]i since 1,4,5-IP3 levels were unchanged by depolarization.