Visualization of the secretory process involved in Ca2+-activated fluid secretion from rat submandibular glands using the fluorescent dye, calcein

The central feature of fluid and electrolyte secretion by salivary acinar c ells is transepithelial Cl- movement as a driving force for the secretion. However, little is known about the membrane localization and regulation by agonists of various anion channels. To characterize the anion transport and fluid secretion, we visualized the secretory process induced by the cholin ergic agonist, carbachol (CCh), using the anionic fluorescent dye, calcein, under a confocal laser scanning microscope. The fluorescence of calcein lo aded into the isolated acini was spread diffusely throughout the cytoplasm and was less intense in the secretory vesicles which occupied the apical po le. Cytoplasmic calcein was released into intercellular canaliculi just aft er the addition of CCh, depending upon a rise in [Ca2+](i) by Ca2+ release from intracellular stores. Thereafter, the formation of watery vacuoles con nected with intercellular canaliculi was visualized in the calcein-loaded a cini, depending upon external Ca2+. Both the calcein release and vacuole fo rmation were inhibited by suppressing the Ca2+-activated K+ efflux. The cal cein release was also affected by the external anion substitution, suggesti ng that calcein is released through an anion channel. in the isolated, perf used glands, CCh-induced fluid secretion was sustained in two phases, where as the loaded calcein was initially and transiently released into the saliv a. By revealing the [Ca2+](i) dependence and sensitivities to channel block ers, our results suggest that the initial phase of CCh-induced fluid secret ion was evoked in association with the release of the organic anion, calcei n, and the late phase of fluid secretion, during which calcein is less perm eable, was associated with the formation of watery vacuoles. Thus, the anio n channels possessing the distinct property of anion permeation may be acti vated in the initial phase and late phase. These results indicate that the anionic fluorescent dye, calcein, is useful fur visualizing the process of Ca2+-dependent fluid secretion, and for clarifying the relation between flu id secretion and anion transport.