A CAPACITATIVE CA2+ INFLUX IS REQUIRED FOR SUSTAINED FLUID SECRETION IN SUBLINGUAL MUCOUS ACINI

The Ca2+ dependence of muscarinic-induced fluid and electrolyte secretion was studied using rat sublingual mucous gland preparations. During stimulation, secretions from vascularly perfused glands were totally inhibited when perfused with a Ca2+-free medium. Fluid secretion correlated with sustained losses of K-42+ and Cl-36-content and sustained increases in Na-22+ content and the intracellular free Ca2+ concentration ([Ca2+]i) in fura-2-loaded acini. The magnitudes of the initial agonist-induced changes in Na+, K+, and Cl- content and [Ca2]i were unaltered in a Ca2+-free medium, whereas extracellular Ca2+ removal resulted in the recovery of these ions during the sustained phase to pre-stimulation levels. The recovery of Cl- content induced by Ca2+ depletion was totally blocked in the presence of bumetanide, an inhibitor of Na+-K+-2Cl- cotransport, while 4,4'-diisothiocyanostilbene-2,2'-disulfonic aicd (DIDS), inhibitor of anion exchange, did not influence Cl-recovery in a HCO3-containing solution (25 mM NaHCO3, 5% CO2). The stimulated increase in [Ca2+]i was not inhibited by the addition of voltage-activated Ca2+ channel blockers (D 888, nifedipine, and diltiazem) or in a Na+-free medium. Studies using the quench of fura-2 by Mn2+ as an index of Ca2+ influx and thapsigargin, an inhibitor of microsomal Ca2+-ATPase, indicate that a capacitative Ca2+ entry pathway mediates Ca2+ entry during stimulation. The above data demonstrate that Ca2+ uptake, which is dependent on the refill status of the agonist-sensitive intracellular Ca2+ pool, is a prerequisite for sustained muscarinic-induced fluid and electrolyte secretion in the rat sublingual mucous gland.