CONFOCAL MICROSCOPIC ANALYSIS OF INTRACELLULAR PH REGULATION IN ISOLATED GUINEA-PIG PANCREATIC DUCTS

pH regulation in isolated guinea pig pancreatic interlobular duct segm ents loaded with the pH-sensitive fluorophore, 5-(6)-carboxy- SNARF-1- acetoxymethyl ester (SNARF-1), was characterized by laser-scanning con focal microscopy. In HCO3--free medium, intracellular pH(pH(i))of duct epithelial cells fell by 0.32 +/- 0.06 pH units in the presence of 0. 5 mM amiloride and by 0.36 +/- 0.08 pH units in the absence of Na+. In the presence of extracellular HCO3-, pH(i) acidified in Na+-free medi um but not in amiloride-containing medium. Superfusion with Cl--free b uffers or with buffers containing 4,4'-diisothiocyanostilbene-2,2'-dis ulfonic acid produced a cytosolic alkalinization of 0.13-0.22 pH units . These observations demonstrate the presence of Na+/H+ exchange, Na+- HCO3- cotransport, and Cl-/HCO3- exchange in guinea pig pancreatic duc ts. pH(i) recovered significantly from an NH4Cl pulse in HCO3--free bu ffers containing amiloride and carbachol (50.4%) or amiloride and secr etin (40.6%). This recovery was blocked by the H+-adenosinetriphosphat ase (H+-ATPase) inhibitor bafilomycin A(1) and by preincubation of duc ts with nocodazole or cytochalasin D. These observations suggest that a vesicular H+-ATPase augments Na+-dependent H+ extrusion during agoni st-stimulated bicarbonate secretion and that activation of this transp ort mechanism involves cytoskeletal elements.