Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts.
pH regulation in isolated guinea pig pancreatic interlobular duct segments loaded with the pH-sensitive fluorophore, 5-(6)-carboxy-SNARF-1-acetoxymethyl ester (SNARF-1), was characterized by laser-scanning confocal microscopy. In HCO3(-)-free medium, intracellular pH (pHi) 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-, pHi acidified in Na(-)-free medium but not in amiloride-containing medium. Superfusion with Cl(-)-free buffers or with buffers containing 4,4'-diisothiocyanostilbene-2,2'-disulfonic 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 ducts. pHi recovered significantly from an NH4Cl pulse in HCO3(-)-free buffers containing amiloride and carbachol (50.4%) or amiloride and secretin (40.6%). This recovery was blocked by the H(+)-adenosinetriphosphatase (H(+)-ATPase) inhibitor bafilomycin A1 and by preincubation of ducts with nocodazole or cytochalasin D. These observations suggest that a vesicular H(+)-ATPase augments Na(+)-dependent H+ extrusion during agonist-stimulated bicarbonate secretion and that activation of this transport mechanism involves cytoskeletal elements.
de Ondarza
J
J
Department of Physiology, Michigan State University, East Lansing 48824-1101, USA.
Hootman
S R
SR
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017920:Antiporters / Q000378:metabolism; D001639:Bicarbonates / Q000494:pharmacology; D027963:Chloride-Bicarbonate Antiporters; D018679:Cholinergic Agonists / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology; D006168:Guinea Pigs; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D018942:Macrolides; D018613:Microscopy, Confocal; D010183:Pancreatic Ducts / Q000166:cytology / Q000378:metabolism*; D012633:Secretin / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
D000900:Anti-Bacterial Agents; D017920:Antiporters; D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D018679:Cholinergic Agonists; D018942:Macrolides; D017923:Sodium-Hydrogen Exchangers; D012633:Secretin; D000584:Amiloride; D006859:Hydrogen; C040929:bafilomycin A1