Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells.
Regulation of cytosolic free Na (Nai) was measured in isolated rabbit gastric glands with the use of a recently developed fluorescent indicator for sodium, SBFI. Intracellular loading of the indicator was achieved by incubation with an acetoxymethyl ester of the dye. Digital imaging of fluorescence was used to monitor Nai in both acid-secreting parietal cells and enzyme-secreting chief cells within intact glands. In situ calibration of Nai with ionophores indicated that SBFI fluorescence (345/385 nm excitation ratio) could resolve 2 mM changes in Nai and was relatively insensitive to changes in K or pH. Measurements on intact glands showed that basal Nai was 8.5 +/- 2.2 mM in parietal cells and 9.2 +/- 3 mM in chief cells. Estimates of Na influx and efflux were made by measuring rates of Nai change after inactivation or reactivation of the Na/K ATPase in a rapid perfusion system. Na/K ATPase inhibition resulting from the removal of extracellular K (Ko) caused Nai to increase at 3.2 +/- 1.5 mM/min and 3.5 +/- 2.7 mM/min in parietal and chief cells, respectively. Na buffering was found to be negligible. Addition of 5 mM Ko and removal of extracellular Na (Nao) caused Nai to decrease rapidly toward 0 mM Na. By subtracting passive Na efflux under these conditions (the rate at which Nai decreased in Na-free solution containing ouabain), an activation curve (dNai/Nai) for the Na/K ATPase was calculated. The pump demonstrated the greatest sensitivity between 5 and 20 mM Nai. At 37 degrees C the pump rate was less than 3 mM/min at 5 mM Nai and 26 mM/min at 25 mM Nai, indicating that the pump has a great ability to respond to changes in Nai in this range. Carbachol, which stimulates secretion from both cell types, was found to stimulate Na influx in both cell types, but did not have detectable effects on Na efflux. dbcAMP+IBMX, potent stimulants of acid secretion, had no effect on Na metabolism.
Negulescu
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PA
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Harootunian
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A
Tsien
R Y
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Machen
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TE
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D003994:Bucladesine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004988:Ethers, Cyclic; D005470:Fluorometry; D005753:Gastric Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D010295:Parietal Cells, Gastric / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013268:Stimulation, Chemical
D001572:Benzofurans; D004988:Ethers, Cyclic; C061665:sodium-binding benzofuran isophthalate; D003994:Bucladesine; D002217:Carbachol; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D015056:1-Methyl-3-isobutylxanthine