EXTRACELLULAR ION GRADIENTS ARE BEING MEASURED AT THE APICAL AND BASOLATERAL POLES OF INDIVIDUAL ISOLATED NECTURUS OXYNTIC CELLS SUSPENDED IN A LIGHTLY BUFFERED (1MM HEPES) BICARBONATE CONTAINING MEDIA (5MM HCO3) USING VIBRATING (FREQUENCY = 0.5 HZ; EXCURSION=10~M) H+ SENSITIVE LIQUID ION-EXCHANGE ELECTRODES. THE MEASURED GRADIENTS CORRECTED FOR ATTENUATION CAUSED BY THE INCLUSION OF MOBIL BUFFERS IN THE BATHING MEDIA WERE USED TO ESTIMATE PROTON FLUX FROM DIFFERENT POLES OF THE CELLS. PROTON EXTRUSION OCCURS UNIFORMLY FROM THE ENTIRE SURFACE OF ISOLATED RESTING OXYNTIC CELLS. THIS BASAL PROTON EXTRUSION IS INHIBITED BY EXPOSURE OF THE CELLS TO DINITROPHENOL (10-5 M), BUT NOT TO AMILORIDE (10-4 M) OR SCH28080 (10-5 M), AN H+/K+-ATPASE INHIBITOR. 5-AMINO-SUBSTITUTED LIPOPHILIC AMILORIDE ANALOGUES, WHICH HAVE STRONGER AFFINITY FOR NA+/H+ EXCHANGERS (NHE) THAN FOR THE EPITHELIAL NA+ CHANNEL WILL BE TESTED IN CONJUNCTION WITH VARYING CONCENTRATIONS OF CIMETIDINE TO IDENTIFY THE SUBTYPE(S) OF NHE RESPONSIBLE FOR THIS METABOLICALLY-DEPENDENT BASAL SECRETION FROM THE TWO POLES OF THE CELL. STIMULATION OF RESTING CELLS WITH EITHER CAMP (10-3 M) OR HISTAMINE (10-4 M) RESULTS IN SPECIFIC AND DRAMATIC SCH28080 INHIBITABLE INCREASES OF UP TO 300% IN PROTON SECRETION FROM THE APICAL MEMBRANE ALONE DUE TO ACTIVATION OF THE H+/K+-ATPASE. CARBACHOL AND IONOMYSIN WILL BE TESTED TO DETERMINE IF FUNCTIONAL MUSCARINIC ACETYLCHOLINE RECEPTORS AND CA2+ MEDIATED INTRACELLULAR SIGNALLING PATHWAYS ALSO EXIST IN THESE CELLS FOR THE ACTIVATION OF THE H+/K+-ATPASE.
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