CONTINUOUS FLUOROMETRIC MEASUREMENT OF INTRACELLULAR PH AND CA2-GLANDAND PANCREAS( IN PERFUSED SALIVARY)

Intracellular pH (pH(i)) has been measured in intact, perfused rat man dibular salivary glands loaded with the fluorescent pH indicator BCECF ',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]. Glands mounted in the cuvette of a conventional bench-top spectrofluorometer were perfus ed for 5 min with the acetoxy methyl ester of BCECF and fluorescence w as measured ratiometrically at 6-s intervals. The mean value of pH(i) in glands perfused with a HCO3--free, N-2-hydroxyethylpiperazine-N'-2- ethanesulphonic acid (HEPES) buffered solution at 37 degrees C was 7.3 6 +/- 0.01 (n = 52) which is comparable with values obtained by P-31 n uclear magnetic resonance (NMR) spectroscopy. NMR data confirmed that the BCECF loading period was accompanied by a transient acidification of the cells, but there was no significant change in the content of th e major phosphorus metabolites. Changes in pH(i) in response to NH4Cl pulses and acetylcholine stimulation were comparable with results repo rted previously for isolated acini. Additional, preliminary experiment s show that the method can also be used to monitor intracellular Ca2(using fura-2) in perfused salivary glands, and can be adapted for stu dies of the isolated, perfused pancreas.