Continuous fluorometric measurement of intracellular pH and Ca²⁺ in perfused salivary gland and pancreas

Intracellular pH (pH_i) has been measured in intact, perfused rat mandibular salivary glands loaded with the fluorescent pH indicator BCECF [2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]. Glands mounted in the cuvette of a conventional bench-top spectrofluorometer were perfused for 5 min with the acetoxymethyl ester of BCECF and fluorescence was 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°C was 7.36±0.01 (n=52) which is comparable with values obtained by ³¹P nuclear 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 the major phosphorus metabolites. Changes in pH in response to NH₄Cl pulses and acetylcholine stimulation were comparable with results reported previously for isolated acini. Additional, preliminary experiments show that the method can also be used to monitor intracellular Ca²⁺ (using fura-2) in perfused salivary glands, and can be adapted for studies of the isolated, perfused pancreas.