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

Abstract

Intracellular pH (pHi) 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 spectro-fluorometer were perfused for 5 min with the acetoxymethyl ester of BCECF and fluorescence was measured ratiometrically at 6-s intervals. The mean value of pHi in glands perfused with a HCO3(-)-free, N-2-hydroxyethylpiperazine-N'-2- ethanesulphonic acid (HEPES)-buffered solution at 37 degrees 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 pHi 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.

TM9SF4 is a novel V-ATPase-interacting protein that modulates tumor pH alterations associated with drug resistance and invasiveness of colon cancer cells

“…For TM9SF4 and ATP6V0A double staining, cells were labeled with anti-TM9SF4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-ATP6V0A2 antibodies (Abcam, Cambridge, UK), respectively, stained with anti-goat Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 (Invitrogen). Evaluation of pHi pHi was measured in cells loaded with the fluorescent probe BCECF-AM according to Seo et al 50 Briefly, after Baf-A1 treatment as described above, BCECF-AM (Invitrogen) was added for 30 min to a final concentration of 1 μM. Fluorescence intensity was measured as described above.…”

Metabolic and ionic responses of trout hepatocytes to anisosmotic exposure

“…Excitation was set to 490·nm and 440·nm, and emission was again recorded above 510·nm. Calibrations were performed by replacing the experimental medium with high K + saline, where the concentrations of NaCl and KCl were reversed, containing the cation ionophores nigericin (10·µmol·l -1 ) and valinomycin (5·µmol·l -1 ) with a pH adjusted to 6.80, 7.20 or 7.60 (Pocock and Richards, 1992;Seo et al, 1994).…”

Interdependence of Ca2+ and proton movements in trout hepatocytes

“…Excitation was set to 490·nm and 440·nm, and emission was again recorded above 510·nm. Calibrations were performed by replacing the experimental medium with high K + saline (where the concentrations of NaCl and KCl were reversed) containing the cation ionophores nigericin (10·mol·l -1 ) and valinomycin (5·mol·l -1 ), with a pH adjusted to 6.80, 7.20 or 7.60 (Pocock and Richards, 1992;Seo et al, 1994). When experimental media of pH values higher or lower than 7.6 were used, calibration media were adjusted to cover the range of pHi values determined.…”