Continuous fluorometric measurement of intracellular pH and Ca2+ in perfused salivary gland and pancreas.
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.
Seo
J T
JT
School of Biological Sciences, University of Manchester, UK.
Steward
M C
MC
Larcombe-McDouall
J B
JB
Cook
L J
LJ
Case
R M
RM
D000109:Acetylcholine / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D005452:Fluoresceins / Q000493:pharmacokinetics; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013363:Submandibular Gland / Q000187:drug effects / Q000378:metabolism*
D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D000109:Acetylcholine; D002118:Calcium