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Membrane potential and whole-cell current were studied in rat pancreatic beta-cells using the 'perforated patch' technique and cell volume measured by a video-imaging method. Exposure of beta-cells to the alpha-ketoaldehyde methylglyoxal (1 mM) resulted in depolarization and electrical activity. In cells voltage-clamped at -70 mV, this effect was accompanied by the development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors of this conductance, 4,4'-dithiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased beta-cell volume to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the alpha-ketoaldehyde. None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect on beta-cell K+ channel activity. It is suggested that the metabolism of methylglyoxal to D-lactate causes beta-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings could be relevant to the stimulatory action of D-glucose, the metabolism of which generates significant quantities of L-lactate.

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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.