Changes in 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein-, fura-2 and autofluorescence in intact rat pancreatic islets in response to nutrients and non-nutrients.
Intracellular pH (pHi) was measured in intact rat islets loaded with the dye 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein. Raising the concentration of glucose from 3 to 13 mM caused a modest, gradual increase in pHi (500:450 fluorescence ratio). The addition of 20 mM lactate caused a gradual decline in pHi which reversed upon withdrawal of lactate. In contrast, the weak acids propionate and acetate (20 mM) induced a rapid, pronounced fall in pHi followed by a gradual recovery. Upon removal of the weak acid, a marked, reversible alkalinization occurred. The addition of 20 mM NH4Cl caused a pronounced intracellular alkalinization, followed by recovery. The subsequent removal of NH4Cl induced a rapid, reversible acidification. The addition of 20 mM KCl did not affect pHi. Epifluorescence at 350 and 380 nm excitation, and the 350:380 fluorescence ratio, an index of cytosolic [Ca2+] ([Ca2+]i), were measured in islets loaded with the calcium indicator fura-2. Approximately 30% of the total fluorescence was estimated to be derived from NAD(P)H autofluorescence. Addition of KCl or acetylcholine to fura-2 loaded islets raised and lowered, respectively, the 350 and 380 signals, thereby causing marked increases in the 350:380 ratio. Neither KCl nor acetylcholine affected cellular NAD(P)H autofluorescence in non-loaded islets. An increase in glucose concentration caused an increase in both the 350 and 380 fluorescence signals and also in the 350:380 ratio. Qualitatively similar, although smaller changes were observed when Ca2+ was omitted from the medium.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cell Physiology Group, School of Biological Sciences, University of Manchester, UK.
Elliott
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D000109:Acetylcholine / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016257:Fura-2; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D007773:Lactates / Q000494:pharmacology; D019344:Lactic Acid; D008297:Male; D009249:NADP / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007773:Lactates; D000643:Ammonium Chloride; D019344:Lactic Acid; D009249:NADP; D011189:Potassium Chloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D005947:Glucose; D000109:Acetylcholine; D002118:Calcium; D016257:Fura-2