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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.

Authors
Journal
Molecular and Cellular Endocrinology
0303-7207
Publisher
Elsevier
Publication Date

Abstract

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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