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 (pH(i)) was measured in intact rat islets loaded with the dye 2',7' -bis(carboxyethyl) 5/(6')-carboxyfluorescein. Raising t he concentration of glucose from 3 to 13 mM caused a modest, gradual i ncrease in pH(i) (500:450 fluorescence ratio). The addition of 20 mM l actate caused a gradual decline in pH(i) which reversed upon withdrawa l of lactate. In contrast, the weak acids propionate and acetate (20 m M) induced a rapid, pronounced fall in pH(i) followed by a gradual rec overy. Upon removal of the weak acid, a marked, reversible alkalinizat ion occurred. The addition of 20 mM NH4Cl caused a pronounced intracel lular alkalinization, followed by recovery. The subsequent removal of NH4Cl induced a rapid, reversible acidification. The addition of 20 mM KCl did not affect pH(i). Epifluorescence at 350 and 380 nm excitatio n, and the 350:380 fluorescence ratio, an index of cytosolic [Ca2+] ([ Ca2+](i)), were measured in islets loaded with the calcium indicator f ura-2. Approximately 30% of the total fluorescence was estimated to be derived from NAD(P)H autofluorescence. Addition of KCl or acetylcholi ne to fura-2 loaded islets raised and lowered, respectively, the 350 a nd 380 signals, thereby causing marked increases in the 350:380 ratio. Neither KCl nor acetylcholine affected cellular NAD(P)H autofluoresce nce in non-loaded islets. An increase in glucose concentration caused an increase in both the 350 and 380 fluorescence signals and also in t he 350:380 ratio. Qualitatively similar, although smaller changes were observed when Ca2+ was omitted from the medium. An increase in glucos e concentration also caused a rise in these (auto)fluorescence signals in non-loaded islets. The addition of 20 mM lactate to islets also in duced increases in the 350 and 380 signal and the 350:380 ratio in the presence or absence of calcium, and to a lesser extent in non-loaded islets. The addition to fura-2 loaded islets of propionate or acetate caused an initial increase followed by a fall in the 350 signal, a pro gressive fall in the 380 signal and a biphasic increase in the 350:380 ratio. In non-loaded islets, these agents caused a progressive fall i n 350 and 380 signals with little change in their ratio. Both addition and withdrawal of NH4Cl resulted in an increase in the 350:380 ratio in fura-2 loaded islets, but there were no changes in autofluorescence . In conclusion, [Ca2+](i) in rat islets is sensitive to changes in pH (i). In addition, the use of fura-2 to measure changes in [Ca2+](i) in pancreatic islets in response to nutrients or agents which alter intr acellular pH could be complicated by accompanying changes in autofluor escence, possibly reflecting altered levels of NAD(P)H.