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

Abstract

Intracellular pH (pH i) 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 pH i (500:450 fluorescence ratio). The addition of 20 mM lactate caused a gradual decline in pH i which reversed upon withdrawal of lactate. In contrast, the weak acids propionate and acetate (20 mM) induced a rapid, prouounced fall in pH i followed by a gradual recovery. Upon removal of the weak acid, a marked, reversible alkalinization occurred. The addition of 20 mM NH 4Cl caused a pronounced intracellular alkalinization, followed by recovery. The subsequent removal of NH 4Cl induced a rapid, reversible acidification. The addition of 20 mM KCl did not affect pH i. Epifluorescence at 350 and 380 nm excitation, and the 350:380 fluorescence ratio, an index of cytosolic [Ca 2+] ([Ca 2+] 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 Ca 2+ was omitted from the medium. An increase in glucose concentration also caused a rise in these (auto)fluorescence signals in non-loaded islets. The addition of 20 mM lactate to islets also induced 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 progressive fall in the 380 signal and a biphasic increse in the 350:380 ratio. In non-loaded islets, these agents caused a progressive fall in 350 and 380 signals with little change in their ratio. Both addition and withdrawal of NH 4Cl resulted in an increase in the 350:380 ratio in fura-2 loaded islets, but there were no changes in autofluorescence. In conclusion, [Ca 2+] i in rat islets is sensitive to changes in pH i. In addition, the use of fura-2 to measure changes in [Ca 2+] i in pancreatic islets in response to nutrients or agents which alter intracellular pH could be complicated by accompanying changes in autofluorescence, possibly reflecting altered levels of NAD(P)H.