Terada, Shin, Isao Muraoka, and Izumi Tabata. Changes in [Ca 2ϩ ]i induced by several glucose transportenhancing stimuli in rat epitrochlearis muscle. .-The purpose of the present investigation was to establish a method for estimating intracellular Ca 2ϩ concentrations ([Ca 2ϩ ]i) in isolated rat epitrochlearis muscles. Epitrochlearis muscles excised from 4-wkold male Sprague-Dawley rats were loaded with a fluorescent Ca 2ϩ indicator, fura 2-AM, for 60-90 min at 35°C in oxygenated

more »

... t buffer. After fura 2 loading and subsequent 20-min incubation, the intensities of 500-nm fluorescence, induced by 340-and 380-nm excitation lights (Ftotal340 and Ftotal380), were measured. The fluorescences specific to fura-2 (Ffura 2340 and Ffura 2380) were calculated by subtracting the non-fura 2-specific component from Ftotal340 and Ftotal380, respectively. The ratio of Ffura 2340 to Ffura 2380 was calculated as R, and the change in the ratio from the baseline value (⌬R) was used as an index of the change in [Ca 2ϩ ]i. In resting muscle, ⌬R was stable for 60 min. Incubation for 20 min with caffeine (3-10 mM) significantly increased ⌬R in a concentration-dependent manner. Incubation with hypoxic Krebs-Henseleit buffer for 10-60 min significantly elevated ⌬R, depending on the duration of the incubation. Incubation with 50 M N-(6-aminohexyl)-5chloro-1-naphthalenesulfonamide for 20 min significantly elevated ⌬R (P Ͻ 0.05). No significant increases in ⌬R were observed during incubation for 20 min with 2 mM 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside or with 2 mU/ml insulin. These results demonstrated that, by using the fura 2-AM fluorescence method, the changes in [Ca 2ϩ ]i can be monitored in the rat epitrochlearis muscle and suggest that the method can be utilized to observe quantitative information regarding [Ca 2ϩ ]i that may be involved in contraction-and hypoxia-stimulated glucose transport activity in skeletal muscle. fura 2; hypoxia; 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside; insulin; caffeine; intracellular Ca 2ϩ concentration