Changes in [Ca2+]i induced by several glucose transport-enhancing stimuli in rat epitrochlearis muscle

The purpose of the present investigation was to establish a method for estimating intracellular Ca(2+) concentrations ([Ca(2+)](i)) in isolated rat epitrochlearis muscles. Epitrochlearis muscles excised from 4-wk-old male Sprague-Dawley rats were loaded with a fluorescent Ca(2+) indicator, fura 2-AM, for 60-90 min at 35 degrees C in oxygenated Krebs-Henseleit buffer. After fura 2 loading and subsequent 20-min incubation, the intensities of 500-nm fluorescence, induced by 340- and 380-nm excitation lights (F(total)340 and F(total)380), were measured. The fluorescences specific to fura-2 (F(fura 2)340 and F(fura 2)380) were calculated by subtracting the non-fura 2-specific component from F(total)340 and F(total)380, respectively. The ratio of F(fura 2)340 to F(fura 2)380 was calculated as R, and the change in the ratio from the baseline value (DeltaR) was used as an index of the change in [Ca(2+)](i). In resting muscle, DeltaR was stable for 60 min. Incubation for 20 min with caffeine (3-10 mM) significantly increased DeltaR in a concentration-dependent manner. Incubation with hypoxic Krebs-Henseleit buffer for 10-60 min significantly elevated DeltaR, depending on the duration of the incubation. Incubation with 50 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide for 20 min significantly elevated DeltaR (P < 0.05). No significant increases in DeltaR were observed during incubation for 20 min with 2 mM 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside or with 2 mU/ml insulin. These results demonstrated that, by using the fura 2-AM fluorescence method, the changes in [Ca(2+)](i) can be monitored in the rat epitrochlearis muscle and suggest that the method can be utilized to observe quantitative information regarding [Ca(2+)](i) that may be involved in contraction- and hypoxia-stimulated glucose transport activity in skeletal muscle.