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 Ca²⁺ concentrations ([Ca²⁺]_i) in isolated rat epitrochlearis muscles. Epitrochlearis muscles excised from 4-wk-old male Sprague-Dawley rats were loaded with a fluorescent Ca²⁺ indicator, fura 2-AM, for 60-90 min at 35°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_total340 and F_total380), 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_total340 and F_total380, 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 (ΔR) was used as an index of the change in [Ca²⁺]_i. In resting muscle, ΔR was stable for 60 min. Incubation for 20 min with caffeine (3-10 mM) significantly increased ΔR in a concentration-dependent manner. Incubation with hypoxic Krebs-Henseleit buffer for 10-60 min significantly elevated ΔR, depending on the duration of the incubation. Incubation with 50 μM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide for 20 min significantly elevated ΔR (P < 0.05). No significant increases in AR were observed during incubation for 20 min with 2 mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside or with 2 mU/ml insulin. These results demonstrated that, by using the fura 2-AM fluorescence method, the changes in [Ca²⁺]_i can be monitored in the rat epitrochlearis muscle and suggest that the method can be utilized to observe quantitative information regarding [Ca²⁺]_i that may be involved in contraction- and hypoxia-stimulated glucose transport activity in skeletal muscle.