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J Appl Physiol (January 24, 2003). doi:10.1152/japplphysiol.00780.2002
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Submitted on August 26, 2002
Accepted on January 2, 2003

Changes in intracellular Ca2+ concentration induced by several glucose transport-enhancing stimuli in rat epitrochlearis muscle

Shin Terada1, Isao Muraoka2, and Izumi Tabata3*

1 Laboratory of Exercise Physiology, Division of Health Promotion and Exercise, National Institute of Health and Nutrition, Shinjuku City, Tokyo, Japan; Department of Physiology and Biomechanics, National Institute of Fitness and Sports, Kanoya, Kagoshima, Japan; Department of Sports Sciences, School of Human Sciences, Waseda University, Tokorozawa, Saitama, Japan
2 Department of Sports Sciences, School of Human Sciences, Waseda University, Tokorozawa, Saitama, Japan
3 Laboratory of Exercise Physiology, Division of Health Promotion and Exercise, National Institute of Health and Nutrition, Shinjuku City, Tokyo, Japan; Department of Physiology and Biomechanics, National Institute of Fitness and Sports, Kanoya, Kagoshima, Japan

* To whom correspondence should be addressed. E-mail: tabata{at}nih.go.jp.

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-week-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 (KHB). 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 ({Delta}R) was used as an index of the change in [Ca2+]i. In resting muscle, {Delta}R was stable for 60 min. Incubation for 20 min with caffeine (3 ~ 10 mM) significantly increased {Delta}R in a concentration-dependent manner. Incubation with hypoxic KHB for 10~60 min significantly elevated {Delta}R, depending on the duration of the incubation. Incubation with 50 µM W-7 for 20 min significantly elevated {Delta}R (p<0.05). No significant increases in {Delta}R were observed during incubation for 20 min with 2 mM AICAR or with 2 mU/ml insulin. These results demonstrated that 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.




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