|Budget Amount *help
¥9,200,000 (Direct Cost : ¥9,200,000)
Fiscal Year 1999 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1998 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1997 : ¥6,000,000 (Direct Cost : ¥6,000,000)
Firstly, we have developed a method for measuring cytosol CaィイD12+ィエD1 concentration in mammal skeletal muscle tissue. Changes in CaィイD12+ィエD1 concentration was estimated using double-beam fluorescent method with fura-2/AM as a fluorescent indicator which penetrates into cytosol of skeletal muscle (emittion: 340 nm and 380 nm, fluorescent 500 nm). Using a small and thin forelimb muscle named epitrochlearis from rat weighing 100-110 g, CaィイD12+ィエD1 concentration in the muscle was evidenced to be measured during long-term incubation and submaximal contraction. However, during tetani contractions, CaィイD12+ィエD1 concentration was not found to be measured due to artifact related to tenani.
Since recent studies suggested that stimulated glucose transport activity by contraction is related to increased CaィイD12+ィエD1 concentration in skeletal muscle, we observed effects of physiological stimuli including insulin, submaximal contraction, and submaximal hypoxia that do not induce tetani, and pharma
cological stimuli that affect CaィイD12+ィエD1 concentration in cell.
We firstly observed elevation of CaィイD12+ィエD1 in the muscle during incubation with low concentration caffeine (3 mM), W-7(50μM), and submaximal hypoxia. As these stimuli are known to increase glucose transport activity in the muscle, we think that the method is valid and valuable to clarify mechanisms related to submaximal contraction-stimulated glucose transport activity in rat skeletal muscle.
There was no change in CaィイD12+ィエD1 during incubation in AICAR known to stimulate AMP kinase that may intervene muscle-contraction induced increase in glucose transport activity. This result suggests that pathway related to CaィイD12+ィエD1 -enhanced glucose transport activity does not exist prior to AMP kinase.
Insulin has been shown not to affect CaィイD12+ィエD1 level in the muscle until 20 min after initiation of incubation. However, we observed that CaィイD12+ィエD1 concentration did increase after 20 min incubation in insulin (2 mU/ml). This result may suggest that this method can also be used for the purpose of elucidating insulin-induced glucose transport activity in skeletal muscle. Less