THE TRIAL STUDY FOR THE MINIMUM INVASIVE DIAGNOSTIC TEST FOR MHS -THE EFFECTS OF CAFFEINE AND HALOTHANE ON CULTURED HUMAN SKELETAL MUSCLE CELL MESURING INTRA CELLULAR Ca CONCENTRATION CONCENTRATION AND GENETIC ANALYSIS-

1.CICR rate and genetic linkage studiesWe have performed genetic linkage analysis with CICR test in 63 subjects who were referred to our hospital for investigation of MHS.Seven of them had a history of f-MH episode and 33had a family history of f-MH，9had a history of mild MH reaction and I O had a family history of mild MH reaction.We performed CICR test in 23 subjects.Ten of 11f-MH subjects，3family members with f-MH and 2 unrelated subjects with mild MH reactions showed the accelerated changes in CICR test.We isolated genomic DNA from all of63 subjects。Analysis for a substitution of cytosine 1840 to thymine which is same mutation of porcine MHS，was performed in all63 subjects。We also checked the several other one point mutations in some but not all.However，we din not find any alterations。From our results，we can say there remains a possibility of other point mutations。However，we found one MHS family，which carried infomative polymorphisms。The proband，blue square o…More ne，manifested f-MH during a neurosurgical operation。CICR test in his daughter and son showed acceralation.In genetic linkage study they showed informative polymorphisms relating to MHS diagnosed with CICR test.In this family，linkage analysis of the RYR1gene was very useful for further examination，including muscle biopsy2。The pilot stud of screening test using caltured human skeletal muscle cellWe cultured normal human skeletal muscle cell(SRMC2874，Clonetics I D1RⅡD1)on glass cover slips and loaded with 5µM Fura-2/AM。Cover slips were mounted on perfusion system which was perfused with HEPES solution。Caffeine(0.25-10 mM)and Halothane(0.1-2 mM)，which were dissolved in HEPES solution in a gas-tight vial，was connected to this perfusion system just before these stimulations.The cover slip was placed on the stage of a fluorescence inverted microscope，combined with ARGUS-50(Hamamatsu Photonics Ii D1R I D1)。The fluorescence intensity ratio with excitation at 340/380 nm and emission at 510 nm was recorded every5sec.As the results，1)Caffeine(0.25-10 mM)stimulations increased[Ca I D12+ii D1]i in a concentration-dependent manner.2)Also，halothane(0.1-2 mM)stimulations increased[Ca I D12+ii D1]i in a concentration-dependent manner。Less：Less

1. CICR rate and genetic linkage studiesWe have performed genetic linkage analysis with CICR test in 63 subjects who were referred to our hospital for investigation of MHS.Eleven of them had a history of f-MH episode and 33 had a family history of f-MH, 9 had a history of mild MH reaction and I O had a family history of mild MH reaction.We performed CICR test in 23 subjects. Ten of 11 f-MH subjects, 3 family members with f-MH and 2 unrelated subjects with mild MH reactions showed the accelerated changes in CICR test.We isolated genomic DNA from all of 63 subjects. Analysis for a substitution of cytosine 1840 to thymine which is same mutation of porcine MHS, was performed in all 63 subjects. We also checked the several other one point mutations in some but not all. However, we din not find any alterations. From our results, we can say there remains a possibility of other point mutations. However, we found one MHS family, which carried infomative polymorphisms. The proband, blue square o … More ne, manifested f-MH during a neurosurgical operation. CICR test in his daughter and son showed acceralation.In genetic linkage study they showed informative polymorphisms relating to MHS diagnosed with CICR test. In this family, linkage analysis of the RYR1 gene was very useful for further examination, including muscle biopsy2. The pilot stud of screening test using caltured human skeletal muscle cellWe cultured normal human skeletal muscle cell (SRMC 2874, CloneticsィイD1RィエD1) on glass cover slips and loaded with 5 μ M Fura-2/AM. Cover slips were mounted on perfusion system which was perfused with HEPES solution. Caffeine (0.25-10mM) and Halothane (0.1-2mM), which were dissolved in HEPES solution in a gas-tight vial, was connected to this perfusion system just before these stimulations. The cover slip was placed on the stage of a fluorescence inverted microscope, combined with ARGUS-50 (Hamamatsu PhotonicsィイD1RィエD1). The fluorescence intensity ratio with excitation at 340/380 nm and emission at 510 nm was recorded every 5 sec.As the results, 1)Caffeine (0.25-10mM) stimulations increased [CaィイD12+ィエD1]i in a concentration- dependent manner. 2)Also ,halothane (0.1-2mM) stimulations increased [CaィイD12+ィエD1]i in a concentration-dependent manner. Less