During exercise, phosphorylase kinase (PhK) is the key regulatory enzyme responsible for maintaining glycogenolytic flux to sustain muscle contraction. The absence of PhK in skeletal muscle results in glycogen storage...During exercise, phosphorylase kinase (PhK) is the key regulatory enzyme responsible for maintaining glycogenolytic flux to sustain muscle contraction. The absence of PhK in skeletal muscle results in glycogen storage disease (GSD) Type IX which is characterized by muscle weakness and rapid fatigue upon exercise. In this study, we have used the phka1 deficient I/LnJ mouse model of GSD to investigate the physiological and genetic adaptations that occur in response to voluntary exercise. When quantified over training periods of either 1, 2, or 5 weeks, I/LnJ mice ran significantly less time/day and distance/day than agematched C57/Bl6 mice. Cumulatively after five weeks, adult I/LnJ mice ran ~1/2 the total time and distance of wild-type mice, 116 ± 6 hours and 211 ±23 kmversus 194 ± 3 hours and 418 ±4 km, respectively. After 5 weeks, C57/Bl6 mice demonstrated an increase in endurance as a result of aerobic training;this observed physiological adaptation was not present in I/LnJ mice. The decrease in total distance run by I/LnJ mice was not due to a reduction in speed;juvenile and adult I/LnJ mice ran ~75% - 80% as fast as C57/Bl6 mice. When transcription of glycolytic genes glucose transporter 4 (scla1), pyruvate dehydrogenase (pdha1), and phosphofructokinase (pfk) were quantified at the end of each training period, no significant differences in expression levels were found between mouse strains, suggesting that non-glycolytic mechanisms work to maintain the muscle function observed in the I/LnJ mice.展开更多
Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiologic...Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NOX) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.展开更多
文摘During exercise, phosphorylase kinase (PhK) is the key regulatory enzyme responsible for maintaining glycogenolytic flux to sustain muscle contraction. The absence of PhK in skeletal muscle results in glycogen storage disease (GSD) Type IX which is characterized by muscle weakness and rapid fatigue upon exercise. In this study, we have used the phka1 deficient I/LnJ mouse model of GSD to investigate the physiological and genetic adaptations that occur in response to voluntary exercise. When quantified over training periods of either 1, 2, or 5 weeks, I/LnJ mice ran significantly less time/day and distance/day than agematched C57/Bl6 mice. Cumulatively after five weeks, adult I/LnJ mice ran ~1/2 the total time and distance of wild-type mice, 116 ± 6 hours and 211 ±23 kmversus 194 ± 3 hours and 418 ±4 km, respectively. After 5 weeks, C57/Bl6 mice demonstrated an increase in endurance as a result of aerobic training;this observed physiological adaptation was not present in I/LnJ mice. The decrease in total distance run by I/LnJ mice was not due to a reduction in speed;juvenile and adult I/LnJ mice ran ~75% - 80% as fast as C57/Bl6 mice. When transcription of glycolytic genes glucose transporter 4 (scla1), pyruvate dehydrogenase (pdha1), and phosphofructokinase (pfk) were quantified at the end of each training period, no significant differences in expression levels were found between mouse strains, suggesting that non-glycolytic mechanisms work to maintain the muscle function observed in the I/LnJ mice.
文摘Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NOX) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.
