K-ATP channel independent effects of pinacidil on ATP production in isolated cardiomyocyte or pancreatic beta-cell mitochondria

Evidence has been presented that mitochondria contain ATP sensitive potassium channels (mK-ATP channels), which may confer tissue protection upon activation. It is, however, not known whether activation of mK-ATP channels has a direct effect on mitochondrial ATP production. This study was performed to define the effect of pinacidil (PIN) on ATP production by oxidative phosphorylation in isolated cardiomyocyte or pancreatic beta-cell mitochondria. Cardiomyocyte mitochondria produced seven times more ATP than beta-cell mitochondria in the presence of pyruvate/malate. PIN inhibited pyruvate/malate-induced mitochondrial ATP production with half maximal effect at 360 microM in both cell types. The inclusion of 5-hydroxydecanoate (5-HD) did not prevent this inhibition. Succinate induced a similar ATP production in cardiomyocyte or beta-cell mitochondria. In beta-cell mitochondria succinate-induced ATP production was inhibited by PIN with half maximal effects at 500 microM PIN. However, in cardiomyocyte mitochondria PIN stimulated succinate-induced ATP production 3-fold with half maximal effect at 100 microM and maximal effect at 200 microM. This PIN-dependent stimulation was mimicked by rotenone. The inclusion of 5-HD could not prevent these PIN effects. In conclusion, PIN may inhibit complex 1 of the respiratory chain without indications of opening mK-ATP channels. In cardiomyocytes with metabolically inhibited succinate dehydrogenase this results in a stimulation of ATP production conferring tissue protection. In beta-cells without a metabolically inhibited succinate dehydrogenase, there is no stimulation by PIN and tissue protection by PIN is not to be expected.     

Sensitive assay of creatine kinase isoenzymes in human serum using M subunit inhibiting antibody and firefly luciferase

A sensitive method for the assay of B subunit and total creatine kinase activity is described. The ATP formation in the creatine kinase reaction is continuously monitored by measuring the bioluminescence obtained with a purified firefly luciferase reagent. The B subunit activity is determined using an M subunit inhibiting antibody resulting in greater than 99.5% and approximately 50% inhibition of MM and MB isoenzymes, respectively. The bioluminescent method correlated well with a similar spectrophotometric method in the assay of B subunit as well as total creatine kinase activity (r greater than or equal to 0.98). However, the bioluminescent assay is considerably more sensitive, allowing the assay of B subunit activity in serum from healthy individuals. This is due to the inherent sensitivity of the bioluminescent assay of ATP, a reduced analytical interference from adenylate kinase and a reduced reagent blank. The within-series precision at 1 U/liter and greater than 52 U/liter corresponded to a C.V. of 14% and 3%, respectively. The method is as rapid and suitable for routine work as spectrophotometric methods. From a clinical point of view the new method is of particular interest in the early diagnosis of small acute myocardial infarctions.