Subcellular distribution of dihydropyridine isothiocyanate binding in guinea-pig ileal smooth muscle

The subcellular distribution of the 3H--2,6-dimethyl-3,5-dicarbomethoxy-4(2-isothiocyano) phenyl-1,4-dihydropyridine (DPSCN) binding to guinea-pig ileal smooth muscle was studied by subcellular fractionation. Initial experiments on subcellular fractionation of 3H-DPSCN-labelled tissues by differential centrifugation showed that there was an excellent correlation between the levels of the label present in a fraction and the plasma membrane marker phosphodiesterase I (r = 0.98) but not between the label and the putative endoplasmic reticulum marker NADPH: cytochrome-c-reductase (r = 0.56) or the inner mitochondrial marker cytochrome-c-oxidase (r = 0.36). Centrifugation of the microsomes on a continuous sucrose density gradient showed an excellent correlation of the migration of the label with phosphodiesterase I activity (r = 0.93) but not with the activities of NADPH: cytochrome-c-reductase (r = 0.66) or cytochrome-c-oxidase (r = 0.44). Treatment of microsomes with digitonin (1 mg/ml) followed by centrifugation on continuous sucrose density gradients increased the weighted mean densities of the phosphodiesterase activity (plasma membrane marker) and the labelling by similar magnitudes (0.04 to 0.06 g/ml). The weighted mean densities of NADPH: cytochrome-c-reductase and the cytochrome-c-oxidase were not altered significantly. It is concluded that in the guinea-pig ileal smooth muscle, DPSCN labels the plasma membrane specifically.