Kinetic constants of isoprenaline and corticosterone for extraneuronal uptake in different cell types from various tissues

1. Extraneuronal uptake of isoprenaline was studied in a number of different, histologically identified, cell types: trachealis smooth muscle cells, vascular smooth muscle cells and myocardial cells. Segments of cat, rat or rabbit trachea, dog coronary artery or cat atria were incubated in isoprenaline, in the presence of U-0521 (100 μmol l-1) to inhibit catechol-O-methyltransferase. The intensities of formaldehyde-induced fluorescence (due to accumulation of isoprenaline) were measured, using microphotometry, in the appropriate cells in histogical sections of the tissue. 2. Endogenous fluorescence in adrenergic nerves was removed by pretreatment of rats with 6-hydroxydopamine and of cats and rabbits with reserpine. Segments of dog coronary artery were incubated with 6-hydroxydopamine in vitro to remove neuronal fluorescence. This treatment was also shown to be effective in guinea-pig trachea without any influence on the determination of the kinetic parameters of isoprenaline uptake in the trachealis smooth muscle cells. 3. Fluorescence in all cell types studied was shown to represent isoprenaline which had accumulated by extraneuronal uptake, in that fluorescence was reduced by drugs which inhibit extraneuronal uptake (corticosterone, normetanephrine, metanephrine and/or phenoxybenzamine), by exposure to a K+-Krebs solution and by incubating tissues in isoprenaline at 0°C rather than 37°C. 4. In each cell type, isoprenaline uptake obeyed Michaelis-Menten saturation kinetics, both in the absence and presence of corticosterone. Corticosterone was a competitive inhibitor of isoprenaline uptake in all the cell types. 5. The apparent Km values of isoprenaline (μmol l-1) were: rat trachealis smooth muscle cells 474; cat trachealis smooth muscle cells 358; rabbit trachealis smooth muscle cells 380; dog coronary artery medial smooth muscle cells 240; cat atrial myocardial cells 254. The apparent Ki values (μmol l-1) of corticosterone in the same cell types were 3.7, 1.9, 2.3, 2.4 and 2.3, respectively. Any accumulation of isoprenaline in the cells which resulted from diffusional entry of the amine has not been subtracted. Therefore these values may be overestimated. 6. Neither the apparent Km nor the apparent Ki values were more than 2-fold apart in different cell types, and thus provide no indication that there are differences in the extraneuronal uptake carrier in different cells (vascular and non-vascular smooth muscle cells and atrial myocardial cells) or in the same cell type (trachealis smooth muscle cells) from different species