Measurement of Ca channel activity of isolated adult rat heart cells using 54Mn

Abstract

Isolated adult rat heart cells incubated with 5 μ m Mn in a medium with 1 m m Ca showed a rapid phase of Mn binding plus a slow phase of Mn uptake. The rapid phase was extracellular binding, as judged by its temperature-insensitive removal by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid. The slow linear phase represented cellular uptake, as judged by its release with digitonin plus the ionophore A23187. Isoproterenol increased the linear rate of Mn uptake and induced spontaneous beating activity in some cells. Both effects were inhibited by nitrendipine. Electrical stimulation of the cells in suspension increased the linear rate of cellular Mn uptake. The increase was potentiated by isoproterenol, and inhibited by nitrendipine or verapamil. Stimulation-dependent Mn uptake (per milligram protein) was greater for cells from 5- to 6-week-old rats than for 8- to 9-month-old female retired breeder rats, in the presence of isoproterenol. Ryanodine increased the stimulation-dependent Mn uptake in the presence of isoproterenol, but not in its absence. We conclude: (i) that cellular uptake of 54Mn is a good probe of Ca channel function; (ii) that isoproterenol promotes Mn influx by the channel in isolated heart cells; (iii) that cells from young rats (5–6 weeks) have a higher β-adrenergically induced Ca channel activity than cells from mature rats (8–9 months); and (iv) that ryanodine promotes Ca channel activity (perhaps indirectly) in the presence of isoproterenol.