Commentary on Gressel's review

Transgenic Research -     

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

Isolated adult rat heart cells incubated with 5 microM Mn in a medium with 1 mM 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(beta-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 54 Mn 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 beta-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.     

Modulation of uncoupler-induced sugar uptake in isolated adult rat heart cells by isoproterenol

When cells (2.4 mg/ml) in the presence of glucose were exposed to 0.15 microM p-trifluoromethoxyphenylhydrazone (FCCP), the time until 50% of the rod-shaped cells had undergone contracture was more than twice as long for cells without isoproterenol as for cells with isoproterenol. The cause of this large effect was revealed in experiments without glucose where 3-O-methylglucose entry, ATP levels, and cellular configuration were measured simultaneously. It was found that the onset of contracture was almost coincident with the decline in total measured ATP, suggesting that, in any cell, contracture was accompanied by a sudden and total ATP loss. In control cells, FCCP stimulated 3-O-methylglucose entry at or before the time this ATP catastrophe occurred. In cells exposed to isoproterenol, however, the stimulation of 3-O-methylglucose entry by FCCP did not occur until after the ATP catastrophe, and the extent of stimulation was reduced. This suggests that, when glucose was present, the FCCP-induced glucose influx was sufficient to significantly delay the onset of contracture in control cells but not in cells treated with isoproterenol. This conclusion was borne out by the observation that the effect of isoproterenol on contracture could be overcome with insulin.