Inhibition of Ca 2+ current in neonatal and adult rat ventricular myocytes by the tyrosine kinase inhibitor, genistein

Abstract

Yokoshiki et al. (Yokoshiki, H., Sumii, K., Sperelakis, N., 1996. Inhibition of L-type calcium current in rat ventricular cells by the tyrosine kinase inhibitor, genistein and its inactive analog, daidzein. J. Mol. Cell. Cardiol. 28, 807–814) reported that genistein and daidzein inhibited L-type Ca 2+ current ( I Ca(L)) in young rat ventricular cells. Therefore, we investigated the developmental differences in the effect of genistein, an inhibitor of tyrosine kinases, on I Ca(L) in freshly-isolated neonatal (3–7 days) and adult (2–5 months) rat ventricular myocytes using whole-cell voltage clamp and single-channel recordings (cell-attached configuration). For whole-cell voltage clamp, I Ca(L) was measured as the peak inward current at a test potential of +10 mV by applying a 300 ms pulse from a holding potential of −40 mV. To isolate I Ca(L), the pipette solution was Cs +-rich and the bath solution was Na +-, K +-free. Ca 2+ (1.8 mM) was used as charge carrier. Bath application of 100 μM genistein (sufficient for maximal effect) decreased the basal I Ca(L) by 43.3% ( n=27) in neonatal cells and by 30.6% ( n=14) in adult cells ( P<0.05). In the current/voltage relationships, the potential of peak I Ca(L) was shifted to the right by genistein by 8.6 mV in neonatal and by 9.3 mV in adult cells. Genistein produced a shift of the steady-state inactivation curve (to the left) in neonatal cells (from −16.0±3.9 mV to −26.1±4.2 mV; P<0.05) and in adult cells (−15.9±3.2 mV to −22.9±3.3 mV; P<0.05); the slope factor was not affected. For single-channel recordings in cell-attached patches, Ca 2+ currents were evoked by applying a 150 ms pulse from a holding potential of −40 mV to a test potential of 0 mV. The pipette solution contained 110 mM Ba 2+ (as charge carrier), and the bath solution contained 150 mM K + (to bring resting potential to near zero). Genistein (50 μM) decreased the open probability of the channels from 2.8% to 0.75% ( P<0.05) in absence of Bay K 8644, and from 24% to 7.9% ( P<0.05) in presence of Bay K 8644; the mean open time and the slope conductance of the currents were not affected. In conclusion, (1) genistein inhibits the basal I Ca(L) in rat ventricular cells and (2) the inhibition of I Ca(L) by genistein is greater in immature cells than in adult cells.