Genistein elicits biphasic effects on L-type Ca2+ current in feline atrial myocytes.

A perforated patch recording method was used to determine the effects of genistein (Gen), a protein tyrosine kinase (PTK) inhibitor, on basal L-type Ca2+ current ( I Ca,L) in feline atrial myocytes. Gen (50 μM) elicited biphasic changes in I Ca,L: an initial inhibition (-55 ± 4%; phase 1) followed by a secondary stimulation (34 ± 9%; phase 2) of I Ca,L. Withdrawal of Gen elicited a further potentiation of I Ca,L (152 ± 19%; phase 3) above control ( n = 46). In general, phase 1 inhibition and phase 3 potentiation varied directly with Gen concentration, and phase 2stimulation exhibited biphasic concentration-dependent changes compared with control. When cells were dialyzed using a ruptured patch recording method, Gen elicited only inhibition of I Ca,L; phases 2 and 3 were abolished. Vanadate (1 mM), an inhibitor of protein tyrosine phosphatase, abolished both Gen-induced inhibition and stimulation of I Ca,L. Daidzein (50 μM), a weakly active analog of Gen, exerted no significant effects on I Ca,L, and withdrawal of daidzein failed to potentiate I Ca,L. In a few cells, Gen elicited a prominent vanadate-sensitive stimulation of I Ca,L in the absence of any significant inhibition of I Ca,L. Gen-induced changes in I Ca,L were unaffected by either 100 μM 1,2-bis(2-aminophenoxy)ethane- N, N, N', N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM) or 1 μM ryanodine, agents that alter intracellular Ca2+; 4 μM H-89 or 50 μM Rp diastereomer of adenosine 3',5'-monophosphothioate (RP-cAMPS), inhibitors of protein kinase A (PKA); 0.1 μM calphostin C or 2 μM chelerythrine, inhibitors of protein kinase C (PKC); or 100 μM N G-monomethyl-l-arginine (l-NMMA), an inhibitor of nitric oxide (NO) synthase. We conclude that in feline atrial myocytes, Gen acts via membrane-bound PTKs to inhibit I Ca,L and via cytosolic PTKs to stimulate I Ca,L. Gen-induced changes in I Ca,L are not related to changes in intracellular Ca2+ or to secondary interactions with either PKA, PKC, or NO signaling pathways. These results indicate that in atrial myocytes I Ca,L is regulated by two independent and competing PTK signaling mechanisms.