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 (ICa,L) in feline atrial myocytes. Gen (50 microM) elicited biphasic changes in ICa,L: an initial inhibition (-55 +/- 4%; phase 1) followed by a secondary stimulation (34 +/- 9%; phase 2) of ICa,L. Withdrawal of Gen elicited a further potentiation of ICa,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 2 stimulation exhibited biphasic concentration-dependent changes compared with control. When cells were dialyzed using a ruptured patch recording method, Gen elicited only inhibition of ICa,L; phases 2 and 3 were abolished. Vanadate (1 mM), an inhibitor of protein tyrosine phosphatase, abolished both Gen-induced inhibition and stimulation of ICa,L. Daidzein (50 microM), a weakly active analog of Gen, exerted no significant effects on ICa,L, and withdrawal of daidzein failed to potentiate ICa,L. In a few cells, Gen elicited a prominent vanadate-sensitive stimulation of ICa,L in the absence of any significant inhibition of ICa,L. Gen-induced changes in ICa,L were unaffected by either 100 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM) or 1 microM ryanodine, agents that alter intracellular Ca2+; 4 microM H-89 or 50 microM Rp diastereomer of adenosine 3',5'-monophosphothioate (RP-cAMPS), inhibitors of protein kinase A (PKA); 0.1 microM calphostin C or 2 microM chelerythrine, inhibitors of protein kinase C (PKC); or 100 microM NG-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 ICa,L and via cytosolic PTKs to stimulate ICa,L. Gen-induced changes in ICa,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 ICa,L is regulated by two independent and competing PTK signaling mechanisms.