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