L-type Ca²⁺current in guinea pig ventricular myocytes treated with modulators of tyrosine phosphorylation

Abstract

Guinea pig ventricular myocytes in whole cell configuration were treated with tyrosine kinase (TK) inhibitors [genistein (Gst), tyrphostin A23 (T23), and tyrphostin A25 (T25)] and with inactive analogs [daidzein, genistin, and tyrphostin A1 (T1)] to measure effects on L-type Ca2+ current ( I Ca,L). Gst inhibited I Ca,L(IC50 = 47 μM) without affecting its time course or shifting the I Ca,L-voltage relationship. At the highest concentration of isoflavone tested (200 μM), I Ca,L was inhibited by 66 ± 7% (Gst), 22 ± 2% (daidzein), and 1 ± 3% (genistin). Inhibition of I Ca,L by the active tyrphostins was significantly larger than inhibition by T1; at 200 μM the inhibitions were 72 ± 6% (T23), 71 ± 6% (T25), and 27 ± 6% (T1). The phosphotyrosine phosphatase inhibitor orthovanadate (1 mM) had a small stimulatory effect (6 ± 2%) on basal I Ca,L and blocked the inhibition of I Ca,L by TK inhibitors. The data suggest a role for the TK-phosphotyrosine phosphatase system in the regulation of cardiac Ca2+ channels.

Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

“…Orthovanadate, which is an inhibitor of protein phosphatases, increases basal tyrosine phosphorylation and is used to antagonize the effects of PTK inhibitor. For example, orthovanadate has been shown to antagonize the inhibitory effect of genistein on L-type Ca 2ϩ currents and delayedrectifier K ϩ currents in guinea pig ventricular myocytes (26,29). In the present study, pretreatment with orthovanadate had no effect on the inhibitory action of genistein on Kv4.3.…”

Cardiac Na+–Ca2+ exchanger current induced by tyrphostin tyrosine kinase inhibitors

“…Under these conditions, 200-ms depolarizations to 0 mV elicited inward I Ca,L that rapidly reached a peak and then inactivated. As previously reported (Ogura et al, 1999), I Ca,L was reversibly inhibited by 100 mM genistein and 100 mM A23 (Figure 1a). These drugs also had reversible effects on holding current at -40 mV (I h ) and end-of-pulse current (I 200 ) at 0 mV ( Figure 1a).…”

“…In an earlier study on the action of TK inhibitors on L-type Ca 2 þ current (I Ca,L ) in guinea-pig ventricular myocytes (Ogura et al, 1999), we observed that both genistein and the structurally and mechanistically different A23 (Levitzki & Gazit, 1995) caused development of outward current at 0 mV. The current appeared to be CFTR Cl À current (I Cl(CFTR) ), and was not further investigated.…”

“…Genistein, tyrphostin A23 (A23), and tyrphostin A25 (A25) are tyrosine kinase (TK) inhibitors (Akiyama et al, 1987;Gazit et al, 1989;Holmes et al, 1996) that are widely used to investigate the involvement of tyrosine phosphorylation in the regulation of ion channels (Cataldi et al, 1996;Wang et al, 1996;Ogura et al, 1999;Albert et al, 2001), pumps (Feraille et al, 1997), and exchangers (Wang et al, 1997;Linck et al, 1998;Kiang et al, 2003). The inhibitors are active on external application, and are typically used at concentrations up to 300 mM, with little effect on the activity of other kinases such as protein kinase A (PKA) and protein kinase C (PKC) (Akiyama et al, 1987;Enright & Booth, 1991;O'Dell et al, 1992).…”

Redox modulation of basal and β‐adrenergically stimulated cardiac L‐type Ca2+ channel activity by phenylarsine oxide

“…Previous studies have demonstrated that the inhibition of tyrosine phosphatase activity by vanadate derivatives has little or no effect on the basal L-type Ca 2 þ current in cardiac myocytes (Wang & Lipsius, 1998;Ogura et al, 1999;Sims et al, 2000;Belevych et al, 2001). On the other hand, inhibition of tyrosine phosphatase activity by PAO, an arsenicbased compound, has been reported to regulate the basal L-type Ca 2 þ channel activity in vascular smooth muscle cells and neurons (Pafford et al, 1995;Wijetunge et al, 1998).…”