Effects of protein tyrosine kinase inhibitors on voltage‐operated calcium channel currents in vascular smooth muscle cells and pp60^c‐src kinase activity

Abstract

1 Tyrosine kinases have been proposed as regulators of voltage-operated calcium channels. The e ects of a range of structurally di erent inhibitors of protein tyrosine kinases (PTK) were examined on voltage-operated calcium channel currents (I Ba ) and pp60 c-src kinase (c-src) activity in vitro. 2 I Ba was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage-clamp techniques. The activity of puri®ed human c-src was measured in vitro using a nonradioactive assay. 3 Bath application of tyrphostin-23 and genistein (non-selective PTK inhibitors), bistyrphostin (a receptor-PTK-selective inhibitor) and PP1 (a src family-selective inhibitor) inhibited I Ba in a concentration-dependent manner over a range of test membrane potentials. Intracellular application of peptide-A, a peptide inhibitor of c-src also inhibited currents. Inhibitor potency series against I Ba was PP1 4 genistein 4 tyrphostin 23 4 bistyrphostin. 4 Tyrphostin-23, genistein, PP1, and peptide-A shifted the steady-state inactivation curves in a hyperpolarized direction without altering their slope. The inhibitors had no signi®cant e ects on I Ba activation calculated from current-voltage relationships. 5 The agents inhibited c-src activity in a concentration-dependent manner. The order of potency was PP1 4 genistein 4 peptide-A 4 tyrphostin-23 4 bistyrphostin. The IC 50 for inhibition of c-src activity was similar to the IC 50 for inhibition of I Ba in all cases. 6 Western blot analysis with a speci®c antibody to c-src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells. 7 A range of structurally dissimilar inhibitors of PTKs inhibit I Ba and c-src activity with similar potency. These data provide further evidence implicating endogenous c-src in the modulation of Ltype calcium channels in vascular smooth muscle cells. British Journal of Pharmacology (2000) 129, 1347 ± 1354 Keywords: Calcium; calcium channel; L-type; smooth muscle; vascular; pp60src; tyrosine kinase Abbreviations: BSA, bovine serum albumin; [Ca 2+ ] i , intracellular calcium concentration; c-src, pp60 src kinase; DMSO, dimethyl sulphoxide; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; ELISA, enzyme linked immunosorbent assay; g, maximum conductance; HRP, horseradish peroxidase; I Ba , voltage-operated calcium channel currents; I non , non-inactivating component of current; I-V, current-voltage; k, slope factor; Mab, monoclonal antibody; PDGF, platelet-derived growth factor; PMSF, phenylmethylsulphonyl¯uoride; PP1, (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine); PSS, modi®ed physiological salt solution; PTK, protein tyrosine kinase; SDS ± PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TBS, Tris bu ered saline; TBS, Tris bu ered saline containing 0.001% Tween 20; TEA, tetraethylammonium; V, membrane potential; V act , membrane potential for half maximal activation; V inact , membrane potential for half maximal inactivation; V r , reversal potential

Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells

“…Even though an active and direct participation of TKs in L-type Ca 2ϩ channel function in different tissues was demonstrated (29,38,49,52), less is known about VDCC regulation through tyrosine phosphorylation in GH3 cells. Cataldi et al (5) showed that TK inhibition reduced L-type Ca 2ϩ channel activity evoked by 55 mM K ϩ in GH3 cells.…”

“…Non-RTKs, such as pp60 c-Src (c-Src), have also been involved in the control of Ca 2ϩ signal transduction pathways, including interaction with Ca 2ϩ channels in different tissues (38,52). To study whether the depolarization-induced activa- Fig.…”

“…However, the nature of the Ca 2ϩ channels involved and a possible regulation through direct channel phosphorylation by RTKs remain controversial and differ among tissues. On the other hand, a direct Ca 2ϩ channel regulation by the c-Src family of cytosolic tyrosine kinases (TKs) has been suggested, but contradictory results have been reported (38,52 (20).…”

Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate

“…It has been reported that agents that increase tyrosine phosphorylation activate a nonselective cation channel (1). Src is implicated in modulation of Ca 2ϩ influx through both store-operated channels (2) and L-type Ca 2ϩ channels (30). Ca 2ϩ release from the ER critically depends on PLC activity.…”

“…The effect of tyrosine kinases on the sustained phase of the Ca 2ϩ response to contractile agonists in smooth muscle is equally unclear. Potentiation (25) or inhibition (10) of this phase of the response by tyrosine kinase inhibitors has been described, and tyrosine kinases have been implicated in regulation of voltagedependent L-type Ca 2ϩ channels (30), nonspecific cation channels (1), and store-operated Ca 2ϩ channels (2). In the present study, we employed several strategies to clarify the role of Src kinases in GPCR-mediated ASM contraction and Ca 2ϩ signaling, focusing mainly on the effects of Src family kinases on Ca 2ϩ release from the endoplasmic reticulum (ER).…”