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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

Increasingly it is recognized that tyrosine phosphorylation plays an important part in the regulation of function in differentiated contractile vascular smooth muscle. Tyrosine kinases and phosphatases are present in large amounts in vascular smooth muscle and have been reported to influence a number of processes crucial to contraction, including ion channel gating, calcium homeostasis and sensitization of the contractile process to [Ca2+]i. This review summarizes current understanding regarding the role of tyrosine phosphorylation in excitation-contraction coupling in blood vessels.