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Effects of protein tyrosine kinase inhibitors on voltage‐operated calcium channel currents in vascular smooth muscle cells and pp60 c‐src kinase activity
Author(s) -
Wijetunge S,
Lymn J S,
Hughes A D
Publication year - 2000
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0703186
Subject(s) - proto oncogene tyrosine protein kinase src , tyrosine kinase , genistein , tyrosine kinase inhibitor , kinase , biology , microbiology and biotechnology , vascular smooth muscle , tyrosine protein kinase csk , calcium channel , tyrosine phosphorylation , chemistry , calcium , sh2 domain , biochemistry , signal transduction , endocrinology , genetics , organic chemistry , cancer , smooth muscle
Tyrosine kinases have been proposed as regulators of voltage‐operated calcium channels. The effects of a range of structurally different 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 . I Ba was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage‐clamp techniques. The activity of purified human c‐src was measured in vitro using a non‐radioactive assay. 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 > genistein > tyrphostin 23 > bistyrphostin. 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 significant effects on I Ba activation calculated from current‐voltage relationships. The agents inhibited c‐src activity in a concentration‐dependent manner. The order of potency was PP1 > genistein > peptide‐A > tyrphostin‐23 > bistyrphostin. The IC 50 for inhibition of c‐src activity was similar to the IC 50 for inhibition of I Ba in all cases. Western blot analysis with a specific antibody to c‐src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells. 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 L‐type calcium channels in vascular smooth muscle cells.British Journal of Pharmacology (2000) 129 , 1347–1354; doi: 10.1038/sj.bjp.0703186