title: 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 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. 2. 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. 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 > genistein > tyrphostin 23 > 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 significant effects 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 > 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. 6. Western blot analysis with a specific 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 L-type calcium channels in vascular smooth muscle cells.
authors.affiliation: Imperial College School of Medicine, National Heart & Lung Institute, St Mary's Hospital, London W2 1NY, UK. v.wijetunge@ic.ac.uk
authors.forename: S
authors.initials: S
authors.lastname: Wijetunge
authors.forename: J S
authors.initials: JS
authors.lastname: Lymn
authors.forename: A D
authors.initials: AD
authors.lastname: Hughes
mesh_terms: D000818:Animals; D001158:Arteries / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D015220:Calcium Channels / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004423:Ear / Q000098:blood supply; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000187:drug effects* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011817:Rabbits; D020032:Tyrphostins / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D020032:Tyrphostins; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C060641:tyrphostin A23