Vol. 63, Issue 4, 808-813, April 2003

Involvements of Voltage-Independent Ca²⁺ Channels and Phosphoinositide 3-Kinase in Endothelin-1-Induced PYK2 Tyrosine Phosphorylation

Yoshifumi Kawanabe, Nobuo Hashimoto, and Tomoh Masaki

Departments of Neurosurgery (Y.K., N.H.) and Pharmacology (T.M.), Kyoto University Graduate School of Medicine, Kyoto, Japan; and Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts (Y.K.)

We demonstrated recently that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels [designated nonselective cation channel (NSCC)-1 and NSCC-2] and a store-operated Ca²⁺ channel (SOCC) in rabbit internal carotid artery vascular smooth muscle cells (ICA VSMCs). These channels can be distinguished by their sensitivity to Ca²⁺ channel blockers 1-(-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365, NSCC-2 is sensitive to both LOE 908 and SK&F 96365, and SOCC is resistant to LOE 908 and sensitive to SK&F 96365. The purpose of the present study was to identify the Ca²⁺ channels involved in the ET-1-induced, proline-rich tyrosine kinase 2 (PYK2) phosphorylation in ICA VSMCs. Based on sensitivity to nifedipine, an L-type voltage-operated Ca²⁺ channel (VOCC) blocker, Ca²⁺ influx through VOCC seems to play a minor role in the ET-1-induced PYK2 phosphorylation. In the presence of nifedipine, PYK2 phosphorylation was abolished by blocking Ca²⁺ influx through NSCC-1, NSCC-2, and SOCC. The phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibited ET-1-induced Ca²⁺ influx through NSCC-2 and SOCC. In addition, these inhibitors blocked PYK2 phosphorylation that depends on Ca²⁺ influx through NSCC-2 and SOCC. These results indicate that 1) Ca²⁺ influx through NSCC-1, NSCC-2, and SOCC plays essential roles in ET-1-induced PYK2 phosphorylation, 2) NSCC-2 and SOCC are stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade, and 3) PI3K is involved in the PYK2 phosphorylation that depends on Ca²⁺ influx through SOCC and NSCC-2.