Abstract
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-(β-[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.
Footnotes
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This study was supported by the Banyu Fellowship Awards in Cardiovascular Medicine (to Y.K.), sponsored by Banyu Pharmaceutical Co., Ltd., and by The Merck Company Foundation (to Y.K.), by a grant from the Smoking Research Foundation, Japan, and by the Uehara Memorial Foundation Fellowship, Tokyo, Japan.
- Abbreviations:
- ET-1
- endothelin-1
- VSMC
- vascular smooth muscle cell
- GPCR
- G-protein coupled receptor
- ETxR
- endothelin type x receptor (x = A or B)
- PYK2
- proline-rich tyrosine kinase 2
- VICC
- voltage-independent Ca2+ channels
- PI3K
- phosphoinositide 3-kinase
- VOCC
- voltage-operated Ca2+channel
- NSCC
- nonselective cation channel
- SOCC
- store-operated Ca2+ channel
- SK&F 96365
- 1-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride
- LOE 908
- (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate
- ICA
- internal carotid artery
- PBS
- phosphate-buffered saline
- BQ 788
- N-cis-2,6-dimethylpiperidinocarbonyl-l-γ-methylleucyl-d-1-methoxycarbonyltrptophanyl-d-Nle
- LY 294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
- BQ123
- cyclo-d-Asp-Pro-d-Val-Leu-d-Trp
- Received October 16, 2002.
- Accepted December 24, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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