Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells.
The mitogenic effect of extracellular ATP was examined in cultured rat aortic smooth muscle cells (VSMCs). ATP, 2-methylthio-ATP, and ADP stimulated [3H]thymidine and [3H]leucine incorporation and cell growth. AMP, adenosine, UTP, and P2x agonists showed little of these effects. Reactive blue 2, a P2Y purinoceptor antagonist, was effective in suppressing the mitogenic effect of ATP and 2-methylthio-ATP, indicating that extracellular ATP-induced VSMC proliferation is mediated by P2Y purinoceptors. The P2Y purinoceptor activation was coupled to a pertussis toxin (PTX)-insensitive G protein (Gq) and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C (PKC), Raf-1, and mitogen-activated protein kinase (MAPK) in VSMCs. In response to ATP, both 42-and 44-kDa MAPKs were activated, and tyrosine was phosphorylated. Western blot analysis using PKC isozyme-specific antibodies indicated that VSMCs express PKC-alpha, PKC-delta, and PKC-zeta. A complete down-regulation of PKC-alpha and PKC-delta was seen after 24-hr treatment with 12-O-tetradecanoylphorbol-13-acetate. When cells were pretreated with 12-O-tetradecanoyl-phorbol-13-acetate for 24 hr and subsequently challenged with ATP, Raf-1 activation and 42-kDa as well as 44-kDa MAPK tyrosine phosphorylation failed to be induced. These results demonstrate that ATP-induced Raf-1 and MAPK activations involve the activation of PKC-alpha and PKC-delta. P2Y purinoceptor stimulation with ATP also caused accumulation of c-fos and c-myc mRNAs. Both Reactive blue 2 and staurosporine significantly blocked this increase by ATP. In conclusion, the mitogenic effect of ATP seemed to be triggered by activation of the Gq protein-coupled P2Y purinoceptor that led to the formation of inositol trisphosphate and activation of PKC. PKC and, in turn, Raf-1 and MAPK were then activated, leading eventually to DNA synthesis and cell proliferation.
Yu
S M
SM
Department of Pharmacology, Chang Gung College of Medicine and Technology, Kwei-San, Tao-Yuan, Taiwan. smyu@cguaplo.cgu.edu
Chen
S F
SF
Lau
Y T
YT
Yang
C M
CM
Chen
J C
JC
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology; D066298:In Vitro Techniques; D007930:Leucine / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013268:Stimulation, Chemical; D013936:Thymidine / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis; D014316:Tritium
D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D014157:Transcription Factors; D014316:Tritium; D000255:Adenosine Triphosphate; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D007930:Leucine; D013936:Thymidine