A(1) adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells.
The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.
Ansari
Habib R
HR
Department of Physiology and Pharmacology, Center for Cardiovascular and Respiratory Sciences, Robert C. Byrd Health Science Center, School of Medicine, West Virginia University, Morgantown, WV 26506, USA.
Teng
Bunyen
B
Nadeem
Ahmed
A
Roush
Kevin P
KP
Martin
Karen H
KH
Schnermann
J
J
Mustafa
S Jamal
SJ
D000241:Adenosine / Q000031:analogs & derivatives; D058907:Adenosine A1 Receptor Agonists; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D054799:Phospholipase C beta / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D043682:Receptor, Adenosine A1 / Q000235:genetics* / Q000378:metabolism*
D058907:Adenosine A1 Receptor Agonists; D002227:Carbazoles; D004791:Enzyme Inhibitors; D005419:Flavonoids; D043682:Receptor, Adenosine A1; C081021:Go 6976; C087857:protein kinase C gamma; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D054799:Phospholipase C beta; C518040:Plcb3 protein, mouse; D000241:Adenosine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one