Endothelin-1 promotes Ca2+ antagonist-insensitive coronary smooth muscle contraction via activation of epsilon-protein kinase C.
Certain forms of coronary artery disease do not respond to treatment with Ca2+ channel blockers, and a role for endothelin-1 (ET-1) in Ca2+ antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1-induced coronary smooth muscle contraction is Ca2+ antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca2+]i was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank's solution (1 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused a transient increase in [Ca2+]i (236+/-14 nmol/L) followed by a maintained increase in [Ca2+]i (184+/-8 nmol/L) and 35% cell contraction. The Ca2+ channel blockers verapamil and diltiazem (10(-6) mol/L) abolished the maintained ET-1-induced [Ca2+]i, but only partially inhibited ET-1-induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and epsilon-PKCV1-2. ET-1 caused translocation of Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1-induced translocation of alpha-PKC, but not that of epsilon-PKC. Phorbol 12-myristate 13-acetate (10(-6) mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca2+ influx, caused 35% cell contraction and increase in [Ca2+]i (291+/-11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of alpha- or epsilon-PKC. In Ca2+-free (2 mmol/L EGTA) Hank's solution, ET-1 caused 15% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that were inhibited by epsilon-PKC V1-2 inhibitory peptide. Thus, a significant component of ET-1-induced contraction of coronary smooth muscle is Ca2+ antagonist-insensitive and involves activation and translocation of Ca2+-independent epsilon-PKC, and may represent a signaling mechanism of Ca2+ antagonist-resistant forms of coronary vasospasm.
Department of Medicine, Veterans Affairs Medical Center, West Roxbury, Mass, USA.
Lashonn L
LL
McNair
Daisy A
DA
Salamanca
Raouf A
RA
Khalil
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003329:Coronary Vasospasm / Q000201:enzymology / Q000503:physiopathology*; D003331:Coronary Vessels / Q000187:drug effects* / Q000502:physiology; D004110:Diltiazem / Q000494:pharmacology; D004195:Disease Models, Animal; D004351:Drug Resistance; D019332:Endothelin-1 / Q000494:pharmacology* / Q000502:physiology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D009281:Naphthalenes / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D051744:Protein Kinase C-epsilon; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects* / Q000502:physiology; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D010446:Peptide Fragments; D014700:Verapamil; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D004110:Diltiazem; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium