Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of epsilon-protein kinase C-dependent pathway.
Certain angina and coronary artery disease forms do not respond to Ca2+ channel blockers, and a role for vasoactive eicosanoids such as PGF2alpha in Ca2+ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF2alpha-induced coronary smooth muscle contraction is Ca2+ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca2+ concentration ([Ca2+]i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca2+), PGF2alpha (10-5 M) caused transient [Ca2+]i increase followed by maintained [Ca2+]i increase and 34% cell contraction. Ca2+ channel blockers verapamil and diltiazem (10-6 M) abolished maintained PGF2alpha-induced [Ca2+]i increase but only partially inhibited PGF2alpha-induced cell contraction to 17%. Verapamil-insensitive PGF2alpha contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and epsilon-PKC V1-2. PGF2alpha caused Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF2alpha-induced alpha-but not epsilon-PKC translocation. PMA (10-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca2+]i increase and epsilon-PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca2+ influx, caused 36% cell contraction and [Ca2+]i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause alpha- or epsilon-PKC translocation. Thus a significant component of PGF2alpha-induced contraction of coronary smooth muscle is Ca2+ antagonist insensitive, involves Ca2+-independent epsilon-PKC activation and translocation, and may represent a signaling mechanism of Ca2+ antagonist-resistant coronary vasospasm.
Harvard Medical School, VA Boston Healthcare-Research, 1400 VFW Parkway, 3/2B123, Boston, MA 02132, USA.
Andrea
A
Dallas
Raouf A
RA
Khalil
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002478:Cells, Cultured; D003329:Coronary Vasospasm / Q000503:physiopathology; D003331:Coronary Vessels / Q000166:cytology / Q000187:drug effects; D015237:Dinoprost / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D007424:Intracellular Fluid / Q000737:chemistry / Q000187:drug effects; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013552:Swine; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D014662:Vasoconstrictor Agents; D015237:Dinoprost; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D002118:Calcium