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 PGF₂ in Ca²⁺ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF₂-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⁺), PGF₂ (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 PGF₂-induced [Ca²⁺]_i increase but only partially inhibited PGF₂-induced cell contraction to 17%. Verapamil-insensitive PGF₂ contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and -PKC V1-2. PGF₂ caused Ca²⁺-dependent -PKC and Ca²⁺-independent -PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF₂-induced -but not -PKC translocation. PMA (10^-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca²⁺]_i increase and -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 - or -PKC translocation. Thus a significant component of PGF₂-induced contraction of coronary smooth muscle is Ca²⁺ antagonist insensitive, involves Ca²⁺-independent -PKC activation and translocation, and may represent a signaling mechanism of Ca²⁺ antagonist-resistant coronary vasospasm. eicosanoids; calcium; vascular smooth muscle