Ca²⁺ antagonist-insensitive coronary smooth muscle contraction involves activation of ϵ-protein kinase C-dependent pathway

Abstract

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.

Estrogen Receptor Subtypes Mediate Distinct Microvascular Dilation and Reduction in [Ca2+]i in Mesenteric Microvessels of Female Rat

“…While Ca 21 is a major determinant of VSM contraction, additional mechanisms, such as Rho-kinase and PKC, may increase the myofilament force sensitivity to [Ca 21 ] i (Dallas and Khalil, 2003;Schubert et al, 2008). E2 inhibits Rhokinase activity in the female rat basilar artery (Chrissobolis et al, 2004), Rho-kinase mRNA expression in coronary VSM Fig.…”

PKCα Deficiency in Mice Is Associated with Pulmonary Vascular Hyperresponsiveness to Thromboxane A2 and Increased Thromboxane Receptor Expression

“…1c). This pulmonary vascular hyperresponsiveness in PKCα -/- mice was, at first sight, contradictory, as PKCα is thought to promote vasoconstriction [40,41]. To analyze whether this hyperresponsiveness is (1) thromboxane-specific or (2) specific for vasoconstriction initiated at the vascular site via direct G protein-coupled receptor activation, we studied pulmonary vascular responsiveness to ET-1 and serotonin as well as HPV.…”

“…As PKCα is proposed to be a promoter of smooth muscle cell contraction [40,41,43], this finding was unexpected. TXA2 is a prostanoid derived from arachidonic acid, which exerts its constrictive effects by binding TP receptors on vascular smooth muscle cells, leading to the inhibition of voltage-gated K + channels, membrane depolarization, the activation of L-type Ca 2+ channels, and ultimately smooth muscle cell contraction [16].…”

Functional medial thickening and folding of the internal elastic lamina in coronary spasm

“…There are a number of studies on the substances that may participate in coronary spasm and on the roles of ECs in the genesis of coronary spasm (1,5,8). Morphological studies on the behavior of medial SMCs that play an important role in spasm, however, have been ignored.…”