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(Hypertension. 2004;43:897.)
© 2004 American Heart Association, Inc.

Scientific Contributions

Endothelin-1 Promotes Ca²⁺ Antagonist-Insensitive Coronary Smooth Muscle Contraction Via Activation of -Protein Kinase C

From the Department of Medicine, Veterans Affairs Medical Center, West Roxbury, and Harvard Medical School, Boston, Mass.

Correspondence to Raouf A. Khalil, MD, PhD, Harvard Medical School, VA Boston Healthcare-Research, 1400 VFW Parkway, 3/2B123, Boston, MA 02132. E-mail raouf_khalil{at}hms.harvard.edu

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 -PKC_V1–2. ET-1 caused translocation of Ca²⁺-dependent -PKC and Ca²⁺-independent -PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1–induced translocation of -PKC, but not that of -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 -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 - or -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 -PKC that were inhibited by -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 -PKC, and may represent a signaling mechanism of Ca²⁺ antagonist-resistant forms of coronary vasospasm.

Key Words: endothelin • calcium • protein kinases

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