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(Hypertension. 2001;37:561.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Oxidized-LDL Enhances Coronary Vasoconstriction by Increasing the Activity of Protein Kinase C Isoforms and

Jena B. Giardina; Dennis J. Tanner; Raouf A. Khalil

From the Department of Physiology and Biophysics and Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson.

Correspondence to Raouf A. Khalil, MD, PhD, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216. E-mail rkhalil{at}physiology.umsmed.edu

Oxidized low-density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerotic coronary vasospasm; however, the cellular mechanisms involved are not fully understood. We tested the hypothesis that ox-LDL enhances coronary vasoconstriction by increasing the activity of specific protein kinase C (PKC) isoforms in coronary smooth muscle. Active stress was measured in de-endothelialized porcine coronary artery strips; cell contraction and [Ca²⁺]_i were monitored in single coronary smooth muscle cells loaded with fura-2; and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies with Western blots. Ox-LDL (100 µg/mL) caused slow but significant increases in active stress to 1.3±0.4x10³ N/m² and cell contraction (10%) that were completely inhibited by GF109203X (10^-6 mol/L), an inhibitor of Ca²⁺-dependent and -independent PKC isoforms, with no significant change in [Ca²⁺]_i. 5-Hydroxytryptamine (5-HT; 10^-7 mol/L) and KCl (24 mmol/L) caused increases in cell contraction and [Ca²⁺]_i that were inhibited by the Ca²⁺ channel blocker verapamil (10^-6 mol/L). Ox-LDL enhanced coronary contraction to 5-HT and KCl with no additional increases in [Ca²⁺]_i. Direct activation of PKC by phorbol 12-myristate13-acetate (PMA; 10^-7 mol/L) caused a contraction similar in magnitude and time course to ox-LDL–induced contraction and enhanced 5-HT– and KCl-induced contraction with no additional increases in [Ca²⁺]_i. The ox-LDL–induced enhancement of 5-HT and KCl contraction was inhibited by Gö6976 (10^-6 mol/L), an inhibitor of Ca²⁺-dependent PKC isoforms. Both ox-LDL and PMA caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and an increase in the particulate/cytosolic PKC activity ratio. Western blots revealed the Ca²⁺-dependent PKC- and the Ca²⁺-independent PKC-, -, and - isoforms. In unstimulated tissues, PKC-- and - were mainly cytosolic, PKC- was mainly in the particulate fraction, and PKC- was equally distributed in the cytosolic and particulate fractions. Ox-LDL alone or PMA alone caused translocation of PKC- from the cytosolic to particulate fraction, whereas the distribution pattern of PKC-, -, and - remained unchanged. 5-HT (10^-7 mol/L) alone and KCl alone did not change PKC activity. In tissues pretreated with ox-LDL or PMA, 5-HT and KCl caused additional increases in PKC- activity. Native LDL did not significantly affect coronary contraction, [Ca²⁺]_i, or PKC activity. These results suggest that ox-LDL causes coronary contraction via activation of the Ca²⁺-independent PKC- and enhances the contraction to [Ca²⁺]_i-increasing agonists by activating the Ca²⁺-dependent PKC-. Activation of PKC- and - may represent a possible cellular mechanism by which ox-LDL could enhance coronary vasospasm.

Key Words: lipoproteins, 5-hydroxytryptamine • calcium • vascular smooth muscle • contraction

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