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 ± 0.4×103 N/m-2 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-myristate 13-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 Cw2+-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-α and the Ca2+-independent PKC-δ. -ε, and -ζ isoforms. In unstimulated tissues, PKC-α- and -ε were mainly cytosolic. PKC-δ was mainly in the particulate fraction, and PKC-ζ was equally distributed in the cytosolic and particulate fractions. Ox-LDL alone or PMA alone caused translocation of PKC-ε from the cytosolic to particulate fraction, whereas the distribution pattern of PKC-α, -δ, and -ζ 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-α 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-ε and enhances the contraction to [Ca2+]i-increasing agonists by activating the Ca2+-dependent PKC-α. Activation of PKC-α and -ε may represent a possible cellular mechanism by which ox-LDL could enhance coronary vasospasm.
|Original language||English (US)|
|Number of pages||8|
|Issue number||2 II|
|State||Published - Mar 19 2001|
- Vascular smooth muscle
ASJC Scopus subject areas
- Internal Medicine