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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

	Abstract

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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

	Introduction

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Coronary vasospasm is often associated with ischemic heart disease, angina, and myocardial infarction.¹ Although the causes of coronary vasospasm are not clear, hypercholesterolemia and atherosclerosis have been implicated as major risk factors. Low-density lipoprotein (LDL), a major carrier of cholesterol in the circulation, becomes very atherogenic if it undergoes oxidative modification by endothelial cells, vascular smooth muscle, or macrophages within the arterial wall.^{2 3 4 5} Oxidized LDL (ox-LDL) is rapidly taken up by macrophages in the arterial wall, thus transforming these cells into foam cells, one of the early signs of atherosclerosis (fatty streak).² Additionally, ox-LDL induces several proatherogenic mechanisms that stimulate basal coronary tone^{6 7 8} and enhance coronary vasoconstriction to vasoactive agonists such as 5-hydroxytryptamine (5-HT).^{8 9 10}

Several studies have suggested that ox-LDL stimulates coronary tone and enhances 5-HT contraction by decreasing endothelium-derived nitric oxide release and reducing endothelium-dependent vascular relaxation.^{6 7 8 9 10} However, whether ox-LDL directly affects coronary smooth muscle reactivity is unclear.¹¹ Also, whether the ox-LDL–induced changes in coronary contraction involve alterations in the mechanisms of smooth muscle contraction is not clearly understood.

Vascular smooth muscle contraction is triggered by increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) resulting from Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space.^{12 13 14 15} Also, the interaction of a vasoconstrictor agonist with its receptor stimulates the breakdown of plasma membrane phospholipids and increases the production of diacylglycerol (DAG).^{16 17} DAG binds to and activates protein kinase C (PKC). PKC is a family of several isoforms that have different enzyme properties and exhibit different subcellular distributions.^{16 17 18 19} PKC is mainly cytosolic under resting conditions and undergoes translocation to the particulate fraction when activated by DAG or phorbol esters.^{16 17} Also, direct activation of PKC by phorbol esters such as phorbol 12-myristate 13-acetate (PMA) causes sustained contraction of vascular smooth muscle with no significant change in [Ca²⁺]_i,^{20 21} suggesting a role for PKC in regulating smooth muscle contraction, at least in part, by increasing the myofilament force sensitivity to [Ca²⁺]_i.

The purpose of this study was to test the hypothesis that ox-LDL causes coronary smooth muscle contraction and enhances the contraction to vasoactive agonists such as 5-HT by increasing the activity of specific PKC isoforms. Because the PKC family includes both Ca²⁺-dependent and -independent isoforms, any ox-LDL– or 5-HT–induced changes in [Ca²⁺]_i may determine which PKC isoform would be activated. Therefore, experiments were designed to investigate (1) whether ox-LDL stimulates contraction and enhances 5-HT–induced contraction in coronary smooth muscle, (2) whether the ox-LDL–induced enhancement of coronary smooth muscle contraction is associated with increases in [Ca²⁺]_i, and (3) whether the ox-LDL–induced enhancement of coronary smooth muscle contraction is associated with increases in the activity of specific PKC isoforms. The effects of ox-LDL were compared with those of the phorbol ester PMA, a direct activator of PKC, and the sensitivity of the effects to the Ca²⁺ channel antagonist verapamil and the PKC inhibitors GF109203X and Gö6976 was also investigated. Because ox-LDL may affect various types of vascular cells, including endothelial cells,^{6 7 8 9 10} the present study was performed on endothelium-denuded porcine coronary artery strips and freshly isolated coronary smooth muscle cells.

	Methods

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Tissue Preparation
Castrated male Yorkshire pigs (30 kg, local breeder) were anesthetized with isoflurane. The heart was rapidly excised, and the left anterior descending coronary artery was carefully dissected under microscopic visualization, cleaned of adipose and connective tissue, and opened by a cut along its longitudinal axis. The endothelium was removed by rubbing of the vessel interior with wet filter paper. The coronary artery was then sectioned into 3x5-mm strips. All procedures followed the guidelines of the Institutional Animal Care and Use Committee.

Isometric Contraction
Coronary strips were transferred to a tissue bath containing Krebs’ solution bubbled with 95% O₂/5% CO₂ at 37°C. One end of the strip was fixed to a glass hook, and the other end was connected to a force transducer (Grass FT03C, Astro-Med). The strips were stretched to L_max (1.5 the initial unloaded length, L) and allowed to equilibrate for 1 hour while the changes in tension were displayed on a Grass 7D polygraph. After two 96-mmol/L KCl contractions followed by washing with normal Krebs’ solution, the tissues were stimulated with ox-LDL (100 µg/mL), PMA (10^-7 mol/L), 5-HT, or KCl either separately or combined. Control tissues were either untreated or treated with native LDL or the inactive 4-PMA. The developed force was corrected for the cross-sectional area of the strip and expressed as active stress (N/m²) with this equation: stress=force/cross-sectional area, where cross-sectional area=wet weight/(tissue densityxstrip length), and tissue density=1.055 g/cm³.

Single Cell Isolation
Coronary artery strips (50 mg) were placed in a tissue digestion mixture containing 5 mg collagenase type II (236 U/mg protein, Worthington), 4 mg elastase (3.25 U/mg protein, Boehringer Mannheim), and 147 µL trypsin inhibitor (10 000 U/mL, Sigma) dissolved in 7.5 mL of Ca²⁺- and Mg²⁺-free Hanks’ solution supplemented with 30% bovine serum albumin.^{22 23 24} The preparation was placed in a 125-mL Pyrex flask in a shaking water bath (80 cycles/min) for 60 minutes at 34°C in an atmosphere of 95% O₂/5% CO₂. The preparation was strained through a nylon mesh and rinsed with 12.5 mL Hanks’ solution, and the fluid containing the first batch of cells was discarded. The remaining tissue was further digested twice for 30 minutes in a digestion medium containing 2.5 mg collagenase, 4 mg elastase, and 122 µL trypsin inhibitor. The preparation was strained again through the nylon mesh and rinsed with 12.5 mL Hanks’ solution, and the fluid containing batches 2 and 3 of cells was poured over glass coverslips placed in wells and cooled to 2°C. The cells were allowed to settle by gravity and adhere to the glass coverslips. The coverslips with the attached cells were removed from the enzyme solution and placed in enzyme-free Hanks’ solution. Increasing concentrations of extracellular Ca²⁺ were gradually added back to the Hanks’ solution to avoid the "calcium paradox."²⁵

Cell Contraction Studies
Coverslips with the attached cells were viewed on a Nikon (Diaphot-300) microscope. Only viable, healthy, and spindle-shaped smooth muscle cells 60 µm in length were selected. Viable cells adhered to the glass coverslips and appeared bright, with a halo along the periphery and without a visible nucleus when viewed with phase-contrast optics. Cell viability was confirmed at the end of each experiment by their consistent and significant contraction (30%) to 5-HT (10^-5 mol/L) or KCl (51 mmol/L). Cell images were acquired with a PXL CCD camera and displayed on a PC with PMIS image analysis software (Photometrics). The number of pixels corresponding to the cell length was transformed into microns with a calibration bar. The cell contraction was expressed as (L_i-L)/L_i, where L_i is the initial cell length and L is the final cell length. All contraction measurements were made at 37°C.

Measurement of [Ca²⁺]_i
Cells were loaded with fura-2 for 30 minutes at 34°C.^{23 24 26} The fura-2 loading solution was made of Hanks’ solution, 1 µmol/L fura-2 acetoxymethyl ester (fura-2/AM) (Molecular Probes), and 0.01% Pluronic F-127 (Sigma). The fura-2–loaded cells were washed and further incubated in Hanks’ solution for 30 minutes to allow complete de-esterification of fura-2/AM. Precautions were taken throughout the procedure to avoid extensive photobleaching of fura-2.²⁷

The fura-2–loaded cells were viewed through a Nikon 100X oil-immersion objective (NA 1.3) on an inverted Nikon (Diaphot-300) microscope. The Ca²⁺ indicator was excited alternately at 340 and 380 nm with a filter wheel that alternates at 0.5 Hz. The emitted light was collected at 510 nm to a photomultiplier tube R928 (Ludl Electronic Products) through a pinhole 1 µm in diameter positioned 1 µm from the plasma membrane and 1 µm from the nucleus. The fluorescent signal was digitized with a module (Mac 2000, Ludl) and analyzed on a PC with data analysis software. The fluorescent signal was background subtracted. The ratio between the fluorescence intensity at 340 and 380 nm (R) was transformed to [Ca²⁺]_i as described by Grynkiewicz et al²⁷ : [Ca²⁺]_i=K_d(Sf₂/Sb₂)[(R-R_min)/(R_max-R)], where R_min and R_max are the minimal and maximal ratios measured by adding fura-2 free acid (50 µmol/L) to Ca²⁺-free (10 mmol/L EGTA) and Ca²⁺-replete (2 mmol/L) solutions, respectively. Sf₂/Sb₂ is the ratio of the 380-nm signal in Ca²⁺-free and Ca²⁺-replete solutions, respectively. K_d is the dissociation constant of fura-2 for Ca²⁺.²⁷ All experiments were performed at 37°C.

Tissue Fractions
Tissue strips (80 mg) at rest or stimulated with ox-LDL or PMA for 1 hour or with 5-HT or KCl for 30 minutes were transferred to ice-cold buffer A containing (mmol/L) Tris · HCl 25 (pH 7.5), EGTA 5, leupeptin 0.02, phenylmethylsulfonyl fluoride 0.2, and dithiothreitol 1. For PKC activity, the tissue was transferred to a homogenization buffer B that had the same composition as buffer A plus 250 mmol/L sucrose. For Western blots, the tissue was transferred to a homogenization buffer containing 20 mmol/L 3-[N-morpholino]propane sulfonic acid, 4% sodium dodecylsulfate, 10% glycerol, 2.3 mg dithiothreitol, 1.2 mmol/L EDTA, 0.02% bovine serum albumin, 5.5 µmol/L leupeptin, 5.5 µmol/L pepstatin, 2.15 µmol/L aprotinin, and 20 µmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride. The tissue was homogenized with a 2-mL tight-fitting homogenizer (Kontes Glass) at 4°C and centrifuged at 100 000 rpm for 20 minutes at 4°C (Ultra-Centrifuge TL-100, Beckman), and the supernatant was used as the cytosolic fraction. The pellet was resuspended in homogenization buffer containing 1% Triton X-100 for 20 minutes, diluted with homogenization buffer to a final concentration of 0.2% Triton, and centrifuged at 100 000 rpm for 20 minutes at 4°C. The supernatant was used as the particulate fraction. Protein concentrations were determined with a protein assay kit (Bio-Rad).

PKC Activity
The cytosolic and particulate fractions were applied to diethylaminoethyl-cellulose columns (0.8x4.0 cm). The columns were washed with buffer A, and the protein was eluted with 0.1 mol/L NaCl. PKC activity in the aliquots was determined with a PKC assay mixture containing 25 mmol/L Tris · HCl (pH 7.5), 10 mmol/L MgCl₂, 200 µg/mL histone IIIS, 80 µg/mL phosphatidylserine, 30 µg/mL diolein, [³²P]ATP (1 to 3x10⁵ cpm/nmol), and 0.5 to 3 µg protein.^{28 29} After 5 minutes’ incubation at 30°C, the reaction was stopped by spotting 25 µL of the assay mixture onto phosphocellulose disks. The disks were washed 3 times for 5 minutes with 5% trichloroacetic acid and placed in 4-mL Ecolite scintillation cocktail, and radioactivity was measured in a liquid scintillation counter.

Immunoblotting
Protein-matched samples of the cytosolic and particulate fractions were subjected to electrophoresis on 8% sodium dodecylsulfate–polyacrylamide gels and then transferred electrophoretically to nitrocellulose membranes. The membranes were incubated in 5% dried milk in phosphate buffered saline (PBS)-Tween at 22°C for 1 hour, washed with PBS-Tween 3 times for 5 minutes, and then incubated in the primary anti-PKC antibody solution at 4°C for 24 hours. Polyclonal antibodies to PKC-, -ß, -, -, -, and - (Gibco) were used. These antibodies have been shown to react with the specific PKC isoforms in porcine endothelial cells and smooth muscle.^{28 29 30 31} The specificity of the antibodies was confirmed by the absence of specific bands when the antibody solution was supplemented with the peptide to which the antibody was raised. We used the same anti-PKC antibody titer (1:500) and protein concentration (10 µg) in all tissue samples. These concentrations gave optimal immunoreactive signals while remaining on the linear portion of the titration curve. The nitrocellulose membranes were washed 5 times for 15 minutes in PBS-Tween and then incubated in horseradish peroxidase–conjugated anti-rabbit secondary antibody for 1.5 hours. The blots were washed with PBS-Tween 5 times for 15 minutes and visualized with enhanced chemiluminescence (Amersham). PBS-Tween contained (mmol/L) Na₂HPO₄ 80, NaH₂PO₄ 20, NaCl 100, and 0.05% Tween. The reactive bands corresponding to PKC isoforms were analyzed by optical densitometry with a GS-700 imaging densitometer (Bio-Rad).

Preparation of Ox-LDL
Native LDL (Sigma) was oxidized by exposure to CuSO₄ (5 µmol/L) in PBS at 37°C for 24 hours.^{4 6} Control incubations were done in the presence of 200 µmol/L EDTA without CuSO₄. Oxidation was terminated by adding EDTA (0.3 mmol/L) and BHT (0.02 mmol/L) and cooling to 4°C. Ox-LDL was separated from the preparation by dialysis against PBS for 24 hours. Oxidation of LDL was confirmed fluorometrically (excitation wavelength, 515 nm; emission wavelength, 553 nm) by measurement of thiobarbituric acid–reactive substances.³² Freshly prepared tetramethyloxypropane, which yields malonaldehyde (MDA), was used as a standard, and results were expressed as nanomole of MDA equivalents. The thiobarbituric acid–reactive substances content of ox-LDL was 1.12±0.08 versus 0.24±0.06 nmol MDA/100 µg protein in the native LDL.

Solutions
Krebs’ solution contained (mmol/L) NaCl 120, KCl 5.9, CaCl₂ 2.5, MgCl₂ 1.2, NaHCO₃ 25, NaH₂PO₄ 1.2, and dextrose 11.5 (pH 7.4). Hanks’ solution contained (mol/L) NaCl 137, KCl 5.4, KH₂PO₄ 0.44, Na₂HPO₄ 0.42, NaHCO₃ 4.17, dextrose 5.55, and HEPES 10 (pH 7.4). For Ca²⁺- and Mg²⁺-containing Hanks’ solution, 1 mmol/L CaCl₂ and 1.2 mmol/L MgCl₂ were added.

Drugs and Chemicals
5-HT (Sigma) and verapamil (Calbiochem) were dissolved in distilled water. PMA (Alexis Laboratory), GF109203X, and Gö6976 (Kamiya) were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in solution was 0.1. All other chemical were of reagent grade or better.

Statistical Analysis
The data were analyzed and presented as mean±SEM and compared by use of Student’s t test for unpaired data with P<0.05 considered significant. The n value represents the number of tissues and cells from different animals.

	Results

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In coronary artery strips incubated in Krebs’ solution (2.5 mmol/L Ca²⁺), ox-LDL (100 µg/mL) caused a slow but significant contraction (Figure 1A). No detectable changes in tension were observed at ox-LDL concentrations <100 µg/mL. The ox-LDL contraction was not inhibited by Gö6976 (10^-6 mol/L), an inhibitor of Ca²⁺-dependent PKC isoforms, but was completely inhibited by GF109203X (10^-6 mol/L), an inhibitor of both Ca²⁺-dependent and -independent PKC isoforms (Figure 1A). 5-HT (10^-7 mol/L) caused a small contraction that was not affected by the PKC inhibitor GF109203X but was completely inhibited by the Ca²⁺ channel blocker verapamil (10^-6 mol/L; Figure 1B). In tissues pretreated with ox-LDL, 5-HT caused a large increase in contraction (Figure 1C). Direct activation of PKC by PMA (10^-7 mol/L) caused a contraction similar to that induced by ox-LDL. The PMA-induced contraction was not inhibited by Gö6976 but was completely inhibited by GF109203X (Figure 1D). Membrane depolarization by KCl (24 mmol/L), which stimulates Ca²⁺ entry from the extracellular space,¹³ caused a small contraction of coronary artery that was not affected by the PKC inhibitor GF109203X but was completely inhibited by the Ca²⁺ channel blocker verapamil (10^-6 mol/L; Figure 1E). In tissues pretreated with PMA, the KCl contraction was significantly enhanced (Figure 1F). The vehicle DMSO (0.1%) did not cause any significant changes in contraction.

View larger version (21K): [in this window] [in a new window]   Figure 1. Contraction in coronary artery strips stimulated with 100 µg/mL ox-LDL (A), 10-7 mol/L 5-HT (B), ox-LDL plus 5-HT (C), 10-7 mol/L PMA (D), 24 mmol/L KCl (E), and PMA plus KCl (F) in the absence or presence of GF109203X (10-6 mol/L) or verapamil (10-6 mol/L). Interruption symbol in A, C, D, and F indicates 1 hour. Concentration-response curves to 5-HT (G) and KCl (H) in tissues untreated or pretreated with ox-LDL (100 µg/mL) or PMA (10-7 mol/L) for 1 hour were constructed. Data points represent the mean±SEM of experiments on 12 to 16 strips from 6 to 8 pigs.

Cumulative data from different tissue strips stimulated with increasing concentrations of 5-HT and KCl were used to construct concentration-response curves. Increasing concentrations of 5-HT and KCl caused concentration-dependent increases in contraction (Figure 1G and 1H). In tissues pretreated with ox-LDL (100 µg/mL) or the PKC activator PMA (10^-7 mol/L) for 1 hour, the 5-HT and KCl concentration-contraction curves were enhanced compared with tissues stimulated with 5-HT alone (Figure 1G) or KCl alone (Figure 1H). Native LDL or the inactive 4-PMA did not cause significant contraction or enhance the contraction to 5-HT or KCl.

Isolated coronary smooth muscle cells were long and spindle shaped (Figure 2). In cells incubated in Hanks’ solution (1 mmol/L Ca²⁺), the resting cell length was 73±6 µm, and the basal [Ca²⁺]_i was 81±2 nmol/L. Ox-LDL (100 µg/mL) caused small but significant cell contraction, with no significant increase in [Ca²⁺]_i (Figure 2A). Activation of PKC by PMA (10^-7 mol/L) also caused small but significant cell contraction with no significant increase in [Ca²⁺]_i (Figure 2D). In contrast, 5-HT (10^-7 mol/L) and KCl (24 mmol/L) caused significant contractions that were associated with significant increases in [Ca²⁺]_i (Figure 2B and 2E). In cells pretreated with ox-LDL or PMA, the 5-HT and KCl contractions were significantly enhanced with no additional increases in [Ca²⁺]_i (Figure 2C and 2F).

View larger version (41K): [in this window] [in a new window]   Figure 2. Coronary smooth muscle cell contraction (top) and [Ca2+]i (bottom) in response to 100 µg/mL ox-LDL (A), 10-7 mol/L 5-HT (B), ox-LDL plus 5-HT (C), 10-7 mol/L PMA (D), 24 mmol/L KCl (E), and PMA plus KCl (F). Interruption symbols in A, C, D, and F indicate a period of 1 hour in which exposure to excitation light was blocked to limit fura-2 photobleaching.

The effects of the Ca²⁺ channel blocker verapamil on ox-LDL–, PMA-, 5-HT–, and KCl-induced changes in cell contraction and [Ca²⁺]_i were investigated. Pretreatment of the cells with verapamil (10^-6 mol/L) did not significantly affect the contraction induced by ox-LDL alone or PMA alone (Figure 3A) and did not significantly change [Ca²⁺]_i under these conditions (Figure 3C), suggesting that Ca²⁺ entry from the extracellular space is not involved. Verapamil completely abolished the contraction (Figure 3A) and [Ca²⁺]_i (Figure 3C) induced by 5-HT (10^-7 mol/L) alone or KCl (24 mmol/L) alone, suggesting that the 5-HT and KCl responses involve Ca²⁺ entry from the extracellular space. Also, verapamil completely inhibited the ox-LDL– and PMA-induced enhancement of 5-HT and KCl contraction (Figure 3B) and the associated changes in [Ca²⁺]_i (Figure 3D), suggesting that the enhanced 5-HT and KCl contraction is dependent on Ca²⁺ entry.

View larger version (44K): [in this window] [in a new window]   Figure 3. Effect of the Ca2+ channel blocker verapamil (10-6 mol/L) and the PKC inhibitors GF109203X (10-6 mol/L) and Gö6976 (10-6 mol/L) on coronary smooth muscle cell contraction (A, B) and [Ca2+]i (C, D) induced by ox-LDL (100 µg/mL), PMA (10-7 mol/L), 5-HT (10-7 mol/L), and KCl (24 mmol/L) applied separately (A, C) or combined (B, D). Data points represent the mean±SEM of experiments on 18 to 24 coronary smooth muscle cells from 6 to 8 pigs.

The effects of PKC inhibitors on ox-LDL, PMA, 5-HT, and KCl responses were also investigated. Pretreatment of the cells with GF109203X (10^-6 mol/L), an inhibitor of both Ca²⁺-dependent and -independent PKC isoforms, for 10 minutes significantly inhibited the contraction induced by ox-LDL alone or PMA alone (Figure 3A) with no significant change in [Ca²⁺]_i (Figure 3C), suggesting the involvement of PKC. In cells stimulated with 5-HT (10^-6 mol/L) alone or KCl alone, GF109203X did not affect contraction (Figure 3A) or [Ca²⁺]_i (Figure 3C), suggesting that PKC is not activated under these conditions. In cells pretreated with ox-LDL or PMA, GF109203X inhibited the enhancement of 5-HT and KCl contraction (Figure 3B) with no significant change in [Ca²⁺]_i (Figure 3D), suggesting the involvement of PKC. Pretreatment of the cells with Gö6976 (10^-6 mol/L), an inhibitor of Ca²⁺-dependent PKC isoforms, for 10 minutes did not affect the contraction (Figure 3A) or [Ca²⁺]_i (Figure 3C) induced by ox-LDL alone, PMA alone, 5-HT (10^-7 mol/L) alone, or KCl alone. However, in cells stimulated with ox-LDL plus 5-HT, ox-LDL plus KCl, PMA plus 5-HT, or PMA plus KCl, Gö6976 caused a significant reduction in contraction (Figure 3B) with no significant change in [Ca²⁺]_i (Figure 3D), suggesting the involvement of a Ca²⁺-dependent PKC isoform.

PKC activity was measured in tissue fractions of coronary smooth muscle. In resting tissues, PKC activity was greater in the cytosolic fraction than the particulate fraction (Figure 4A) with a particulate/cytosolic PKC activity of 0.47±0.11 (Figure 4B). Ox-LDL and the PKC activator PMA caused significant increases in PKC activity in the particulate fraction, decreases in the cytosolic fraction (Figure 4A), and an increase in the P/C PKC activity ratio (Figure 4B). Native LDL and the inactive 4-PMA did not cause any significant change in PKC activity. 5-HT (10^-7 mol/L) alone (Figure 4A and 4B) or KCl alone did not increase PKC activity. However, in tissues pretreated with ox-LDL or PMA, 5-HT (Figure 4A and 4B) and KCl caused additional increases in PKC activity. Gö6976 (10^-6 mol/L), an inhibitor of Ca²⁺-dependent PKC isoforms, did not affect the PKC activity induced by ox-LDL alone or PMA alone but abolished the additional increase in PKC activity caused by 5-HT (Figure 4B) or KCl in tissues pretreated with ox-LDL or PMA. GF109203X (10^-6 mol/L), an inhibitor of Ca²⁺-dependent and -independent PKC isoforms, completely inhibited the ox-LDL– and PMA-stimulated PKC activity and the additional increase in PKC activity caused by 5-HT (Figure 4B) or KCl in tissues pretreated with ox-LDL or PMA.

View larger version (38K): [in this window] [in a new window]   Figure 4. PKC activity in the cytosolic and particulate fraction (A) and the particulate/cytosolic (P/C) PKC activity ratio (B) in coronary smooth muscle under resting conditions or after stimulation with 100 µg/mL ox-LDL, 10-7 mol/L PMA, 10-7 mol/L 5-HT, ox-LDL plus 5-HT, and PMA plus 5-HT. Other tissues were pretreated with the PKC inhibitor Gö6976 (10-6 mol/L) or GF109203X (10-6 mol/L) for 10 minutes and then stimulated with ox-LDL, PMA, 5-HT, ox-LDL plus 5-HT, or PMA plus 5-HT. Data points represent the mean±SEM of experiments on 18 to 24 tissue samples from 6 to 8 pigs.

Western blots in tissue fractions revealed the Ca²⁺-dependent PKC- and the Ca²⁺-independent PKC-, -, and - isoforms. In resting tissues, PKC- was mainly cytosolic (Figure 5A), PKC- was mainly in the particulate fraction, PKC- was mainly cytosolic (Figure 5B), and PKC- was equally distributed in the cytosolic and particulate fraction. In the presence of ox-LDL alone, PKC- showed significant translocation from the cytosolic to particulate fraction (Figure 5B), while the distribution pattern of PKC- (Figure 5A), -, and - remained unchanged. 5-HT (10^-7 mol/L) alone (Figure 5A and 5B) or KCl alone did not significantly change the distribution of PKC isoforms. In tissues pretreated with ox-LDL, 5-HT (10^-7 mol/L) (Figure 5A) or KCl caused additional translocation of PKC- from the cytosolic to the particulate fraction. Direct activation of PKC by PMA (10^-7 mol/L) alone caused changes in the distribution of PKC- that were similar to those observed in tissues treated with ox-LDL alone. Also, in tissues pretreated with PMA, 5-HT (10^-7 mol/L) and KCl caused additional translocation of PKC- from the cytosolic to the particulate fraction similar to that observed in tissues stimulated with ox-LDL plus 5-HT or ox-LDL plus KCl (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 5. Distribution of PKC- (A) and PKC- (B) in the cytosolic (cyt) and particulate (part) fractions of coronary smooth muscle under resting conditions and during stimulation with ox-LDL (100 µg/mL), 5-HT (10-7 mol/L), and ox-LDL plus 5-HT. Data points represent the mean±SEM of experiments on 6 to 8 tissue samples from 6 to 8 pigs.

	Discussion

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The present study showed that ox-LDL caused significant contraction of coronary smooth muscle with no changes in [Ca²⁺]_i, suggesting activation of other mechanisms of smooth muscle contraction that may increase the myofilament force sensitivity to basal levels of [Ca²⁺]_i. Although the ox-LDL contraction appeared to be relatively small, this contraction could be of considerable significance because only minimal circumferential coronary vasoconstriction is often necessary to critically reduce the luminal cross-sectional area in the setting of coronary vasospasm.³³ Also, if the ox-LDL–stimulated Ca²⁺ sensitizing pathway is combined with another agonist, which by itself causes small increases in coronary artery contraction and [Ca²⁺]_i, the resulting synergistic effect could dangerously enhance coronary vasoconstriction and lead to severe coronary vasospasm.

Several studies have shown that direct activation of PKC by phorbol esters causes significant and sustained contraction of smooth muscle with no significant change in [Ca²⁺]_i,^{20 21} suggesting a role for PKC in regulating smooth muscle contraction, at least in part, by increasing the myofilament force sensitivity to [Ca²⁺]_i. The present results suggest that ox-LDL increases the myofilament sensitivity to [Ca²⁺]_i by activating PKC because (1) ox-LDL contraction was not associated with any significant increase in [Ca²⁺]_I; (2) ox-LDL contraction was not inhibited by the Ca²⁺ channel blocker verapamil; (3) direct activation of PKC by phorbol ester caused a contraction similar to that of ox-LDL with no change in [Ca²⁺]_I; (4) ox-LDL contraction was completely inhibited by GF109203X, an inhibitor of both Ca²⁺-dependent and -independent PKC isoforms; and (5) ox-LDL stimulated PKC activity, an effect that was completely inhibited by GF109203X. Thus, the contraction induced by ox-LDL alone does not require significant increases in [Ca²⁺]_i, but appears to require activation of PKC. Our present findings that ox-LDL activates PKC in coronary smooth muscle cells are consistent with reports that ox-LDL activates PKC in other cell types such as macrophages^{34 35} and vascular endothelial cells.³⁶ The observations that the contraction and PKC activation induced by ox-LDL were completely inhibited by GF109203X, an inhibitor of both Ca²⁺-dependent and -independent PKC isoforms, but not by Gö6976, a relatively specific inhibitor of the Ca²⁺-dependent PKC isoforms, raise the possibility that the responses induced by ox-LDL alone involve a Ca²⁺-independent PKC isoform. This is consistent with the observation that ox-LDL alone caused translocation of the Ca²⁺-independent PKC- but not the Ca²⁺-dependent PKC-.

Small concentrations of 5-HT alone or KCl alone caused significant coronary smooth muscle contraction that was associated with significant increase in [Ca²⁺]_i. The 5-HT (10^-7 mol/L)– and KCl (24 mmol/L)–induced contraction and [Ca²⁺]_i appear to be due mainly to Ca²⁺ entry from the extracellular space because they were completely inhibited by the Ca²⁺ channel blocker verapamil and were not associated with any significant change in PKC activity or the distribution of PKC isoforms.

In cells pretreated with ox-LDL, the 5-HT and KCl contractions were significantly enhanced with no additional increases in [Ca²⁺]_i. Although the enhanced 5-HT and KCl contraction with ox-LDL did not involve additional increases in [Ca²⁺]_i, it appears to require extracellular Ca²⁺ because it was completely inhibited by the Ca²⁺ channel blocker verapamil. Also, the enhancement of 5-HT and KCl contraction by ox-LDL appears to involve PKC because (1) direct activation of PKC by PMA caused similar enhancement of 5-HT and KCl contraction, (2) the enhancement of 5-HT and KCl contraction by ox-LDL was associated with an 2-fold increase in PKC activity, and (3) the enhancement of 5-HT– and KCl-induced contraction and PKC activity by ox-LDL was inhibited by PKC inhibitors. Thus, the enhanced 5-HT– and KCl-induced contraction and PKC activity in tissues pretreated with ox-LDL appear to require both Ca²⁺ and PKC, which raises the possibility that it involves a Ca²⁺-dependent PKC isoform. This is consistent with the observation that the enhancement of 5-HT and KCl contraction by ox-LDL or PMA was associated with activation and translocation of the Ca²⁺-dependent PKC-.

The present results have shown that ox-LDL alone activates the Ca²⁺-independent PKC-. However, when [Ca²⁺]_i is slightly increased above basal levels by low concentrations of 5-HT or KCl, ox-LDL causes not only activation of the Ca²⁺-independent PKC- but also additional activation of the Ca²⁺-dependent PKC-. These observations are analogous to most in vivo conditions, in which there would be some degree of synergistic effects produced by tonic activation of other Ca²⁺-dependent vasoconstrictor pathways in addition to the PKC-dependent pathways activated by ox-LDL. An important question is why ox-LDL could not activate PKC- at basal levels of [Ca²⁺]_i while causing significant activation of PKC- when [Ca²⁺]_i is slightly increased above basal levels by low concentrations of 5-HT or KCl. This could be related to the threshold [Ca²⁺]_i required for activation of Ca²⁺-dependent PKC isoforms. This is consistent with previous reports that a threshold increase in [Ca²⁺]_i is required for activation of PKC- in ferret and porcine vascular smooth muscle cells.^{26 28}

The molecular mechanisms of the ox-LDL–induced activation of PKC are not clear at the present time but could be related to 3 things. First, ox-LDL could increase the formation of DAG, which binds to all DAG-sensitive isoforms of PKC, and this will activate only Ca²⁺-independent isoforms of PKC in the absence of elevated Ca²⁺ but all isoforms of PKC in the presence of elevated Ca²⁺. This possible mechanism can be tested by measuring the effects of ox-LDL on DAG formation. Second, ox-LDL could change the lipid composition of the plasma membrane and thereby change the activity of the lipid-sensitive PKC. This is supported by reports that lysophosphatidylcholine, a lysophospholipid contained in ox-LDL, could change the activity of PKC.³⁷ Third, ox-LDL could directly interact with the PKC molecule. These are only few suggested mechanisms that should represent important areas for future investigations.

In summary, ox-LDL causes coronary smooth muscle contraction with no significant increase in [Ca²⁺]_i but is associated with an increase in the activity of the Ca²⁺-independent PKC- isoform. Small concentrations of 5-HT and KCl cause coronary smooth muscle contraction that is associated with significant increases in [Ca²⁺]_i but not PKC activity. Ox-LDL enhances coronary smooth muscle contraction to 5-HT and KCl with no additional increases in [Ca²⁺]_i and is associated with an increase in PKC- activity. The results suggest that ox-LDL causes coronary vasoconstriction via activation of the Ca²⁺-independent PKC- and enhances the contraction to [Ca²⁺]_i-increasing agonists by activating the Ca²⁺-dependent PKC-. The activation of PKC- and PKC- may represent a possible cellular mechanism by which ox-LDL could enhance coronary vasospasm.

	Acknowledgments

 
This work was supported by grants from the American Heart Association (grant-in-aid, Southeast Affiliate) and the National Institutes of Health (HL-51971 and HL-52696).

Received October 25, 2000; accepted December 11, 2000.

	References

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