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(Hypertension. 2001;37:497.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Endothelin-1 Enhances Eicosanoids-Induced Coronary Smooth Muscle Contraction by Activating Specific Protein Kinase C Isoforms

Zohreh N. Sirous; John B. Fleming; Raouf A. Khalil

From the Department of Physiology and Biophysics and the 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 North State Street, Jackson, MS 39216. E-mail: rkhalil{at}physiology.umsmed.edu

	Abstract

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Endothelin-1 (ET-1), a potent vasoconstrictor, has been implicated in the pathogenesis of coronary vasospasm by enhancing coronary vasoconstriction to vasoactive eicosanoids; however, the cellular mechanisms involved are unclear. We investigated whether physiological concentrations of ET-1 enhance coronary smooth muscle contraction to vasoactive eicosanoids by activating specific protein kinase C (PKC) isoforms. Cell contraction was measured in single smooth muscle cells isolated from porcine coronary arteries, intracellular free Ca²⁺ ([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 anti-PKC antibodies using Western blots. In Hanks’ solution (1 mmol/L Ca²⁺), ET-1 (10 pmol/L) did not increase basal [Ca²⁺]_i (81±2 nmol/L), but it did cause cell contraction (9%) that was inhibited by GF109203X (10^–6 mol/L), an inhibitor of Ca²⁺-dependent and Ca²⁺-indpendent PKC isoforms. The vasoactive eicosanoid prostaglandin F₂ (PGF₂, 10^–7 mol/L) caused increases in cell contraction (11%) and [Ca²⁺]_i (108±7 nmol/L) that were inhibited by the Ca²⁺ channel blocker diltiazem (10^–6 mol/L). Pretreatment with ET-1 (10 pmol/L) for 10 minutes enhanced cell contraction to PGF₂ (35%) with no additional increase in [Ca²⁺]_i (112±8 nmol/L). Direct activation of PKC by phorbol 12,13-dibutyrate (PDBu, 10^–7 mol/L) caused cell contraction (10%) and enhanced PGF₂ contraction (33%) with no additional increase in [Ca²⁺]_i (115±7 nmol/L). The ET-1–induced enhancement of PGF₂ contraction was inhibited by Gö6976 (10^–6 mol/L), an inhibitor of Ca²⁺-dependent PKC isoforms. Both ET-1 and PDBu caused an increase in PKC activity in the particulate fraction and a decrease in the cytosolic fraction and increased the particulate/cytosolic PKC activity ratio. Western blots revealed the Ca²⁺-dependent -PKC and the Ca²⁺-independent -, -, and -PKC isoforms. In resting tissues, - and -PKC were mainly cytosolic, -PKC was mainly in the particulate fraction, and -PKC was equally distributed in the cytosolic and particulate fraction. ET-1 (10 pmol/L) alone or PDBu (10^–7 mol/L) alone caused translocation of -PKC from the cytosolic to the particulate fraction, localized -PKC more in the particulate fraction, but did not change the distribution of -PKC. PGF₂ (10^–7 mol/L) alone did not change PKC activity. In tissues pretreated with ET-1 or PDBu, PGF₂ caused additional increases in -PKC activity. Thus, the enhancement of PGF₂-induced coronary smooth muscle contraction by physiological concentrations of ET-1 involves activation and translocation of -PKC in addition to - and -PKC isoforms, and this may represent one possible cellular mechanism by which ET-1 could enhance coronary vasoconstriction to vasoactive eicosanoids in coronary vasospasm.

Key Words: endothelin • prostaglandin • calcium • muscle, smooth, vascular • myocardial contraction

	Introduction

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Coronary vasospasm is often associated with ischemic heart disease leading to different forms of angina or myocardial infarction, and excessive coronary vasoconstriction in response to endogenous vasoconstrictors has been suggested as one potential cause of ischemic heart disease.¹ Some have suggested that endothelin-1 (ET-1), one of the most potent vasoconstrictors described,² may act as an endogenous modulator of coronary vascular tone and may play a role in the setting of coronary vasospasm.^{3 4 5} In addition, local release of vasoactive eicosanoids such as prostaglandin F₂ (PGF₂) in response to cardiac tissue injury causes significant coronary vasoconstriction that may dangerously interfere with adequate coronary blood flow.^{5 6 7}

Although previous studies have shown that both ET-1 and PGF₂ are potent coronary vasoconstrictors, the effects of ET-1 on smooth muscle contraction have often been evaluated separately from the effects of PGF₂.^{3 4 5 6 7} Also, in most mechanistic studies, high unphysiological concentrations of ET-1 and PGF₂ have often been used to activate maximally the possible mechanisms of smooth muscle contraction. This is in sharp contrast to the in vivo conditions, where the coronary artery is usually exposed to more than one vasoconstrictor at the same time, and the increases in the concentration of vasoconstrictor agonists are usually within the physiological range. Although high concentrations of ET-1 alone or PGF₂ alone are predicted to and have been shown to cause significant coronary contraction,^{3 4 5 6 7} it is not clear whether physiological concentrations of ET-1 enhance coronary vasoconstriction to small concentrations of PGF₂. In addition, although high concentrations of ET-1 alone or PGF₂ alone are predicted to activate maximally one or more mechanisms of smooth muscle contraction, the cellular mechanisms involved in the possible ET-1 induced enhancement of coronary vasoconstriction to the vasoactive eicosanoid PGF₂ are unclear.

It is widely accepted that vascular smooth muscle contraction is triggered by increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) due to Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space.^{8 9 10 11} Other studies have also shown that the interaction of a vasoconstrictor agonist with its specific receptor is coupled to increased breakdown of plasma membrane phospholipids and increased production of diacylglycerol.^{12 13} Diacylglycerol binds to and activates protein kinase C (PKC). PKC is a family of several isoforms that have different enzyme properties, substrates, and functions and exhibit different subcellular distributions.^{12 13 14 15} PKC is mainly cytosolic under resting conditions and undergoes translocation to the particulate fraction when it is activated by endogenous diacylglycerol or exogenous phorbol esters.^{12 13} Also, direct activation of PKC by phorbol esters such as phorbol 12,13-dibutyrate (PDBu) causes sustained contraction of vascular smooth muscle with no significant change in [Ca²⁺]_i.^{16 17} This suggests that PKC may have a role in regulating smooth muscle contraction, at least in part, by increasing the myofilament force sensitivity to [Ca²⁺]_i.

The purpose of the present study was to test the hypothesis that physiological concentrations of ET-1 enhance coronary smooth muscle contraction to the vasoactive eicosanoid PGF₂ by increasing the activity of specific PKC isoforms. Because the PKC family includes both Ca²⁺-dependent and Ca²⁺-indpependent isoforms, any ET-1 or PGF₂ induced changes in coronary smooth muscle [Ca²⁺]_i may determine which PKC isoform would be activated. Therefore, experiments were designed to investigate (1) whether physiological concentrations of ET-1 enhance PGF₂-induced contraction in coronary smooth muscle, (2) whether the ET-1 enhancement of PGF₂-induced coronary smooth muscle contraction is associated with increases in [Ca²⁺]_i, and (3) whether the ET-1 enhancement of PGF₂-induced contraction is associated with increases in the activity of specific PKC isoforms in coronary smooth muscle. The effects of ET-1 were compared with those of the phorbol ester PDBu, a direct activator of PKC, and sensitivity to the effects to the Ca²⁺ channel antagonist diltiazem and the PKC inhibitors GF109203X and Gö6976 was also investigated.

	Methods

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Tissue Preparation
Castrated male Yorkshire pigs (30 kg) from a local breeder were anesthetized by isoflurane inhalation. The heart was rapidly excised and placed in Krebs’ solution. The left anterior descending coronary artery was dissected, cleaned of connective and adipose tissue, and opened by cutting along its longitudinal axis. The endothelium was removed by rubbing the vessel interior with wet filter paper. The tunica media was carefully dissected from the tunica adventitia under microscopic visualization using sharp-tipped forceps and then sectioned into 2x2-mm strips. All procedures followed the guidelines of the Institutional Animal Care and Use Committee.

Single Cell Isolation
Coronary artery strips (50 mg) were placed in a tissue digestion mixture containing collagenase type II (236 U/mg protein, Worthington), elastase grade II (3.25 U/mg protein, Boehringer Mannheim), and trypsin inhibitor type II (10 000 U/mL, Sigma) in 7.5 mL of Ca²⁺- and Mg²⁺-free Hanks’ solution supplemented with 30% bovine serum albumin (Sigma).^{18 19 20} The tissue was incubated 3 times in the tissue digestion mixture to yield 3 batches of cells. For the first batch, the tissue was incubated with 5 mg of collagenase, 4 mg of elastase, and 147 µL of trypsin inhibitor for 60 minutes. For batches 2 and 3, the collagenase was reduced to 2.5 mg, the trypsin inhibitor was reduced to 122 µL, and the incubation period was reduced to 30 minutes. The tissue preparation was placed in a shaking water bath at 34°C in an atmosphere of 95% O₂ and 5% CO₂. The preparation was rinsed with 12.5 mL of Hanks’ solution, 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. Ca²⁺ was gradually added back to the preparation to avoid the "calcium paradox."²¹

Contraction Studies
Coverslips with the attached cells were placed on the stage of an inverted Nikon (Diaphot-300) microscope and viewed using a Nikon 100x oil immersion objective. The cell isolation procedure yielded smooth muscle cells of variable lengths. Only viable, healthy, spindle-shaped cells 60 µm in length were selected. Viable, healthy 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. The viability of the smooth muscle cells was confirmed by their consistent and significant contraction in response to ET-1 and PGF₂. The cells were further characterized and consistently showed significant immunofluorescence signal when fixed and labeled with anti-smooth muscle myosin antibody. Cell images were acquired using a PXL CCD camera and displayed on a computer using PMIS image analysis software (Photometrics). The number of pixels corresponding to the cell length in the cell image was transformed into microns using a calibration bar. The magnitude of cell contraction was expressed as the final cell length as a fraction of the initial cell length. All contraction measurements were made at 37°C. The changes in cell contraction in response to ET-1, PGF₂, and the phorbol ester PDBu were measured.

Measurement of [Ca²⁺]_i
Single coronary smooth muscle cells were loaded with the Ca²⁺ indicator fura-2 for 30 minutes at 34°C.^{19 20} The fura-2 loading solution was made of normal Hanks’ solution, 1 µmol/L of the cell permeant fura-2 acetoxymethyl ester (Molecular Probes), and 0.01% Pluronic F-127 (Sigma). The fura-2–loaded cells were washed twice and further incubated in Hanks’ solution for at least 30 minutes to allow complete de-esterification of the fura-2 acetoxymethyl ester. Precautionary measures were taken throughout the procedure to avoid extensive photobleaching of fura-2.²²

The fura-2–loaded cells were viewed through a Nikon Fluor 100x oil-immersion objective (NA 1.3) on an inverted Nikon (Diaphot-300) microscope. The Ca²⁺ indicator was excited alternately at 340±5 nm and 380±6 nm using a filter wheel that alternates at a frequency of 0.5 Hz. The emitted light was collected at 510 nm to a photomultiplier tube R928 (Ludl Electronic Products) through a pinhole aperture 1 µm in diameter positioned 1 µm from the plasma membrane and 1 µm from the nucleus. The fluorescent signal was digitized using a module (Mac 2000, Ludl) and analyzed on a computer using data analysis software. The fluorescent signal was background-subtracted. Spectral shifts that result from the binding of Ca²⁺ to fura-2 make it possible to use the ratio method, thus rendering the measurements of [Ca²⁺]_i less sensitive to changes in cell thickness or the extent of dye loading and photobleaching. The ratio between the fluorescence intensity at 340 nm and 380 nm (R) was transformed to the corresponding levels of [Ca²⁺]_i as described by Grynkiewicz et al as follows²² :

where R_min and R_max represent the minimal and maximal fluorescence ratios; they were measured by adding fura-2 pentapotassium salt (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 signal in Ca²⁺-free and Ca²⁺-replete solutions, respectively. K_d is the dissociation constant of fura-2 for Ca²⁺ and was established at 224 nmol/L under these experimental conditions.²² All experiments were performed at 37°C. The changes in [Ca²⁺]_i in response to ET-1, PGF₂, and the phorbol ester PDBu were measured.

Tissue Fractions
Tissue strips (80 mg) at rest or stimulated with ET-1, PGF₂, or PDBu for 30 minutes were rapidly transferred to ice-cold equilibrating buffer A containing (in mmol/L): Tris-HCl 25 (pH 7.5), EGTA 5, leupeptin 0.02, phenylmethylsulfonylfluoride 0.2, and dithiothreitol 1. To measure PKC activity, the tissue was transferred to homogenization buffer B, which has 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 of dithiothreitol, 1.2 mmol/L ethylenediamine tetraacetic acid, 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 using 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). The supernatant was used as the cytosolic fraction. The pellet was resuspended in a 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 in tissue fractions were determined using a protein assay kit (Bio-Rad).

PKC Activity
The cytosolic and particulate fractions were applied to diethylaminoethyl-cellulose columns (0.8x4.0 cm; Bio-Rad). 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 by measuring the incorporation of ³²P from [³²P]ATP (ICN) into histone IIIS.^{23 24} The assay mixture contained 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 of protein. After 5 minutes of incubation at 30°C, the reaction was stopped by spotting 25 µL of the assay mixture onto phosphocellulose discs. The discs were washed 3x5 minutes with 5% trichloroacetic acid and placed in a 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 PBS-Tween at 22°C for 1 hour, washed with PBS-Tween for 3x5 minutes, and then incubated in the primary anti-PKC antibody solution at 4°C overnight. Polyclonal antibodies to -, ß-, -, -, -, and -PKC (Gibco) were used. These antibodies have been shown to react with the specific PKC isoforms in porcine aortic endothelial cells and in airway and coronary smooth muscle cells.^{23 24 25 26} The specificity of the antibodies was confirmed by the observation that the peptide controls were successful only with the peptide to which the antibodies were raised and not with other sequences of the PKC molecule. To maintain constant labeling conditions, we used the same anti-PKC antibody titer (1:500) and protein concentration (10 µg) in all tissue samples. These antibody titer and protein concentrations gave optimal immunoreactive signals while remaining on the linear portion of the titration curve. The nitrocellulose membranes were washed 5x15 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 for 5x15 minutes and visualized with enhanced chemiluminescence detection system (Amersham). PBS-Tween contained the following (in mmol/L): Na₂HPO₄ 80, NaH₂PO₄ 20, and NaCl 100 plus 0.05% Tween. The reactive bands corresponding to PKC isoforms were analyzed quantitatively by optical densitometry using a GS-700 imaging densitometer (Bio-Rad).

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

Drugs and Chemicals
ET-1, PGF₂ (Sigma), and diltiazem (Calbiochem) were dissolved in distilled water. PDBu (Alexis Laboratory.), GF109203X, and Gö6976 (Kamiya) were dissolved in DMSO. The final concentration of DMSO in solution was 0.1%. All other chemicals were of reagent grade or better.

Statistical Analysis
The data were analyzed and presented as the mean±SEM and compared using Student’s t test for unpaired data. P<0.05 was considered significant.

	Results

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The cell isolation procedure produced relatively long cells (Figure 1). 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. High concentrations of ET-1 (10^-7 mol/L) caused significant cell contraction (74.9±2.8%) and an initial increase in [Ca²⁺]_i to 496±37 nmol/L followed by a maintained increase to 167±7 nmol/L (Figure 1A). High concentrations of PGF₂ (10^–4 mol/L) also caused significant cell contraction (73±2.3%) and a transient increase in [Ca²⁺]_i to 423±29 nmol/L and a maintained increase to 181±5 nmol/L (Figure 1B).

View larger version (32K): [in this window] [in a new window]   Figure 1. Representative cell contraction (upper panels) and [Ca2+]i (lower panels) in coronary smooth muscle cells activated with 10–7 mol/L ET-1 (A), 10–4 mol/L PGF2 (B), 10–11 mol/L ET-1 (C), 10–7 mol/L PGF2 (D), and 10–11 mol/L ET-1 plus 10–7 mol/L PGF2 (E).

Small concentrations of ET-1 (10^–11 mol/L) caused a small but significant cell contraction (9.2±2.3%), with no significant increases in [Ca²⁺]_i (Figure 2C). However, small concentrations of PGF₂ (10^–7 mol/L) caused small increases in both contraction (11.3±2.2%) and [Ca²⁺]_i (108±7 nmol/L; Figure 1D). In cells pretreated with ET-1 (10^–11 mol/L) for 10 minutes, PGF₂ (10^–7 mol/L) caused a large contraction (34.9±3.2%), with no additional increases in [Ca²⁺]_i (112±7 nmol/L; Figure 1E).

View larger version (19K): [in this window] [in a new window]   Figure 2. Coronary smooth muscle contraction (A) and [Ca2+]i (B) in response to increasing concentrations of ET-1 alone (open circles) or PGF2 alone (closed circles). Other cells were pretreated with 10–11 mol/L ET-1 (open triangles) or 10–7 mol/L PDBu (closed triangles) for 10 minutes and then stimulated with increasing concentrations of PGF2; then, cell contraction and [Ca2+]i were measured. Data points represent mean±SEM of experiments on 18 to 24 cells from 6 to 8 pigs. L indicates final cell length; Li, initial cell length.

Cumulative data from different cells stimulated with increasing concentrations of ET-1 or PGF₂ were used to construct concentration-response curves (Figure 2). ET-1 alone caused concentration-dependent increases in cell contraction and [Ca²⁺]_i. PGF₂ alone caused concentration-dependent increases in contraction and [Ca²⁺]_i, although PGF₂ was less potent than ET-1. In cells pretreated with ET-1 (10^–11 mol/L) for 10 minutes, the PGF₂ concentration-contraction curve was significantly enhanced (Figure 2A), with no additional increases in [Ca²⁺]_i (Figure 2B). Direct activation of PKC by PDBu (10^–7 mol/L) caused cell contraction (10%) with no significant change in [Ca²⁺]_i (115±7 nmol/L). In cells pretreated with PDBu (10^–7 mol/L) for 10 minutes, PGF₂ contraction was enhanced to levels similar to those observed in cells pretreated with ET-1 and then stimulated with PGF₂ (Figure 2A), with no additional increase in [Ca²⁺]_i (Figure 2B).

The effects of the Ca²⁺ channel blocker diltiazem on the responses of ET-1, PGF₂, and ET-1 plus PGF₂ were investigated. Pretreatment with diltiazem (10^–6 mol/L) for 10 minutes did not affect the cell contraction (Figure 3A) or [Ca²⁺]_i (Figure 3B) induced by ET-1 (10^–11 mol/L) alone, suggesting that Ca²⁺ entry from the extracellular space is not involved. Diltiazem completely abolished the contraction (Figure 3A) and [Ca²⁺]_i (Figure 3B) induced by PGF₂ (10^–7 mol/L) alone, suggesting that the PGF₂ responses involve Ca²⁺ entry. Diltiazem also significantly inhibited the ET-1 induced enhancement of PGF₂ contraction (Figure 3A), suggesting that the enhanced response is dependent on Ca²⁺ entry.

View larger version (33K): [in this window] [in a new window]   Figure 3. Effect of Ca2+ channel blocker diltiazem (10–6 mol/L) and PKC inhibitors GF109203X (10–6 mol/L) and Gö6976 (10–6 mol/L) on coronary smooth muscle contraction (A) and [Ca2+]i (B) induced by ET-1 (10–11 mol/L), PGF2 (10–7 mol/L), and ET-1 (10–11 mol/L) plus PGF2 (10–7 mol/L). Data points represent mean±SEM of experiments on 18 to 24 cells from 6 to 8 pigs.

The effects of PKC inhibitors on the responses of ET-1, PGF₂, and ET-1 plus PGF₂ were also investigated. Pretreatment with GF109203X (10^–6 mol/L), an inhibitor of both Ca²⁺-dependent and Ca²⁺-independent PKC isoforms, for 10 minutes significantly inhibited the cell contraction induced by ET-1 (10^–11 mol/L) alone (Figure 3A), with no significant change in [Ca²⁺]_i (Figure 3B), suggesting the involvement of PKC. In cells stimulated with PGF₂ (10^–7 mol/L) alone, GF109203X did not affect contraction (Figure 3A) or [Ca²⁺]_i (Figure 3B), suggesting that PKC is not activated during stimulation by PGF₂ (10^–7 mol/L) alone. In cells pretreated with ET-1, GF109203X inhibited the enhancement of PGF₂ contraction (Figure 3A), with no significant change in [Ca²⁺]_i (Figure 3B), suggesting the involvement of PKC. Pretreatment with Gö6976 (10^–6 mol/L), an inhibitor of Ca²⁺-dependent PKC isoforms, for 10 minutes did not affect the cell contraction (Figure 3A) or [Ca²⁺]_i (Figure 3B) induced by ET-1 (10^–11 mol/L) alone or PGF₂ (10^–7 mol/L) alone. However, in cells stimulated with ET-1 (10^–11 mol/L) plus PGF₂ (10^–7 mol/L), Gö6976 caused a significant reduction in contraction (Figure 3A), with no significant change in [Ca²⁺]_i (Figure 3B), 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 0.46±0.05 (Figure 4B). ET-1 (10^–11 mol/L) caused time-dependent increases in PKC activity in the particulate fraction, a decrease in the cytosolic fraction (Figure 4A), and an increase in the particulate/cytosolic PKC activity ratio (Figure 4B). PGF₂ (10^–7 mol/L) alone did not cause any significant increase in PKC activity (Figure 4A and 4B). In tissues pretreated with ET-1 (10^–11 mol/L), PGF₂ (10^–7 mol/L) caused additional increases in PKC activity (Figures 4A and 4B). Direct activation of PKC by PDBu (10^–7 mol/L) caused a significant increase in PKC activity that was roughly similar to that in tissues stimulated with ET-1 (10^–11 mol/L) alone (Figure 4C). In tissues pretreated with PDBu (10^–7 mol/L), PGF₂ (10^–7 mol/L) caused additional increases in PKC activity (Figure 4C). Gö6976 (10^–6 mol/L), an inhibitor of Ca²⁺-dependent PKC isoforms, did not affect the PKC activity induced by ET-1 alone or PDBu alone, but it abolished the additional increase in PKC activity caused by PGF₂ in tissues pretreated with ET-1 or PDBu (Figure 4C). GF109203X (10^–6 mol/L), an inhibitor of Ca²⁺-dependent and Ca²⁺-independent PKC isoforms, inhibited the ET-1– and PDBu–stimulated PKC activity and the additional increase in PKC activity caused by PGF₂ in tissues pretreated with ET-1 or PDBu (Figure 4C).

View larger version (24K): [in this window] [in a new window]   Figure 4. PKC activity in cytosolic and particulate fraction (A) and particulate/cytosolic (P/C) PKC activity ratio (B) in coronary smooth muscle cells stimulated with 10–11 mol/L ET-1, 10–7 mol/L PGF2, and 10–11 mol/L ET-1 plus 10–7 mol/L PGF2. Other tissues were pretreated with PKC inhibitor Gö6976 (10–6 mol/L) or GF109203X (10–6 mol/L) for 10 minutes and then stimulated with 10–11 mol/L ET-1, 10–7 mol/L PDBu, 10–7 mol/L PGF2, 10–11 mol/L ET-1 plus 10–7 mol/L PGF2, or 10–7 mol/L PDBu plus 10–7 mol/L PGF2, and changes in PKC activity ratio were measured (C). Data points represent 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 -, -, and -PKC isoforms. In resting tissues, -PKC was mainly cytosolic (Figure 5A), -PKC seemed to appear slightly more in the particulate fraction (Figure 5B), -PKC was mainly cytosolic (Figure 5C), and -PKC was equally distributed in the cytosolic and particulate fractions (Figure 5D). In the presence of ET-1 (10^–11 mol/L) alone, the distribution of -PKC did not change (Figure 5A), -PKC was more localized in the particulate fraction (Figure 5B), -PKC showed significant translocation from the cytosolic to the particulate fraction (Figure 5C), and the distribution of -PKC was unchanged (Figure 5D). PGF₂ (10^–7 mol/L) alone did not significantly change the distribution of PKC isoforms. In tissues pretreated with ET-1 (10^–11 mol/L), PGF₂ (10^–7 mol/L) caused additional translocation of -PKC from the cytosolic to the particulate fraction (Figure 5A). Direct activation of PKC by PDBu (10^–7 mol/L) alone caused changes in the distribution of - and -PKC that were similar to those observed in tissues treated with ET-1 (10^–11 mol/L) alone. In tissues pretreated with PDBu (10^–7 mol/L), PGF₂ (10^–7 mol/L) caused additional translocation of -PKC from the cytosolic to the particulate fraction in a manner similar to that observed in tissues stimulated with ET-1 plus PGF₂ (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 5. Distribution of -PKC (A), -PKC (B), -PKC (C), and -PKC (D) in cytosolic and particulate fractions of coronary smooth muscle cells under resting conditions and during stimulation with 10–11 mol/L ET-1, 10–7 mol/L PGF2, and 10–11 mol/L ET-1 plus 10–7 mol/L PGF2. Data points represent 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 ET-1 at concentrations 10^–10 mol/L causes significant contraction of coronary smooth muscle cells. At these high concentrations, ET-1–induced contraction is associated with a significant increase in [Ca²⁺]_i. These results are consistent with previous reports that vascular smooth muscle contraction in response to ET-1 is triggered by increases in [Ca²⁺]_i due to Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space.^{27 28 29 30} ET-1 at very small and physiological concentrations (10^–11 mol/L) still caused a significant contraction of coronary smooth muscle cells that was not associated with any increases in [Ca²⁺]_i, suggesting activation of other mechanisms of smooth muscle contraction that may increase the myofilament force sensitivity even to basal levels of [Ca²⁺]_i. Although the ET-1 contraction at physiological concentrations seemed 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 significant coronary vasospasm.³¹ Also, if ET-1 coronary contraction is mediated by a Ca²⁺-sensitizing pathway rather than by increasing [Ca²⁺]_i, vasodilators functioning solely by lowering [Ca²⁺]_i would be ineffective in overcoming this form of coronary vasospasm. Furthermore, if the ET-1–stimulated Ca²⁺ sensitizing pathway is combined with another agonist, which by itself causes only small increases in coronary smooth muscle 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,^{16 17} 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 ET-1, at small and physiological concentrations, increases the myofilament sensitivity to [Ca²⁺]_i by activating PKC because (1) ET-1 contraction was not associated with any significant increase in [Ca²⁺]_i; (2) ET-1 contraction was not inhibited by the Ca²⁺ channel blocker diltiazem; (3) direct activation of PKC by phorbol ester caused a contraction similar to that of ET-1, with no significant change in [Ca²⁺]_i; (4) ET-1 contraction was completely inhibited by GF109203X, an inhibitor of both Ca²⁺-dependent and Ca²⁺-independent PKC isoforms; and (5) ET-1 caused an increase in PKC activity that was completely inhibited by GF109203X. Thus, the contraction induced by small concentrations of ET-1 alone does not require significant increases in [Ca²⁺]_i, but it seems to involve the activation of PKC. The observations that the contraction and PKC activation induced by ET-1 were completely inhibited by GF109203X, an inhibitor of both Ca²⁺-dependent and Ca²⁺-independent PKC isoforms, but not by Gö6976, a relatively specific inhibitor of the Ca²⁺-dependent isoforms, raises the possibility that the responses induced by ET-1 alone involve a Ca²⁺-independent PKC isoform. This is consistent with the observation that ET-1 alone caused a translocation of the Ca²⁺-independent -PKC and, to a lesser extent, -PKC but not the Ca²⁺-dependent -PKC.

Small concentrations of PGF₂ alone caused a cell contraction that was associated with a significant increase in [Ca²⁺]_i. PGF₂-induced contraction and [Ca²⁺]_i seem to be mainly due to Ca²⁺ entry from the extracellular space because they were inhibited by the Ca²⁺ channel blocker diltiazem and were not associated with any significant change in PKC activity or the distribution of PKC isoforms.

In cells pretreated with ET-1, the PGF₂ contraction was significantly enhanced, with no additional increases in [Ca²⁺]_i. These results are in agreement with reports that ET-1 enhances vascular smooth muscle contraction to other agonists such as 5-hydroxytryptamine.^{32 33 34} Although the enhanced PGF₂ contraction in cells pretreated with ET-1 did not involve additional increases in [Ca²⁺]_i, it seems to require extracellular Ca²⁺ because it was completely inhibited by diltiazem. In addition, the enhancement of PGF₂ contraction by ET-1 seems to involve PKC because (1) direct activation of PKC by PDBu caused similar enhancement of PGF₂ contraction, (2) the enhancement of PGF₂ contraction by ET-1 was associated with an 2-fold increase in PKC activity, and (3) the enhancement of PGF₂-induced contraction and PKC activity by ET-1 were inhibited by PKC inhibitors. Thus, the enhanced PGF₂-induced contraction and PKC activity in tissues pretreated with ET-1 seem to require both Ca²⁺ and PKC, which raises the possibility that the contraction involves a Ca²⁺-dpendent PKC isoform. This is consistent with the observation that the enhancement of PGF₂ contraction by ET-1 was associated with the activation and translocation of the Ca²⁺-dependent -PKC.

The question arises regarding why ET-1 did not activate -PKC at basal levels of [Ca²⁺]_i while causing significant activation of -PKC when [Ca²⁺]_i was slightly increased above basal levels. This could be related, at least in part, to the level of [Ca²⁺]_i required for the activation of Ca²⁺-dependent PKC isoforms. This is consistent with previous reports that a threshold increase in [Ca²⁺]_i is required for the activation of -PKC in vascular smooth muscle cells from the ferret and the pig.^{23 35}

In summary, physiological concentrations of ET-1 cause coronary smooth muscle contraction, with no significant increase in [Ca²⁺]_i but with significant increases in the activity of the Ca²⁺-independent - and -PKC. Small concentrations of PGF₂ cause coronary smooth muscle contraction that is associated with a significant increase in [Ca²⁺]_i but not in PKC activity. Physiological concentrations of ET-1 significantly enhance coronary smooth muscle contraction to PGF₂, with no additional increases in [Ca²⁺]_i, and are associated with an increase in -PKC activity. Thus, the enhancement of PGF₂-induced coronary smooth muscle contraction by physiological concentrations of ET-1 involves activation and translocation of -PKC in addition to - and -PKC isoforms. The additional activation of -PKC may represent one possible cellular mechanism by which ET-1 may cause exaggerated coronary vasoconstriction to vasoactive eicosanoids in the setting of coronary vasospasm.

	Acknowledgments

 
This study 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; first decision December 8, 2000; accepted December 18, 2000.

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