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(Hypertension. 2002;39:543.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Endothelin-1–Induced Enhancement of Coronary Smooth Muscle Contraction via MAPK-Dependent and MAPK-Independent [Ca²⁺]_i Sensitization Pathways

Ashley E. Cain; Dennis M. 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, Miss.

Correspondence to Raouf A. Khalil, MD, PhD, Department of Physiology & 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) has been implicated in coronary vasospasm by enhancing coronary vasoconstriction to vasoactive eicosanoids, and a role for protein kinase C (PKC) activation has been suggested. However, the cellular mechanisms downstream from PKC activation are unclear. We investigated whether physiological concentrations of ET-1 enhance coronary smooth muscle contraction by activating a PKC-mediated signaling pathway involving tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca²⁺]_i was measured in fura-2 loaded cells, and tissue fractions were examined for reactivity with anti-phosphotyrosine (P-Tyr) and anti-MAPK antibodies using immunoprecipitation and immunoblot analysis. In Hanks’ solution (1 mmol/L Ca²⁺), ET-1 (10 pmol/L) did not increase basal [Ca²⁺]_i (81±2 nmol/L) but caused cell contraction (10%) that was inhibited by calphostin C (10^-6 mol/L), inhibitor of PKC, tyrphostin (10^-6 mol/L), inhibitor of tyrosine kinase, and PD098059 (10^-6 mol/L), inhibitor of MAPK kinase. The vasoactive eicosanoid prostaglandin F₂ (PGF₂; 10^-7 mol/L) caused increases in cell contraction (11%) and [Ca²⁺]_i (122±9 nmol/L) that were inhibited by the Ca²⁺ channel blocker verapamil (10^-6 mol/L) but not by calphostin C, tyrphostin, or PD098059. Pretreatment with ET-1 for 10 minutes enhanced cell contraction to PGF₂ (33%) with no additional increase in [Ca²⁺]_i (124±10 nmol/L). Activation of PKC by phorbol 12-myristate 13-acetate (PMA; 10^-7 mol/L) caused cell contraction and enhanced PGF₂ contraction (32%) with no additional increase in [Ca²⁺]_i (126±9 nmol/L). The ET-1– and PMA-induced enhancement of PGF₂ contraction was abolished by verapamil or calphostin C but not by tyrphostin or PD098059. ET-1 and PMA caused significant increases in tyrosine phosphorylation of MAPK that were inhibited by calphostin C, tyrphostin, and PD098059. PGF₂ did not cause any additional increases in tyrosine phosphorylation of MAPK in tissues untreated or pretreated with ET-1 or PMA. Thus, physiological concentrations of ET-1 activate a Ca²⁺-independent PKC-mediated signaling pathway that involves tyrosine phosphorylation and activation of MAPK. The enhancement of PGF₂-induced coronary smooth muscle contraction by ET-1 involves additional activation of a Ca²⁺-sensitive PKC-mediated pathway but not tyrosine phosphorylation or activation of MAPK. The MAPK-dependent and MAPK-independent signaling pathways represent possible cellular mechanisms by which ET-1 could enhance coronary vasoconstriction to vasoactive eicosanoids in coronary vasospasm.

Key Words: endothelin • prostaglandins • calcium • protein kinases • muscle, smooth, vascular

	Introduction

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Coronary vasospasm is often associated with ischemic heart disease, leading to different forms of angina and myocardial infarction, and excessive coronary vasoconstriction in response to endogenous vasoconstrictors has been suggested as one potential cause.^1,2 Endothelin-1 (ET-1), a potent vasoconstrictor, has been suggested to act as an endogenous modulator of coronary vascular tone and to play a role in the setting of coronary vasospasm.^3–6 Also, 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.^7,8

Although several 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 separate from PGF₂.^4–10 Also, in most mechanistic studies, high unphysiological concentrations of ET-1 and PGF₂ have been used to maximally activate the possible mechanisms of smooth muscle contraction.^7–10 This is in sharp contrast to the in vivo conditions in which the coronary artery is exposed to more than 1 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 have been shown to cause significant coronary contraction,^4–10 little information is available on whether physiological concentrations of ET-1 enhance coronary vasoconstriction to small concentrations of PGF₂. Also, the cellular mechanisms involved in the possible ET-1–induced enhancement of coronary vasoconstriction to the vasoactive eicosanoid PGF₂ have not been clearly elucidated.

It is widely accepted that vascular smooth muscle contraction is triggered by increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) as a result of Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space.^11–13 Also, the interaction of a vasoconstrictor agonist with its specific receptor is coupled to increased breakdown of plasma membrane phospholipids, increased production of diacylglycerol, and activation of protein kinase C (PKC).^14–16 PKC is a family of several Ca²⁺-dependent and Ca²⁺-independent isoforms that have different enzyme properties, substrates, functions, and subcellular distribution.^14,15,17,18 Direct activation of PKC by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), has been shown to cause sustained contraction of vascular smooth muscle with no significant change in [Ca²⁺]_i, suggesting a role for PKC in regulating smooth muscle contraction, at least in part, by increasing the myofilament force sensitivity to [Ca²⁺]_i.^19,20 In addition, tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) by MAPK kinase has been suggested to play a role in smooth muscle sensitization to [Ca²⁺]_i.^21–28

Although few studies have suggested that physiological concentrations of ET-1 could enhance vascular smooth muscle contraction to 5-hydroxytryptamine and the vasoactive eicosanoid PGF₂^29–32 and that the enhancement of PGF₂-induced smooth muscle contraction by ET-1 may involve activation and translocation of specific PKC isoforms,³² the signaling mechanisms downstream from PKC activation are not clearly understood.

The purpose of the present study was to test the hypothesis that physiological concentrations of ET-1 enhance coronary smooth muscle contraction to vasoactive eicosanoids by activating a Ca²⁺-sensitive or Ca²⁺-insensitive PKC-mediated signaling pathway involving tyrosine phosphorylation and activation of MAPK. Experiments were designed to investigate the effect of physiological concentrations of ET-1 either alone or in combination with PGF₂ on coronary smooth muscle cell contraction, [Ca²⁺]_i, and tyrosine phosphorylation of MAPK. The effects of ET-1 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, the PKC inhibitor calphostin C, the tyrosine kinase inhibitor tyrphostin, and the MAPK kinase inhibitor PD098059 were investigated.

	Methods

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Tissue Preparation
Castrated male Yorkshire pigs (30 kg) from a local breeder were anesthetized by inhalation of isoflurane. 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, 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% BSA (Sigma).^33–35 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 and 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."³⁶

Cell Contraction
Coverslips with the attached cells were placed on the stage of an inverted Nikon (Diaphot-300) microscope. The cell isolation procedure yielded smooth muscle cells of variable lengths. Only viable, healthy, and 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 significant contraction in response to ET-1 and PGF₂. Cell images were acquired using a PXL CCD camera and displayed on a PC 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 (L) as a fraction of the initial cell length (L_i). All contraction measurements were made at 37°C. The changes in cell contraction in response to ET-1, PGF₂, and the phorbol ester PMA 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.^34,35,37 The fura-2 loading solution was made of normal Hanks’ solution, 1 µmol/L of the cell permeant fura-2 acetoxymethyl ester (fura-2/AM; 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/AM. Precautionary measures were taken throughout the procedure to avoid extensive photobleaching of fura-2.³⁸

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 nm and 380 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) 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 PC using data analysis software. The fluorescent signal was background subtracted. Spectral shifts that result from binding of Ca²⁺ to fura-2 made 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 al³⁸: [Ca²⁺]_i=K_d(Sf₂/Sb₂)[(R-R_min)/(R_max-R)], where R_min and R_max represent the minimal and maximal fluorescence ratios and 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⁺. All experiments were performed at 37°C. The changes in [Ca²⁺]_i in response to ET-1, PGF₂, and the phorbol ester PMA were measured.

Tissue Homogenate
Coronary artery strips (80 mg) at rest or stimulated for 15 minutes with ET-1, PMA, or PGF₂ either separately or combined were rapidly transferred to ice-cold equilibrating buffer A containing (in mmol/L) 25 Tris-HCl (pH 7.5), 5 EGTA, 0.02 leupeptin, 0.2 phenylmethylsulfonyl fluoride, and 1 dithiothreitol. The tissue was transferred to a homogenization buffer containing 20 mmol/L 3-[N-morpholino]propane sulfonic acid, 4% SDS, 10% glycerol, 2.3 mg of dithiothreitol, 1.2 mmol/L EDTA, 0.02% BSA, 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, the homogenate was centrifuged at 10 000g for 2 minutes, and the supernatant was used as the whole-tissue homogenate. Protein concentration was determined using a protein assay kit (Bio-Rad).

Immunoprecipitation
Protein-matched samples of the tissue homogenate were incubated with a protein A-Sepharose slurry to preclear the sample of nonspecific binding to protein A. For phosphotyrosine (P-Tyr) immunoprecipitation, 2 µg of the anti–P-Tyr antibody (Transduction Labs) was added to the sample and allowed to bind for 24 hours at 4°C. Immune complexes were recovered by incubation with 50 µL of 10% protein A-Sepharose beads, and the samples were mixed for 2 hours at 4°C. The beads were centrifuged, washed 3 times with 200 µL of fresh homogenization buffer, and resuspended in a final volume of 60 µL. To this, 60 µL of 2x sample buffer was added. The samples were boiled for 5 minutes, and the immunoprecipitates were separated by SDS-PAGE in preparation for the immunoblot analysis.

Immunoblotting
Samples of the immunoprecipitate were subjected to electrophoresis on 8% SDS polyacrylamide gels then transferred electrophoretically to nitrocellulose membranes. The membranes were incubated in 5% BSA in PBS-Tween at 22°C for 1 hour, washed with PBS-Tween 3x5 minutes, then incubated in the primary anti-MAPK antibody solution (1:1000; Upstate Biotechnology) at 4°C overnight. To maintain the labeling conditions constant, we used the same antibody titer (1:1000) 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 then incubated in horseradish peroxidase–conjugated anti-rabbit IgG for 1.5 hours. The blots were washed with PBS-Tween 5x15 minutes and visualized with enhanced chemiluminescence detection system (Amersham). PBS-Tween contained 80 mmol/L Na₂HPO₄, 20 mmol/L NaH₂PO₄, 100 mmol/L NaCl, and 0.05% Tween. The reactive bands corresponding to MAPK were analyzed quantitatively by optical densitometry using a GS-700 imaging densitometer (Bio-Rad).

Solutions
Krebs solution contained (in mmol/L) 120 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 25 NaHCO₃, 1.2 NaH₂PO₄, and 11.5 dextrose at pH 7.4. Hanks’ solution contained (in mol/L) 137 NaCl, 5.4 KCl, 0.44 KH₂PO₄, 0.42 Na₂HPO₄, 4.17 NaHCO₃, 5.55 dextrose, and 10 HEPES at 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
ET-1, PGF₂ (Sigma), and verapamil (Calbiochem) were dissolved in distilled water. PMA (Alexis Laboratory), calphostin C (Kamiya), PD098059 (Research Biochemicals Int), and tyrphostin (Gibco) 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 with P<0.05 considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
The cell isolation procedure produced relatively long coronary smooth muscle cells (Figure 1). 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. Small concentrations of ET-1 (10^-11 mol/L) caused a small cell contraction (9.8±1.6%) with no significant increase in [Ca²⁺]_i (Figure 1A). Activation of PKC by PMA (10^-7 mol/L) also caused small cell contraction (10.1±1.7%) with no significant increase in [Ca²⁺]_i. Small concentrations of PGF₂ (10^-7 mol/L) caused small increases in both contraction (11.4±1.9%) and [Ca²⁺]_i (122±9 nmol/L; Figure 1B). In cells pretreated with ET-1 (10^-11 mol/L) for 10 minutes, PGF₂ (10^-7 mol/L) caused large contraction (33.4±3.1%) with no additional increases in [Ca²⁺]_i (124±10 nmol/L; Figure 1C). Also, in cells pretreated with PMA (10^-7 mol/L) for 10 minutes, PGF₂ (10^-7 mol/L) caused large contraction (33.3±2.4%) with no additional increases in [Ca²⁺]_i (126±9 nmol/L). Similar enhancements of cell contraction with no additional increases in [Ca²⁺]_i were observed in cells pretreated with PGF₂ (10^-7 mol/L) for 10 minutes then stimulated with ET-1 (10^-11 mol/L) or PMA (10^-7 mol/L).

View larger version (52K): [in this window] [in a new window]   Figure 1. Representative cell contraction (top panels) and [Ca2+]i (bottom panels) in coronary smooth muscle cells activated with 10-11 mol/L ET-1 (A), 10-7 mol/L PGF2 (B), and 10-11 mol/L ET-1 plus 10-7 mol/L PGF2 (C) in the absence or presence of 10-6 mol/L verapamil (D), calphostin C (E), tyrphostin (F), or PD098059 (G).

Pretreatment of the cells with the Ca²⁺ channel blocker verapamil (10^-6 mol/L) for 10 minutes did not affect cell contraction (Figures 1D and 2A) or [Ca²⁺]_i (Figures 1D and 2B) induced by ET-1 (10^-11 mol/L) alone or PMA (10^-7 mol/L) alone, suggesting that Ca²⁺ entry from the extracellular space is not involved. Verapamil completely abolished the contraction (Figure 2A) and [Ca²⁺]_i (Figure 2B) induced by PGF₂ (10^-7 mol/L) alone, suggesting that the PGF₂ responses involve Ca²⁺ entry. Also, verapamil abolished the ET-1– and PMA-induced enhancement of PGF₂ contraction (Figures 1D and 2A), suggesting that the enhanced response is dependent on Ca²⁺ entry.

View larger version (48K): [in this window] [in a new window]   Figure 2. Effect of the Ca2+ channel blocker verapamil (10-6 mol/L), the PKC inhibitor calphostin C (10-6 mol/L), the tyrosine kinase inhibitor tyrphostin (10-6 mol/L), and the MAPK kinase inhibitor PD098059 (10-6 mol/L) on coronary smooth muscle contraction (A) and [Ca2+]i (B) induced by ET-1 (10-11 mol/L), PMA (10-7 mol/L), PGF2 (10-7 mol/L), ET-1 (10-11 mol/L) plus PGF2 (10-7 mol/L), and PMA (10-7 mol/L) plus PGF2 (10-7 mol/L). Data points represent the mean±SEM of experiments on 18 to 24 cells from 6 to 8 pigs.

Pretreatment of the cells with the PKC inhibitor calphostin C (10^-6 mol/L) for 10 minutes inhibited the cell contraction induced by ET-1 (10^-11 mol/L) alone (Figures 1E and 2A) or PMA alone with no significant change in [Ca²⁺]_i (Figures 1E and 2B), suggesting the involvement of PKC. In cells stimulated with PGF₂ (10^-7 mol/L) alone, calphostin C did not affect contraction (Figure 2A) or [Ca²⁺]_i (Figure 2B), suggesting that PKC is not activated during stimulation by PGF₂ (10^-7 mol/L) alone. In cells pretreated with ET-1 or PMA, calphostin C inhibited the enhancement of PGF₂ contraction (Figures 1E and 2A) without affecting [Ca²⁺]_i (Figure 2B), suggesting that the enhanced contractile response involves PKC.

Pretreatment of the cells with tyrphostin (10^-6 mol/L), inhibitor of tyrosine kinase, for 10 minutes significantly inhibited the cell contraction induced by ET-1 alone or PMA alone (Figures 1F and 2A) with no significant change in [Ca²⁺]_i (Figures 1F and 2B), suggesting the involvement of tyrosine kinase. In cells stimulated with PGF₂ (10^-7 mol/L) alone, tyrphostin did not affect cell contraction (Figure 2A) or [Ca²⁺]_i (Figure 2B), suggesting that tyrosine phosphorylation is not involved under these conditions. In cells pretreated with ET-1 or PMA, tyrphostin only partially inhibited the enhancement of PGF₂ contraction (Figures 1F and 2A) without affecting [Ca²⁺]_i (Figure 2B), suggesting that the enhanced contraction is not completely dependent on tyrosine kinase.

MAPK is one of the protein substrates that is phosphorylated at both the threonine and tyrosine residues by MAPK kinase.^24,39–41 Pretreatment of the cells with PD098059 (10^-6 mol/L), inhibitor of MAPK kinase, for 10 minutes significantly inhibited the cell contraction induced by ET-1 alone or PMA alone (Figures 1G and 2A) with no significant change in [Ca²⁺]_i (Figures 1G and 2B), suggesting the involvement of MAPK. In cells stimulated with PGF₂ (10^-7 mol/L) alone, PD098059 did not affect cell contraction (Figure 2A) or [Ca²⁺]_i (Figure 2B), suggesting that MAPK is not activated under these conditions. In cells pretreated with ET-1 or PMA, PD098059 only partially inhibited the enhancement of PGF₂ contraction (Figures 1G and 2A) without affecting [Ca²⁺]_i (Figure 2B), suggesting that the enhanced contraction is not completely dependent on MAPK.

Immunoblots in whole-tissue homogenate of unstimulated coronary artery revealed significant immunoreactive bands at 44 and 42 kDa corresponding to ERK-1 and ERK-2 MAPKs (Figure 3). In control unstimulated tissues, the immunoblots of immunoprecipitated P-Tyr did not show significant reactivity with anti-MAPK antibody, suggesting minimal tyrosine phosphorylation of MAPK under resting conditions. In tissues stimulated with ET-1 alone or PMA alone, significant amounts of MAPK could be detected in the immunoblots of immunoprecipitated P-Tyr, suggesting tyrosine phosphorylation of MAPK (Figure 3). In tissues untreated or pretreated with ET-1 or PMA, PGF₂ (10^-7 mol/L) did not cause any additional increases in the amount of MAPK detected in the immunoblots of immunoprecipitated P-Tyr (Figure 3). In tissues pretreated with calphostin C, tyrphostin, or PD098059 for 10 minutes and stimulated with ET-1 or PMA, the amount of MAPK detected in the immunoblots of immunoprecipitated P-Tyr was significantly reduced (Figure 4).

View larger version (35K): [in this window] [in a new window]   Figure 3. Tyrosine phosphorylation of MAPK in coronary smooth muscle. Control unstimulated coronary artery strips were homogenized, and the whole tissue homogenate was examined for ERK-1 (p44) and ERK-2 (p42) MAPKs using anti-MAPK antibody (1:1000) and immunoblot analysis. In other experiments, coronary artery strips were stimulated with ET-1 (10-11 mol/L), PMA (10-7 mol/L), PGF2 (10-7 mol/L), ET-1 plus PGF2, or PMA plus PGF2 for 15 minutes. The whole-tissue homogenate was prepared, P-Tyr was immunoprecipitated with anti–P-Tyr antibody, and the immunoprecipitate was examined for reactivity with anti-MAPK antibody (1:1000) using immunoblot analysis. Cumulative optical density measurements of p44 (top panel) and p42 (bottom panel) MAPK are presented as the mean±SEM of experiments in 6 to 8 tissue samples from 6 to 8 pigs.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of inhibitors of PKC, tyrosine kinase, and MAPK kinase on ET-1–induced tyrosine phosphorylation of MAPK in coronary smooth muscle. Coronary artery strips were stimulated with ET-1 (10-11 mol/L) in the absence or presence of 10-6 mol/L calphostin C, tyrphostin, or PD098059. The whole-tissue homogenate was prepared, P-Tyr was immunoprecipitated with anti–P-Tyr antibody, and the immunoprecipitate was examined for reactivity with anti-MAPK antibody (1:1000) using immunoblot analysis. Cumulative optical density measurements of p44 (top panel) and p42 (bottom panel) MAPK are presented as the mean±SEM of experiments in 6 to 8 tissue samples from 6 to 8 pigs.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study showed that ET-1 at small and physiological concentrations (10^-11 mol/L) caused significant contraction in coronary smooth muscle cells with no detectable increases in [Ca²⁺]_i. Although some studies have shown that picomolar concentrations of ET-1 increase [Ca²⁺]_i in cultured human coronary smooth muscle cells,⁴² the present data are consistent with previous reports that these small ET-1 concentrations do not significantly increase [Ca²⁺]_i in freshly isolated porcine coronary smooth muscle cells.³² The difference in the results could be related to differences in the cell preparation or the species studied. We should note that in the present study, [Ca²⁺]_i was measured in an area of homogeneous fluorescence 1 µm in diameter, 1 µm from the plasma membrane, and 1 µm from the nucleus. Although small concentrations of ET-1 did not cause any detectable increases in [Ca²⁺]_i in this representative part of the cell, small concentrations of PGF₂ significantly increased [Ca²⁺]_i in the same cell compartment and thus increased the level of confidence in the [Ca²⁺]_i measurements. However, because [Ca²⁺]_i was measured in only 1 part of the cell, we cannot rule out the possibility that ET-1 might change [Ca²⁺]_i in other cell compartments. Therefore, analysis of the effect of ET-1 not only on the average [Ca²⁺]_i but also on the subcellular distribution of [Ca²⁺]_i should be examined further in future investigations.

The observation that small ET-1 concentrations cause significant contraction of coronary smooth muscle cells with no detectable increases in [Ca²⁺]_i suggests 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 reduce critically the luminal cross-sectional area in the setting of significant coronary vasospasm.⁴³ Also, if the ET-1–induced coronary contraction is mediated by a [Ca²⁺]_i-sensitizing pathway rather than by increasing [Ca²⁺]_i, then it is predicted that vasodilators that function solely by lowering [Ca²⁺]_i would be less effective in overcoming this form of coronary vasospasm. Furthermore, if the ET-1–induced stimulation of a [Ca²⁺]_i-sensitizing pathway is combined with another agonist, which by itself causes only small increases in coronary smooth muscle contraction and [Ca²⁺]_i, then the resulting synergistic effect could dangerously enhance coronary vasoconstriction and lead to severe coronary vasospasm.

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 verapamil, (3) direct activation of PKC by phorbol ester caused a contraction similar to that of ET-1 with no significant change in [Ca²⁺]_i, and (4) ET-1 contraction was completely inhibited by the PKC inhibitor calphostin C. Thus, the contraction induced by small concentrations of ET-1 alone does not require significant increases in [Ca²⁺]_i but seems to involve activation of PKC, suggesting activation of a Ca²⁺-independent PKC-mediated pathway. These results are consistent with previous reports that small concentrations of ET-1 increase PKC activity and promote activation and translocation of the Ca²⁺-independent -PKC isoform.³²

Although basal [Ca²⁺]_i levels (80 nmol/L) seem to be sufficient for ET-1 and PMA to induce coronary smooth muscle contraction and PKC activation, the threshold [Ca²⁺]_i required for the observed ET-1– and PMA-induced responses is unclear. We have previously shown that incubation of coronary smooth muscle in Ca²⁺-free solution reduces [Ca²⁺]_i to 35 nmol/L.¹⁰ We have also shown that ET-1 and phorbol esters could cause significant coronary contraction and PKC activation in Ca²⁺-free solution, which further support the activation of Ca²⁺-insensitive PKC isoforms under these conditions.¹⁰ However, additional sequestration of cytosolic Ca²⁺ can still be achieved by loading the cells with BAPTA. Whether ET-1 and PMA could still cause coronary contraction and PKC activation in these vanishing amounts of cytosolic Ca²⁺ is unclear and should represent important areas for future investigations.

In addition to ET-1 induced activation of PKC, the ET-1 contraction seems to involve tyrosine phosphorylation of MAPK because (1) ET-1 contraction was inhibited by the tyrosine kinase inhibitor tyrphostin, (2) ET-1 contraction was inhibited by the MAPK kinase inhibitor PD098059, and (3) a significant amount of MAPK could be detected in the immunoblots of immunoprecipitated P-Tyr in tissues stimulated with ET-1 and was inhibited in tissues pretreated with tyrphostin and PD098059. The observation that the amount of MAPK detected in the immunoblots of immunoprecipitated P-Tyr in tissues stimulated with ET-1 was inhibited by the PKC inhibitor calphostin C suggests that PKC is involved and is acting upstream from tyrosine phosphorylation of MAPK. This is further supported by the observations that direct activation of PKC by phorbol ester caused similar increases in tyrosine phosphorylation of MAPK that were inhibited by calphostin C, tyrphostin, and PD098059. The present results are consistent with a previously hypothesized Ca²⁺-independent PKC-mediated signaling cascade involving activation of MAPK kinase and MAPK and leading to phosphorylation of the actin-binding protein caldesmon and enhancement of smooth muscle contraction.^24,44 This is also supported by a study showing that ET-1 induces caldesmon phosphorylation in porcine coronary artery.⁴⁵ However, a clear relation between the ET-1–induced caldesmon phosphorylation and the smooth muscle contraction could not be conclusively demonstrated in that study.⁴⁵

Small concentrations of PGF₂ (10^-7 mol/L) alone caused cell contraction that was associated with a significant increase in [Ca²⁺]_i. The PGF₂-induced contraction and [Ca²⁺]_i seem to be due mainly to Ca²⁺ entry from the extracellular space because they were inhibited by the Ca²⁺ channel blocker verapamil but not by calphostin C, tyrphostin, or PD098059. The PGF₂-induced [Ca²⁺]_i profile seems to be dependent on the concentration used. We have previously shown that high concentrations of PGF₂ (10^-4 mol/L) cause an initial transient increase in [Ca²⁺]_i followed by maintained increase in [Ca²⁺]_i.³² The biphasic [Ca²⁺]_i profile is generally attributed to initial activation of the inositol 1,4,5-trisphosphate–induced Ca²⁺ release mechanism followed by maintained Ca²⁺ entry from the extracellular space. In contrast, smaller concentrations of PGF₂ seem preferentially to stimulate the Ca²⁺ entry pathway. These data are consistent with previous studies in coronary smooth muscle cells that have shown that small concentrations of PGF₂ cause small increases in [Ca²⁺]_i that are inhibited by the Ca²⁺ channel antagonist diltiazem.³²

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.^29–31 Although the enhanced PGF₂ contraction in cells pretreated with ET-1 was not associated with additional increases in [Ca²⁺]_i, it seemed to require extracellular Ca²⁺ because it was completely inhibited by verapamil. Also, the enhancement of PGF₂ contraction by ET-1 seems to involve PKC because (1) direct activation of PKC by PMA caused similar enhancement of PGF₂ contraction and (2) the enhancement of PGF₂-induced contraction by ET-1 or PMA was inhibited by the PKC inhibitor calphostin C. Thus, the enhanced PGF₂-induced contraction in tissues pretreated with ET-1 seems to require both Ca²⁺ and PKC, which raises the possibility that it involves a Ca²⁺-dependent PKC isoform. This is consistent with previous reports that the enhancement of PGF₂ contraction by ET-1 is associated with activation and translocation of the Ca²⁺-dependent -PKC³² and that a threshold increase in [Ca²⁺]_i is required for activation of -PKC in vascular smooth muscle cells of the ferret and the pig.^9,37 However, the enhanced PGF₂-induced contraction in tissues pretreated with ET-1 or PMA does not seem to be completely dependent on tyrosine phosphorylation of MAPK because (1) the ET-1– and PMA-induced enhancements of PGF₂ contraction were only partially inhibited by the tyrosine kinase inhibitor tyrphostin or the MAPK kinase inhibitor PD098059, and (2) the ET-1– and PMA-induced enhancements of PGF₂ contraction were not associated with additional increases in the amount of MAPK detected in the immunoblots of immunoprecipitated P-Tyr. Thus, the enhancement of PGF₂ contraction by ET-1 seems to require activation of a Ca²⁺-sensitive PKC pathway but not tyrosine phosphorylation of MAPK. This PKC-dependent but MAPK-independent pathway may involve other signaling mechanisms downstream from PKC and should represent important areas of future investigation. One potential signaling mechanism is that PKC may increase the phosphorylation and translocation of other intracellular protein substrates, such as the actin-binding protein calponin, and thereby lead to enhancement of smooth muscle contraction.^46,47

In summary, physiological concentrations of ET-1 activates a Ca²⁺-insensitive PKC-mediated signaling pathway involving tyrosine phosphorylation and activation of MAPK. The enhancement of PGF₂-induced coronary smooth muscle contraction by physiological concentrations of ET-1 involves activation of a Ca²⁺-sensitive PKC-mediated pathway but not tyrosine phosphorylation and activation of MAPK. The MAPK-dependent and MAPK-independent signaling pathways represent possible cellular mechanisms by which ET-1 could enhance coronary vasoconstriction to vasoactive eicosanoids in the setting of 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 September 23, 2001; first decision November 2, 2001; accepted November 13, 2001.

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