Departments of Physiology and Medicine, Virginia Commonwealth University Medical School, Richmond, Virginia 23298
Submitted 23 April 2004 ; accepted in final form 12 June 2004
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ABSTRACT |
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endothelin receptor type A; endothelin receptor type B; myosin phosphatase targeting subunit
A subtype of ETB receptor (ETB1) mediates the ability of ET-1 and ET-3 to stimulate nitric oxide (NO) formation in endothelial cells via Ca2+/calmodulin-dependent activation of endothelial NO synthase (eNOS) and/or via Gi-dependent activation of Akt and phosphorylation of eNOS (14, 24). ET-1 released from endothelial cells or via the circulation interacts with ETA and/or ETB2 receptors to cause contraction of vascular smooth muscle cells. The signaling pathway in vascular or visceral smooth muscle involves stimulation of phosphoinositide (PI) hydrolysis and Ca2+ release, presumed to occur via coupling of ETA or ETB to Gq and, less often, to Gi (7, 11, 36, 37). A more complex pathway is present in iris sphincter and ciliary smooth muscle, where ET-1 activates cytoplasmic phospholipase 2 (cPLA2) and induces prostaglandin E2 (PGE2)-dependent Ca2+ mobilization and cAMP formation (1, 15). In other regions, for example, intestinal muscle and internal anal sphincter, ET-1 induces a transient nerve-mediated relaxation followed by a sustained contraction (4, 10, 12, 25).
Gohla et al. (11) provided the first evidence of specific G proteins activated by ET-1, using cultured aortic smooth muscle cells. ET-1 was shown to activate mainly Gq and G13 and to stimulate 20-kDa myosin light chain (MLC20) phosphorylation and contraction; the latter were blocked by inhibitors of PI hydrolysis, MLC kinase (MLCK), RhoA, and Rho kinase, suggesting the involvement of MLCK and Rho kinase pathways. The specificity of endothelin receptors mediating these pathways and the downstream components of RhoA-dependent pathway involved in inactivation of MLC phosphatase and stimulation of MLC20 phosphorylation were not further analyzed.
Recent studies with other agonists in vascular and visceral smooth muscle suggest that the MLCK- and RhoA-dependent pathways are temporally distinct and mediated by specific receptors and G proteins (18, 19, 31, 40, 41). In the present study, we have examined the role of ETA and ETB receptors in the regulation of each pathway and have shown that both ETA and ETB mediate Gq-dependent activation of MLCK and transient stimulation of MLC20 phosphorylation and contraction, whereas only ETA mediates G13-dependent activation of RhoA, resulting in Rho kinase-mediated phosphorylation of the myosin phosphatase targeting subunit MYPT1 and sustained MLC20 phosphorylation and contraction. The second limb of the RhoA pathway, involving PKC-mediated phosphorylation of CPI-17 and inhibition of MLC phosphatase, was not detected. Its absence reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A) via a pathway involving ETB-dependent, p38 MAPK-mediated activation of PP2A.
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MATERIALS AND METHODS |
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Expression of ET receptors. Specific primers were designed based on homologous sequences in human, rat, and mouse cDNAs for ETA and ETB. ETA primers were 5'-CCGGAGAGATACAGCGCTAATC-3' (forward) and 5'-CAGCTTGCAGAGAAACACTCC-3' (reverse); the expected size of the PCR product was 414 bp. ETB primers were 5'-GACATCGCCCGCAAGCCGGTGCGG-3' (forward) and 5'-GTAGGTGTTAATGGGTATGTC-3' (reverse); the expected size of the PCR product was 478 bp. Total RNA (5 µg) isolated from cultured circular and longitudinal smooth muscle cells was reversibly transcribed and amplified by PCR under standard conditions as described previously (43, 47). The PCR products were separated by electrophoresis in 1.2% agarose gel in the presence of ethidium bromide, visualized by ultraviolet fluorescence, and recorded using a ChemiImager 4400 fluorescence system. The PCR products were purified and sequenced.
Selective protection of ETA and ETB receptors. A technique for selective receptor protection previously used to determine the coexistence and function of various G protein-coupled receptors was used to characterize the signaling pathways mediated by ETA and ETB receptors (29, 30). The technique involves protection of one receptor subtype with selective ET-1 agonists or antagonists, followed by inactivation of all unprotected receptors with a low concentration of N-ethylmaleimide (NEM; 5 µM). Freshly dispersed muscle cells were incubated with an ETA or ETB antagonist at 31°C for 2 min, followed by addition of NEM for 20 min. The cells were centrifuged twice at 150 g for 10 min and resuspended in control HEPES medium for 60 min. The contractile response to ET-1 of cells treated in this fashion was compared with the response of untreated cells. As previously shown (29, 30), muscle cells incubated with NEM without protective agent did not contract in response to receptor-linked agonists, but they responded fully to ionomycin and KCl.
Identification of endothelin-activated G proteins.
Activation of specific G proteins was determined from agonist-induced increase in G binding to guanosine 5'-O-(3-thiotriphosphate) (GTP
S) as described previously (29, 30, 32, 35, 47). Cells were homogenized in 20 mM HEPES (pH 7.4), the homogenates were centrifuged at 4°C for 30 min at 30,000 g, and the membranes were solubilized in 20 mM HEPES buffer containing 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Solubilized membranes were incubated for 20 min at 37°C with 100 nM [35S]GTP
S in 10 mM HEPES with ET-1 in the presence or absence of ETB or ETA antagonist. The reaction was stopped with 10 volumes of 100 mM Tris·HCl medium (pH 8.0) containing 20 µM GTP, and the membranes were incubated for 2 h on ice in wells precoated with a specific G
antibody. After membranes were washed with phosphate buffer, the radioactivity from each well was counted by liquid scintillation.
Assay of phospholipase C- activity.
Total inositol phosphates were measured using anion exchange chromatography as described previously (31, 47). Freshly dispersed smooth muscle cells were labeled for 4 h with myo-2-[3H]inositol (0.5 µCi/ml) in inositol-free HEPES medium. Muscle cells (2 x 106 cells/ml) were treated with ET-1 (1 µM) for 60 s in 1 ml of 25 mM HEPES buffer (pH 7.4) consisting of (in mM) 115 NaCl, 5.8 KCl, 2.1 KH2P04, 2 CaCl2, 0.6 MgCl2, and 14 glucose. The reaction was terminated by the addition of 940 µl of chloroform-methanol-HCl (50:100:1). After extraction with 340 µl of chloroform and 340 µl of H2O, the aqueous phase was applied to DOWEX AG-1 columns; [3H]inositol phosphates were then eluted, and radioactivity was determined by liquid scintillation.
Measurement of Ca2+ release. 45Ca2+ release was measured in permeabilized muscle cells as described previously (47). The cells were permeabilized by treatment with saponin (35 µg/ml) for 5 min and, after being washed, were resuspended in a medium containing 100 nM Ca2+, 10 µM antimycin, 10 µCi/ml 45Ca2+, 1.5 mM ATP, and ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine kinase). Steady-state Ca2+ uptake was measured after incubation for 60 min (2.38 ± 0.26 nmol Ca2+/106 cell), and Ca2+ release was determined from the decrease in steady-state 45Ca2+ cell content during the first 30 s after addition of ET-1.
Assay for RhoA activity. Activated RhoA was measured in freshly dispersed muscle cells with the use of a technique using Rhotekin as described previously (32). Muscle cells lysates (100 µg of protein) were incubated with glutathione-agarose slurry of Rhotekin at 4°C for 45 min. The beads were washed three times with the washing buffer containing 50 mM Tris·HCl (pH 7.2), 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 0.1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. GTP-bound RhoA was solubilized in Laemmli sample buffer and analyzed by 15% SDS-PAGE followed by Western blot and chemiluminescence.
Assay for phospholipase D activity. Phospholipase D (PLD) activity was determined by formation of the PLD-specific product phosphatidylethanol (PEt) as described previously (28). Smooth muscle cells (2 x 106 cells/ml) were incubated for at 31°C for 3 h in HEPES medium with [3H]myristic acid (2 µCi/ml) and then with 150 mM ethanol. The cells were centrifuged, resuspended in fresh medium, and stimulated with ET-1 for 5 min. The reaction was terminated with chloroform-methanol-HCl (100:200:2, vol/vol/vol), and the organic phase was extracted and analyzed for [3H]PEt by thin-layer chromatography. [3H]PEt was identified using unlabeled standards and visualized under ultraviolet light at 357 nm. The spots corresponding to [3H]PEt were scraped and counted by liquid scintillation.
Assay for Rho kinase activity.
Rho kinase activity was determined by immunokinase assay in cell extracts as described previously (31). The immunoprecipitates were washed with phosphorylation buffer and incubated for 5 min on ice with 5 µg of myelin basic protein. Kinase assays were initiated by the addition of 10 µCi of [-32P]ATP (3,000 Ci/mmol) and 20 µM ATP, followed by incubation for 10 min at 37°C. 32P-labeled myelin basic protein was absorbed onto phospho-cellulose disks, and free radioactivity was removed by repeated washing with 75 mM phosphoric acid. Phosphorylation was determined by liquid scintillation.
Assay for PKC activity. PKC activity was measured in the particulate fraction as described previously (27, 31). Muscle cells (2 x 106 cells/ml) were incubated with ET-1 for 5 min in the presence or absence of ETA or ETB antagonist, and the reaction was terminated by rapid freezing. After homogenization, PKC activity was measured in the membrane pellet by phosphorylation of myelin basic protein, and the results are expressed as cpm per milligram of protein per minute.
Immunoblot analysis of phosphorylated MLC20, MYPT1, and CPI-17. Phosphorylated MLC20 was determined by immunoblot using phospho-specific antibodies to MLC20 (Ser19; sc-19849), MYPT1 (Thr696; sc-17556), and CPI-17 (Thr38; sc-17560) as described previously (31, 47). The proteins were resolved by SDS-PAGE and electrophoretically transferred on to polyvinylidene difluoride membranes. The membranes were incubated for 12 h with appropriate antibody and then for 1 h with a horseradish peroxidase-conjugated secondary antibody. The bands were identified by enhanced chemiluminescence.
Measurement of contraction in dispersed smooth muscle cells. Muscle cell contraction was measured in freshly dispersed muscle cells by scanning micrometry as described previously (27, 30, 31, 47). A cell aliquot containing 104 muscle cells/ml was added to 0.1 ml of medium containing ET-1, and the reaction was terminated with 1% acrolein. Time course measurements were taken at intervals for 5 min. The mean cell length of 50 muscle cells treated with ET-1 was measured using scanning micrometry and was compared with the mean length of untreated muscle cells (mean control cell length: 128 ± 5 µm).
Materials.
Collagenase and soybean trypsin inhibitor were obtained from Worthington Biochemical (Freehold, NJ). Western blotting and chromatography materials were obtained from Bio-Rad Laboratories (Hercules, CA). RhoA antibody was purchased from Upstate Biotechnology (Lake Placid, NY). MLC20 phospho-antibody, CPI-17 phospho-antibody, MYPT1 phospho-antibody, Rho kinase antibody, and polyclonal antibodies to Gi1 G
i2, G
i3, G
13, and G
s were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). [
-32P]ATP was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). myo-[3H]inositol and [35S]GTP
S were obtained from DuPont NEN (Boston, MA). Okadaic acid, U-73122, ML-9, Y-27632, and bisindolylmaleimide were obtained from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Both ETA and ETB receptors were detected by RT-PCR on RNA extracted from cultured rabbit circular and longitudinal muscle cells in first passage using primers based on conserved sequences of human, mouse, and rat cDNAs (Fig. 1). As shown previously (43), the use of smooth muscle cells in first passage ensured the absence of contaminants from neural, endothelial, and interstitial cells.
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Exposure of muscle cells isolated separately from the circular and longitudinal muscle layers of intestine to ET-1 caused an immediate contraction that attained a peak within 30 s and was sustained for up to 10 min of observation (Fig. 2). The initial peak contraction at 30 s was concentration dependent and was partly inhibited by selective ETA (BQ-123) and ETB (BQ-788) antagonists and abolished by a combination of the two antagonists (Figs. 2 and 3). The effect of each antagonist on the initial contraction was concentration dependent (Fig. 3). In contrast, the sustained contraction determined at 5 min was abolished by the ETA antagonist but was not affected by the ETB antagonist (Fig. 3). Studies with other agonists have shown that the initial and sustained contractions are mediated by distinct G proteins and signaling pathways (27, 31, 47).
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ET-1 selectively activated Gq and G13 in solubilized membranes derived separately from circular and longitudinal muscle cells, causing a significant increase in the binding of [35S]GTPS to G
q (365 ± 43 and 371 ± 31% in circular and longitudinal muscle cells, respectively) and G
13 (248 ± 28 and 285 ± 36% in circular and longitudinal muscle cells, respectively) but not in binding to G
i1, G
i2, G
i3, or G
s (Table 1). The increase in [35S]GTP
S binding to G
q was partly inhibited by ETA and ETB antagonists (54 ± 3 and 47 ± 5%, respectively) and completely inhibited (94 ± 4%) by a combination of the two antagonists (Fig. 5). In contrast, the increase in [35S]GTP
S binding to G
13 was completely inhibited (91 ± 6%) by the ETA antagonist but was not affected by the ETB antagonist [8 ± 5%; not significant (NS)] (Fig. 5). The results implied that ET-1 interacts with both ETA and ETB receptors: ETA receptors are coupled to activation of G
q and G
13, whereas ETB receptors are coupled to activation of G
q only.
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We previously showed that agonist-induced initial contraction is mediated by Ca2+/calmodulin-dependent activation of MLCK and phosphorylation of MLC20. The initial increase in intracellular Ca2+ concentration ([Ca2+]i) in intestinal circular muscle is mediated by D-myo-inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release, whereas the increase in [Ca2+]i in longitudinal muscle is mediated by arachidonic acid-dependent Ca2+ influx, which triggers Ca2+-induced Ca2+ release via ryanodine receptors/Ca2+ channels (21, 22, 27).
In circular muscle cells, ET-1 caused a threefold increase in PI hydrolysis within 1 min that was partly inhibited by ETA and ETB antagonists (67 ± 4 and 48 ± 6% inhibition, respectively) and abolished by a combination of both antagonists (94 ± 6%) (Fig. 6A). PI hydrolysis was not affected by pretreatment of the cells for 60 min with pertussis toxin (400 ng/ml), consistent with the absence of Gi activation by ET-1 in these cells (Table 1). In permeabilized circular muscle cells, ET-1 stimulated prompt Ca2+ release from sarcoplasmic stores (32 ± 4% decrease in 45Ca2+ cell content within 1 min), which was completely blocked by heparin (0.1 µg/ml) (3 ± 6%; NS), indicating that it was mediated by activation of IP3 receptor/Ca2+ channels (Fig. 6B).
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Signaling pathways mediating sustained contraction and MLC20 phosphorylation by ET-1.
We recently showed that agonist (e.g., acetylcholine, sphingosine-1-phosphate)-induced sustained contraction and MLC20 phosphorylation are Ca2+ independent and mediated cooperatively via Rho kinase phosphorylation of MYPT1 and PKC phosphorylation of CPI-17, resulting in inhibition of MLC phosphatase (31, 47). In this study, ET-1 activated RhoA in naive circular and longitudinal muscle cells and in cells where only ETA receptors were preserved; RhoA activity was not stimulated in cells where only ETB receptors were preserved (Fig. 8). As expected, ET-1 activated all regulatory mediators downstream of RhoA, including Rho kinase, PLD, and PKC in both circular muscle cells (Rho kinase: 465 ± 53%; PLD: 281 ± 36%; PKC: 382 ± 47%) and longitudinal muscle cells (Rho kinase: 507 ± 62%; PLD: 335 ± 42%; PKC: 428 ± 59%) (Fig. 9). Dephosphorylation of phosphatidic acid, the primary product of phosphatidylcholine hydrolysis by PLD, yields diacylglycerol, which stimulates PKC activity (28). The ETA antagonist BQ-123, but not the ETB antagonist BQ-788, inhibited all three enzymatic activities.
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We examined the possibility that the absence of CPI-17 phosphorylation by PKC could reflect dephosphorylation by an activated PP2A. In support of this notion, ET-1 induced CPI-17 phosphorylation in the presence of a low concentration of okadaic acid (1 nM), which selectively blocks PP2A. CPI-17 phosphorylation was inhibited by bisindolylmaleimide, implying that it was mediated by PKC (Fig. 11). CPI-17 phosphorylation was also observed after blockade of ETB but not ETA receptors and in the presence of the p38 MAPK inhibitor SB-203580, implying that the upstream pathway that resulted in activation of PP2A involved stimulation of p38 MAPK via ETB receptors (Fig. 11).
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DISCUSSION |
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The present study provides a detailed analysis of the signaling pathways initiated by ETA and ETB receptors in smooth muscle. A model depicting these pathways is shown in Fig. 12. ET-1 activated Gq and G13, but not Gi1, Gi2, Gi3, or Gs, and stimulated contraction and MLC20 phosphorylation. The response consisted of two phases, a transient Ca2+-dependent phase and a sustained Ca2+-independent phase, mediated by distinct receptors, G proteins, and signaling pathways. The close parallelism between the responses in circular and longitudinal muscle cells, except for the well-known difference in Ca2+ mobilization, provided further corroboration.
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In contrast, the sustained phase of MLC20 phosphorylation and contraction was mediated selectively by ETA receptors via a pathway involving sequential activation of G13, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 and inhibition of MLC phosphatase. Although PLD and PKC, which are also downstream of RhoA (31), were activated, CPI-17 was not phosphorylated and thus did not contribute to inhibition of MLC phosphatase. In this respect, the response to ET-1 differed from the response to other G13/RhoA-coupled agonists, such as acetylcholine and sphingosine-1-phosphate, which cause inhibition of MLC phosphatase via phosphorylation of both MYPT1 and CPI-17 in these cells (31, 47). The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by PP2A. PP2A was activated via a pathway involving ETB-dependent stimulation of p38 MAPK activity. The existence of this "silent" pathway, suggested by studies in other cell types linking p38 MAPK to activation of PP2A (23, 42, 45), was confirmed by measurement of CPI-17 phosphorylation in the presence of selective kinase and phosphatase inhibitors. CPI-17 phosphorylation was unmasked in the presence of the ETB antagonist BQ-788, but not the ETA antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent.
Our results differ from those of Niiro et al. (33) and Kitazawa et al. (19) in intact rabbit femoral artery and vas deferens muscle strips. Both groups showed that ET-1 caused phosphorylation of CPI-17 at Thr38. ET-1 caused phosphorylation of MYPT1 also, but only at Thr799, a site different from the critical phosphorylation site (Thr696) responsible for inhibition of MLC phosphatase. The effects of Rho kinase and PKC inhibitors on ET-1-induced CPI-17 phosphorylation, MLC20 phosphorylation, and muscle contraction were not examined in these studies. It is possible that the participation of MYPT1 and/or CPI-17 in inhibition of MLC phosphatase and stimulation of MLC20 phosphorylation in response to ET-1 is tissue specific. Alternatively, ETB receptors that mediate activation of PP2A and dephosphorylation of CPI-17 may not be expressed in these tissues.
In summary, ET-1 induced an initial contraction in intestinal smooth muscle mediated additively by ETA and ETB receptors and a sustained contraction mediated exclusively by ETA receptors. The two phases of response involved distinct receptors, G proteins, and signaling pathways (Fig. 12). The initial response reflected Ca2+-dependent activation of MLCK and phosphorylation of MLC20 via both receptors. The sustained response reflected activation of RhoA by ETA receptors only, leading to Rho kinase-mediated phosphorylation of MYPT1, inhibition of MLC phosphatase, and phosphorylation of MLC20. PKC-dependent activation of the endogenous MLC phosphatase inhibitor CPI-17 was masked by an ETB-mediated cascade involving activation of PP2A by p38 MAPK and dephosphorylation of CPI-17.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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