doi: 10.1161/hq1201.100264
© 2001 American Heart Association, Inc.
Vascular Biology |
Activation of p38 Mitogen-Activated Protein Kinases by Endothelin and Noradrenaline in Small Arteries, Regulation by Calcium Influx and Tyrosine Kinases, and Their Role in Contraction
From the Department of Medicine, University of Manchester, Manchester Royal Infirmary, Manchester, UK.
Correspondence to Dr Jacqueline Ohanian, Department of Medicine, University of Manchester, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK. E-mail johanian{at}man.ac.uk
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Abstract |
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Small-artery responses to vasoconstrictor agonists are important for vascular function. To investigate the signaling pathways involved in contraction, we studied the activation and regulation of p38 mitogen-activated protein kinases (p38MAPKs) and heat shock protein (HSP) kinase by endothelin and noradrenaline in rat mesenteric arteries. Both vasoconstrictors activated p38
and/or p38ß but not p38
or p38
, leading to increased HSP kinase activity. p38MAPK activation by noradrenaline was maximum between 2 and 10 minutes and was wholly dependent on calcium influx but insensitive to the tyrosine kinase inhibitor herbimycin A. In contrast, endothelin induced a biphasic response, with activation at 2 and 10 minutes. The early activity was wholly dependent on calcium influx and inhibited by herbimycin A. The later activity was only 50% calcium dependent, was insensitive to herbimycin A, but was 50% inhibited by genistein, a nonselective tyrosine kinase inhibitor. With both agonists, p38MAPK activity returned to basal by 30 minutes. SB203580, a p38MAPK inhibitor, blocked agonist-induced HSP kinase activity, and herbimycin A inhibited activation by endothelin but not by noradrenaline. In addition, SB203580 inhibited noradrenaline-induced contraction but had little effect on contraction to endothelin. These data show that vasoconstrictors use different upstream activators of p38MAPK in vascular tissue and that the p38MAPK pathway is selectively implicated in the contractile response to noradrenaline in small arteries.
Key Words: vasoconstrictors • vascular smooth muscle • heat shock proteins • signal transduction
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Introduction |
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Vascular tone is an important determinant of peripheral resistance and blood pressure, and abnormalities in small-artery contractility contribute to pathological states such as vasospasm and hypertension.1 The major mechanism of smooth muscle contraction is an increase in cytoplasmic calcium and phosphorylation of the regulatory light chains of myosin. However, there is considerable evidence indicating that vasoconstrictors activate multiple ancillary pathways that modulate the contractile response (see reviews2,3). Among the many pathways activated, protein kinase C,2 Rho family G proteins,3,4 nonreceptor tyrosine kinases,5 and extracellular signal–regulated kinases (ERK1/2)2,6 have been shown to play a role in smooth muscle contraction. Recently, stress-activated protein kinases have also been implicated in sustained contraction through regulation of the phosphorylation of heat shock protein (HSP)27.7
HSP27 belongs to a family of small HSPs that includes HSP20, myotonic dystrophy kinase–binding proteins, and crystallins.8 Increased phosphorylation of HSP27 has been reported in response to a variety of vasoconstrictors in smooth muscle,9–12 and inhibition of HSP27 phosphorylation or interference with its function reduces contraction.9 HSPs may also play a role in vascular diseases such as hypertension. For instance, stress-induced hypertension in rats increased the expression and phosphorylation of HSP27 in vascular tissue13 as did surgical stress.14 HSP27 phosphorylation is catalyzed by mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2), which is, in turn, a substrate for stress-activated protein kinases, p38MAPKs.10
p38MAPKs are members of the MAPK superfamily.15 Four p38MAPKs have been cloned, (SAPK1), ß (SAPK2), (SAPK3), and (SAPK4), of which the most extensively studied are p38 and p38ß; this is due in part to the availability of SB203580, a specific inhibitor of their kinase activity.16 p38MAPKs are activated in response to cellular stress,16 ischemia/reperfusion,17 and also G protein–coupled receptors (GPCRs).18–20 Upstream regulators of p38MAPKs include Src family tyrosine kinases,21 p21-activated kinases, and small G proteins of the Rho family.22 p38MAPKs are directly activated by phosphorylation on Thr180 and Tyr182 by kinases MKK3 and MKK6.23
Activation of p38MAPKs by GPCR agonists in smooth muscle is implicated in arachidonic acid release,20 HSP27 phosphorylation,10 and possibly contraction.7,12,24 In addition, there is evidence that growth factors and cytokines regulate smooth muscle cell migration through the activation of p38MAPK and the phosphorylation of HSP27.25 These studies point to an important functional role for p38MAPKs in smooth muscle; however, comparatively little is known regarding the mechanisms that control the activation of this pathway. In response to vasopressin,26 protein kinase C appears to be upstream from p38MAPKs, whereas angiotensin II uses a redox-sensitive mechanism.24,27 Also, tyrosine kinases are implicated possibly through GPCR agonist transactivation of the epidermal growth factor receptor28,29 or Src family tyrosine kinases.21 However, these studies have been conducted with smooth muscle cells, and the mechanisms involved in intact tissues have not yet been fully characterized.
We have investigated the role of nonreceptor tyrosine kinases and calcium in p38MAPK activation by GPCRs. Our results show that in rat mesenteric small arteries, noradrenaline (NA) and endothelin-1 (ET-1) activate p38MAPK and/or p38MAPKß, leading to increased HSP27 kinase activity. However, these agonists use different mechanisms such that NA-induced p38MAPK activity is independent of Src tyrosine kinase activity and wholly dependent on calcium influx, whereas the response to ET-1 is partially dependent on a herbimycin A–sensitive tyrosine kinase and calcium influx. In addition, we show that the p38MAPK pathway is selectively implicated in the contractile response to NA.
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Methods |
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Preparation of Small Arteries
Adult female Sprague-Dawley rats (200 to 300 g, Charles River UK Ltd, Kent, England) were used for all experiments. All procedures performed were carried out by trained personnel in accordance with institutional guidelines and the UK Animals (Scientific Procedures) Act of 1986. The mesentery was excised and kept in ice-cold physiological salt solution30 until dissection. Mesenteric small arteries (<400-µm internal diameter) were cleaned of adjoining fat and connective tissues and equilibrated in culture medium (medium 199) for 45 minutes at 37°C before use in all subsequent stages. For each sample, 4 to 6 mesenteric small-artery arcades were used, yielding
50 to 80 µg protein.
Tissue Extracts for Western Blotting and p38MAPK In Vitro Kinase Assays
Small arteries were homogenized in ice-cold lysis buffer, and solubilized proteins were stored at -80°C. Preliminary experiments showed no difference in kinase activity between fresh and frozen samples (data not shown). p38MAPK activity in the extracts (40 µg protein quantified by the Bradford assay) was determined by using glutathioine S-transferase (GST)-MAPKAPK2 fusion protein bound to glutathione-Sepharose beads as a substrate.31 A control incubation without small-artery homogenate was included in each assay to measure MAPKAPK2 autophosphorylation. The reaction products were resolved on 10% SDS-PAGE and stained with Coomassie blue to confirm equivalent substrate loading. The gel was imaged, and radioactivity incorporated into GST-MAPKAPK2 was counted directly. Background signal due to MAPKAPK2 autophosphorylation constituted <13% of the total kinase activity and was corrected by subtraction from the sample counts. The data were expressed as fold change from control. Forty micrograms of the extract was used in the assay; to the remainder of the sample, SDS sample buffer (diluted 1:5 [vol/vol]) was added. The samples were boiled for 5 minutes and stored at -20°C.
Src Kinase Assay
Immunoprecipitates were prepared from small-artery homogenates by using anti–v-Src antibody (Clone 327, Oncogene Research Products). Src kinase activity in the immunoprecipitates was determined in 48 µL of kinase reaction buffer containing 0.25 mg/mL acid-denatured enolase, 20 µmol/L ATP, and 18 µCi [-32P]ATP at 30°C for 20 minutes. Reactions were stopped by the addition of SDS sample buffer and boiling. The incorporation of 32P into enolase was measured by direct counting after separation of the reaction products by SDS-PAGE and transfer to the nitrocellulose membrane. The presence of equivalent amounts of Src in the immunoprecipitates was confirmed by immunoblotting with the use of anti–c-Src (sc-18, Santa Cruz). The data were expressed as percent change from control.
HSP27 Kinase Activity Assay
Tissue extracts (40 µg protein) were incubated with GST-HSP27 fusion protein bound to glutathione-Sepharose beads in kinase buffer (25 mmol/L MOPS, 25 mmol/L ß-glycerophosphate, 15 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L NaF, 1 mmol/L vanadate, 4 mmol/L dithiothreitol, 40 µmol/L ATP, and 10 µCi [-32P]ATP, pH 7.2) in a final volume of 60 µL for 20 minutes at 30°C. The reaction was terminated by the addition of 0.5 mL ice-cold PBS (pH 7.0). The beads were washed twice with ice-cold PBS, resuspended in SDS sample buffer, boiled for 5 minutes, and processed as described for MAPKAPK2.
Immunodetection
Western blot analysis of tissue extracts (20 µg protein) was performed as previously described.30,32
Small-Artery Contraction
Contractile responses were determined from segments of small artery (320-µm internal diameter) by pressure myography, as described previously.33 Vessel segments were incubated with vehicle, 0.01% dimethyl sulfoxide (DMSO), 1 µmol/L SB203580, or 1 µmol/L SB202474 for 15 minutes before the recording of cumulative concentration-response curves to ET-1 (0.03 to 300 nmol/L) and NA (0.1 to 15 µmol/L). Lumen diameter was measured 2 minutes after the addition of the agonist, immediately before the addition of the next concentration. NA concentration-response curves in the presence of vehicle and inhibitor were obtained from a single vessel segment; for ET-1, individual segments were used for each treatment. In a second set of experiments, the contractile response to ET-1 in the absence of extracellular calcium was recorded. Vessel segments were incubated in calcium-free HEPES buffer containing 1 mmol/L EGTA for 15 minutes and stimulated twice with 15 µmol/L NA for 2 minutes to empty the intracellular calcium stores before the addition of 100 nmol/L ET-1. DMSO (0.01%), 1 µmol/L SB203580, or 1 µmol/L SB202474 was included throughout the protocol. The contractile response to ET-1 was recorded over a 10-minute period.
For detailed online methodologies, please see http://atvb.ahajournals.org.
Statistical Analysis
Comparisons between groups were made by Student t test or ANOVA with a Bonferroni correction for multiple comparisons; GraphPad Prism software was used. A value of P<0.05 was considered statistically significant.
Materials
Many of the reagents used were obtained from sources previously described.30,32 SB203580, SB202474, and ET-1 were purchased from Calbiochem. MAPKAPK2 (amino acid residues 46 to 400), a gift from Prof C. Marshall (Chester Beatty Laboratories, Institute of Cancer Research, London, UK), and HSP27, a gift from Prof Saklatvala (Kennedy Institute Rheumatology Division, London, UK), were expressed as GST fusion proteins in Escherichia coli and purified by using glutathione Sepharose 4B beads according to the manufacturer’s protocol (Pharmacia Biotech). The radioactivity in gels and on membranes was counted directly by using an InstantImager (Packard). Polyclonal p38MAPK and phospho-p38MAPK (Thr180/Tyr182) antibodies that recognize p38MAPK and p38MAPKß were a gift from New England Biolabs (Beverly, Mass). Polyclonal pan- and phospho-specific anti-SAPK3 and anti-SAPK4 antibodies were a gift from Prof Phil Cohen (MRC Protein Phosphorylation Unit, Dundee, UK).
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Results |
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Rat Mesenteric Small Arteries Express Multiple p38MAPK Isoforms
Immunochemical analysis showed the presence of p38
(RK/SAPK2a) and/or p38ß (SAPK2b), p38 (SAPK3), and p38 (SAPK4) in small-artery extracts (Figure 1A). The p38/ß band had an apparent molecular mass of 41 kDa, in agreement with canine smooth muscle p38MAPK.19 The molecular mass of SAPK3 (p38) and SAPK4 (p38) was 39 kDa.
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Vasoconstrictor Hormones Activate p38MAPK
Using antibodies that recognize activated p38MAPK when phosphorylated on Thr180 and Tyr182, we detected increased phosphorylation of p38 and/or p38ß after stimulation with 100 nmol/L ET-1 and 15 µmol/L NA (Figure 1B and 1C). There was no detectable change in SAPK3 or SAPK4 phosphorylation (data not shown). To further investigate the activation of p38MAPK, we used an in vitro pull-down assay with GST-MAPKAPK2 as a substrate that preferentially measures p38 and p38ß activity (p38/SAPK3 and p38/SAPK4 have low affinity for this substrate34). To confirm the specificity of this assay, we used immunoblot analysis of GST-MAPKAPK2 pull downs to show the presence of p38 and/or p38ß (Figure 1D); neither c-Jun N-terminal kinase 1/2 nor ERK1/2 was detected (data not shown). p38 and p38ß in GST-MAPKAPK2 pull downs from nonstimulated artery homogenates showed very low levels of phosphorylation (Figure 1D). In contrast, after agonist stimulation, there was a marked increase in phosphorylated p38 and/or p38ß (Figure 1D); reprobing of the membranes with anti-p38/ß antibody demonstrated equivalent amounts of kinase from both preparations (Figure 1D).
ET-1 (100 nmol/L) and NA (15 µmol/L) increased p38MAPK activity within 2 minutes of stimulation, and the response to both agonists had returned to basal levels after 30 minutes (Figure 2). ET-1 induced a biphasic response with an initial peak of activity at 2 minutes, followed by a second peak of equivalent magnitude at 10 minutes (Figure 2A). NA induced a single-phase response, with peak activity at 5 to 10 minutes (Figure 2B). The magnitude of the response, an 4-fold peak increase, was similar for both agonists (Figure 2).
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Activation of p38MAPK by Vasoconstrictors Is Dependent on Extracellular Calcium
Elevation of intracellular calcium is a major component of vasoconstrictor signaling. Therefore, we incubated vessels in calcium-free medium (0 mmol/L calcium/1 mmol/L EGTA-HEPES buffer) for 10 minutes before agonist stimulation. This treatment completely blocked p38MAPK activity induced by NA (Figure 2B). An early but statistically insignificant increase in activity was observed (Figure 2B), possibly reflecting incomplete removal of calcium or release from intracellular stores. In contrast, only the initial phase of p38MAPK activity in response to ET-1 was completely blocked by the removal of extracellular calcium, whereas the second phase was reduced by 50% (Figure 2A).
Activation of p38MAPK by Vasoconstrictors Is Dependent on Tyrosine Kinase Activity
In mesenteric small arteries, ET-1 induces a rapid increase in protein tyrosine phosphorylation that is blocked by tyrosine kinase inhibitors.30 Accordingly, we investigated whether the tyrosine kinase inhibitor genistein affected the response to ET-1. Fifteen minutes of preincubation with 100 µmol/L genistein inhibited p38MAPK activation by 100 nmol/L ET-1 (10 minutes). Daidzein, a negative analogue of genistein, slightly reduced basal p38MAPK activity but was without effect on ET-1–stimulated activity (p38MAPK activity as fold change from control, where control=1: ET-1 2.11±0.29-fold [P<0.05 compared with control by Student t test], genistein 1.15±0.1-fold, genistein+ET-1 1.62±0.07-fold, daidzein 0.77±0.01-fold [P<0.05 compared with control by Student t test], and daidzein+ET-1 2.60±0.39-fold [P<0.05 compared with control by Student t test]; mean±SEM, n=3).
Activation of Src Family Tyrosine Kinases by ET-1 and NA
Src tyrosine kinases are implicated in p38MAPK activation by GPCRs.21,35 As shown in Figure 3A and 3B, ET-1 (100 nmol/L) and NA (15 µmol/L) induced a rapid (detectable at 30 to 45 seconds) and transient (reduced below basal at 5 minutes) increase in Src kinase activity. Removal of extracellular calcium prevented Src activation by NA but not by ET-1 (Figure 3C).
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Effect of Herbimycin A on NA-Induced and ET-1–Induced p38MAPK Activity
The rapid activation of Src by NA and ET-1 suggested that these tyrosine kinases lie upstream from p38MAPK. Incubation with herbimycin A, an Src-selective inhibitor, blocked the first phase of p38MAPK activation in response to ET-1 but was without effect on the second phase (Figure 4A). In contrast, herbimycin A had no effect on the response to NA (Figure 4B). Herbimycin A completely inhibited ET-1–induced and NA-induced Src kinase activity (Figure 3C), confirming Src inhibition in our preparation. These results implicate a herbimycin A–sensitive tyrosine kinase in ET-1–induced but not NA-induced p38MAPK activation.
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Activation of HSP27 Kinase Activity by NA and ET-1
To determine whether HSP phosphorylation may occur in response to p38MAPK activation, we assayed for endogenous HSP27 kinase activity after NA and ET-1 stimulation. Kinase activity present in extracts from nonstimulated arteries was increased after 2 minutes of stimulation with NA and ET-1 (Figure 5). The basal and stimulated endogenous HSP27 kinase activities were inhibited by a 15-minute preincubation with 1 µmol/L SB203580, demonstrating that p38MAPK lay upstream from this activity (Figure 5A). SB202474, a negative analogue of SB203580, had no effect on HSP kinase activity (Figure 5A). Furthermore, herbimycin A (1 µmol/L) inhibited ET-1–induced HSP27 kinase activity but not NA-induced activity (Figure 5B), in agreement with effects of this inhibitor on p38MAPK activation (Figure 4Aand 4B).
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Role of p38MAPK Activation in Small-Artery Contraction
We have shown previously that tyrosine kinase activity is important for the tonic phase of ET-1–induced contraction in small arteries.30 Furthermore, our present observations that p38MAPK activation in response to agonists is dependent on tyrosine kinase activity and calcium influx suggests that this pathway may be involved in contraction. Accordingly, we studied the effect of p38MAPK inhibition on the contractile response of segments of rat mesenteric artery to NA and ET-1. In the presence of SB203580 (1 µmol/L), there was a significant rightward shift of the concentration response curve to NA (Figure 6A), an increase in the EC50 (for log EC50, NA+0.1% DMSO 1.61±0.26 µmol/L and NA+1 µmol/L SB203580 2.77±0.25, P<0.05; mean±SEM, n=5) and a reduction of maximum contraction (Figure 6A), implicating p38MAPK activity in NA-induced contraction. SB202474 was without effect (Figure 6B). The ET-1 concentration response curve was also shifted rightward in the presence of SB203580 and SB202474, but there was no reduction in maximum contraction (Figure 7A). Additionally, we determined whether either compound affected ET-1–stimulated contraction in the absence of extracellular calcium. ET-1 induced a small and sustained contraction in the absence of extracellular calcium, which was inhibited by SB202474 but not SB203580 (Figure 7B). These data show that the effect of SB202474 on ET-1–induced contraction is unlikely to be due to p38MAPK inhibition. That conclusion is supported by the fact that SB202474 does not directly inhibit p38MAPK activity in the in vitro kinase assay (for GST-MAPKAPK2 phosphorylation with use of 40 µg rat mesenteric artery homogenate, 0.01% DMSO 20±9 cpm, 1 µmol/L SB202474 35±15 cpm, and 1 µmol/L SB203580 0 cpm; mean±SEM, n=3) or inhibit basal or ET-1–induced HSP27 kinase activity (Figure 5A).
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Discussion |
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We have demonstrated that NA and ET-1 activate p38MAPK
and/or p38MAPKß in rat mesenteric arteries, leading to an increase in HSP kinase activity. Inhibition of this response with SB203580 inhibited NA-induced contraction but had little effect on the contractile response to ET-1. In addition, the mechanisms of p38MAPK activation differed between agonists, with NA having a requirement for calcium but not herbimycin A–sensitive tyrosine kinases and with ET-1 being partially dependent on extracellular calcium and tyrosine kinases.
Identification of 4 p38MAPK isoforms in rat mesenteric small arteries confirms and extends the observations of Hedges et al,19 who identified p38 in canine smooth muscle. The present study shows GPCR activation of p38 and/or p38ß isoforms in smooth muscle, with no detectable activation of the SAPK3/p38 or SAPK4/p38 isoforms. Activation of p38 and/or p38ß by NA and ET-1 occurred within 2 minutes and returned to baseline by 20 to 30 minutes. Similar rapid activation of p38MAPK by GPCR agonists has been observed in smooth muscle cells,20 tissue,19 and rat heart.18 However, the biphasic activation (by ET-1) observed in the present study has not been reported previously.
Removal of extracellular calcium completely inhibited NA activation of p38MAPK (Figure 2B), showing that this was a calcium-dependent response. In addition, NA activated Src tyrosine kinases in a rapid and calcium-dependent manner, suggesting that Src may be upstream from p38MAPK. However, inhibition of Src kinase with herbimycin A did not affect NA-induced p38MAPK activity, showing that this tyrosine kinase is not involved. There is evidence that calcium may regulate MAPK activity through a calmodulin-dependent protein kinase cascade. For instance, in PC12 and vascular smooth muscle cells, calmodulin is necessary for ERK activation by membrane depolarization and angiotensin II, respectively,36–38 and constitutively active calmodulin-kinase IV increased p38MAPK activity when expressed in PC12 cells.39 Further studies are required to show whether NA is acting through such a mechanism in smooth muscle.
ET-1 appeared to activate p38MAPK by at least 2 mechanisms: calcium-dependent and tyrosine kinase–dependent pathways. Removal of extracellular calcium completely inhibited the first phase of ET-1–induced p38MAPK activity and partially inhibited the second phase (Figure 2A). Similar to NA, ET rapidly activated Src, but this activation was independent of extracellular calcium; therefore, increased Src activity in the absence of calcium was not sufficient to activate p38MAPK. Herbimycin A inhibited Src and the early phase of p38MAPK activity in response to ET-1, suggesting that Src activity was required for a calcium-dependent step in the activation sequence. A possible candidate is the calcium-dependent cytosolic tyrosine kinase PYK2 (also known as RAFTK and CADTK).40 Recently, it has been shown in mesangial cells that Src activity is necessary for PYK2 and subsequent p38MAPK activation in response to ET-1.35 Furthermore, in rat intact small arteries, NA and ET-1 induce rapid tyrosine phosphorylation of PYK2 (J. and V. Ohanian, unpublished data, 2001), suggesting that PYK2 may be involved in GPCR activation of p38MAPK in smooth muscle. However, our data do not exclude the possibility that a herbimycin A–sensitive tyrosine kinase other than Src is involved in the early phase of ET-1–stimulated p38MAPK activity. The second phase of the response was partially dependent on calcium and tyrosine kinase activity, although not a herbimycin A–sensitive kinase. These data again implicate PYK2, although further experiments are required to confirm the involvement of this kinase.
Inhibition of p38MAPK activity with SB203580 reduced the sensitivity and the maximum contractile response of small-artery segments to NA. This observation is in agreement with a recent report in canine pulmonary artery showing that SB203580 inhibited phenylephrine-induced contraction.7 In contrast, SB203580 had only a minor effect on ET-1–induced contraction in the presence of calcium, and it was unclear whether this was due to p38MAPK inhibition or nonrelated effects of pyridinyl imidazoles, because SB202474, a negative analogue, inhibited ET-1–induced contraction with equal potency despite having no effect on endogenous HSP27 kinase activity or NA-induced contraction. These data demonstrate that the p38MAPK pathway is selectively involved in NA responses, although the mechanism by which p38MAPK might regulate contraction is not known. Recent evidence suggests that small HSPs, downstream effectors of p38MAPKs, are involved in smooth muscle contraction (HSP27) and relaxation (HSP20). HSP27 is strongly expressed in smooth muscle, and increased phosphorylation has been associated with smooth muscle contraction.9–11 The mechanism(s) by which HSP27 modulates contractility is not clear but may involve effects on the actin cytoskeleton, because increased HSP27 phosphorylation has been shown to affect actin microfilament dynamics41 and because changes in the association of HSP27 with actin have been observed during sustained smooth muscle contraction.42,43 In addition, there is evidence that phosphorylation of HSP20 is important for cyclic nucleotide–dependent smooth muscle relaxation44–46 and that phosphorylated HSP27 inhibits this mechanism,47 suggesting that the balance between these 2 HSPs may regulate the level of contraction. We detected in rat small-artery homogenates endogenous HSP27 kinase activity that was stimulated by NA and ET-1. The activation was dependent on p38MAPK activity, as shown by inhibition of the response by SB203580 and the differential sensitivity to herbimycin A. These observations suggest that HSP27 phosphorylation could occur in response to NA and ET-1 in intact small arteries. However, although SB203580 inhibited the increase in HSP27 kinase activity in response to both agonists, it inhibited only NA-induced contraction, suggesting that the role of p38MAPK in NA-induced contraction may be through a different mechanism (eg, regulation of calcium influx, as reported for bradykinin).48 Furthermore, these data show that HSP27 kinase activity and, presumably, HSP27 phosphorylation are not essential for small-artery contraction.
In summary, we have shown that calcium-dependent tyrosine kinase activity appears to be a major regulator of p38MAPK activation in intact small arteries, although ET-1 is able to activate additional mechanisms that are not dependent on calcium but are mediated by tyrosine kinases. Finally, we have shown that the p38MAPK pathway is selectively involved in the NA-induced constriction of small arteries.
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Acknowledgments |
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This study was funded by the British Heart Foundation. We are grateful to Dr Clare Austin for assistance with myography.
Received August 8, 2001; accepted August 23, 2001.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Brenner R, Perez GJ, Bonev AD, Eckman DR, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, Aldrich RW. Vasoregulation by the ß1 subunit of the calcium-activated potassium channel. Nature. 2000; 407: 870–876.[Medline] [Order article via Infotrieve]
2.
Horowitz A, Menice CB, Laporte R, Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev. 1996; 76: 967–1003.
3. Somlyo AP, Somlyo AV. From pharmacomechanical coupling to G-proteins and myosin phosphatase. Acta Physiol Scand. 1998; 164: 437–448.[Medline] [Order article via Infotrieve]
4. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997; 389: 990–994.[Medline] [Order article via Infotrieve]
5. Hughes AD, Wijetunge S. Role of tyrosine phosphorylation in excitation-contraction coupling in vascular smooth muscle. Acta Physiol Scand. 1998; 164: 457–470.[Medline] [Order article via Infotrieve]
6. Gerthoffer WT, Yamboliev IA, Pohl J, Haynes R, Dang S, McHugh J. Activation of MAP kinases in airway smooth muscle. Am J Physiol. 1997; 272: 244–252.
7. Yamboliev IA, Hedges JC, Mutnick JL, Adam LP, Gerthoffer WT. Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway. Am J Physiol. 2000; 278: H1899–H1907.
8.
Benjamin IJ, McMillan DR. Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res. 1998; 83: 117–132.
9. Bitar KN, Kaminski MS, Hailat N, Cease KB, Strahler JR. Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin. Biochem Biophys Res Commun. 1991; 181: 1192–1200.[Medline] [Order article via Infotrieve]
10. Larsen JK, Yamboliev IA, Weber LA, Gerthoffer WT. Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle. Am J Physiol. 1997; 273: L930–L940.[Medline] [Order article via Infotrieve]
11. Beall A, Epstein A, Woodrum D, Brophy CM. Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins. Biochim Biophys Acta. 1999; 1449: 41–49.[Medline] [Order article via Infotrieve]
12. Muller E, Burger-Kentischer A, Neuhofer W, Fraek ML, Marz J, Thurau K, Beck FX. Possible involvement of heat shock protein 25 in the angiotensin II-induced glomerular mesangial cell contraction via p38 MAP kinase. J Cell Physiol. 1999; 181: 462–469.[Medline] [Order article via Infotrieve]
13. Fuchs LC, Giulumian AD, Knoepp L, Pipkin W, Dickinson M, Hayles C, Brophy C. Stress causes decrease in vascular relaxation linked with altered phosphorylation of heat shock proteins. Am J Physiol. 2000; 279: R492–R498.
14. Udelsman R, Blake MJ, Stagg CA, Li DG, Putney DJ, Holbrook NJ. Vascular heat shock protein expression in response to stress: endocrine and autonomic regulation of this age-dependent response. J Clin Invest. 1993; 91: 465–473.[Medline] [Order article via Infotrieve]
15. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol. 1997; 9: 180–186.[Medline] [Order article via Infotrieve]
16. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature. 1994; 372: 739–745.[Medline] [Order article via Infotrieve]
17. Bogoyevitch MA, Andersson MB, Gillespie BJ, Clerk A, Glennon PE, Fuller SJ, Sugden PH. Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy. Biochem J. 1996; 314: 115–121.[Medline] [Order article via Infotrieve]
18. Lazou A, Sugden PH, Clerk A. Activation of mitogen-activated protein kinases (p38-MAPKs, SAPK/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart. Biochem J. 1998; 332: 459–465.[Medline] [Order article via Infotrieve]
19. Hedges JC, Yamboliev IA, Ngo M, Horowitz B, Adam LP, Gerthoffer WT. p38 mitogen-activated protein kinase expression and activation in smooth muscle. Am J Physiol. 1998; 275: C527–C534.[Medline] [Order article via Infotrieve]
20. Husain S, Abdellatif AA. Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2in cat iris sphincter smooth muscle cells. Biochem J. 1999; 342: 87–96.[Medline] [Order article via Infotrieve]
21.
Nagao M, Yamauchi J, Kaziro Y, Itoh H. Involvement of protein kinase C and Src family tyrosine kinase in Gq/11-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. J Biol Chem. 1998; 273: 22892–22898.
22. Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol. 1999; 11: 211–218.[Medline] [Order article via Infotrieve]
23.
Enslen H, Raingeaud J, Davis RJ. Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J Biol Chem. 1998; 273: 1741–1748.
24. Meloche S, Landry J, Huot J, Houle F, Marceau F, Giasson E. p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle. Am J Physiol. 2000; 279: H741–H751.
25.
Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, Gerthoffer WT. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem. 1999; 274: 24211–24219.
26.
Ito T, Kozawa O, Tanabe K, Niwa M, Matsuno H, Sakai N, Ito H, Kato K, Uematsu T. p38 MAP kinase is required for vasopressin-stimulated HSP27 induction in aortic smooth muscle cells. Hypertension. 2000; 35: 673–678.
27.
Ushio-Fukai M, Alexander RW, Akers M, Griendling KK. p38 mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II: role in vascular smooth muscle cell hypertrophy. J Biol Chem. 1998; 273: 15022–15029.
28. Kanda Y, Mizuno K, Kuroki Y, Watanabe Y. Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway. Br J Pharmacol. 2001; 132: 1657–1664.[Medline] [Order article via Infotrieve]
29.
Eguchi S, Dempsey PJ, Frank GD, Motley ED, Inagami T. Activation of MAPKs by angiotensin II in vascular smooth muscle cells: metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK. J Biol Chem. 2001; 276: 7957–7962.
30. Ohanian J, Ohanian V, Shaw L, Bruce C, Heagerty AM. Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric arteries. Brit J Pharmacol. 1997; 120: 653–661.[Medline] [Order article via Infotrieve]
31. Westwick JK, Brenner DA. Methods for analyzing c-jun kinase. Methods Enzymol. 1995; 255: 342–359.[Medline] [Order article via Infotrieve]
32. Ward DT, Ohanian J, Heagerty AM, Ohanian V. Phospholipase D-induced phosphatidate production in intact small arteries during noradrenaline stimulation: involvement of both G-protein and tyrosine phosphorylation linked pathways. Biochem J. 1995; 307: 451–456.[Medline] [Order article via Infotrieve]
33. Izzard AS, Bund SJ, Heagerty AM. Myogenic tone in mesenteric arteries from spontaneously hypertensive rats. Am J Physiol. 1996; 270: H1–H6.[Medline] [Order article via Infotrieve]
34. Kumar S, McDonnell PC, Gum RJ, Hand AT, Lee JC, Young PR. Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun. 1997; 235: 533–538.[Medline] [Order article via Infotrieve]
35.
Sorokin A, Kozlowski P, Graves L, Philip A. Protein-tyrosine kinase Pyk2 mediates endothelin-induced p38 MAPK activation in glomerular mesangial cells. J Biol Chem. 2001; 276: 21521–21528.
36. Egea J, Espinet C, Comella JX. Calmodulin modulates mitogen-activated protein kinase activation in response to membrane depolarization in PC12 cells. J Neurochem. 1998; 70: 2554–2564.[Medline] [Order article via Infotrieve]
37.
Egea J, Espinet C, Comella JX. Calcium influx activates extracellular-regulated kinase/mitogen-activated protein kinase pathway through a calmodulin-sensitive mechanism in PC12 cells. J Biol Chem. 1999; 274: 75–85.
38.
Eguchi S, Matsumoto T, Motley ED, Utsunomiya H, Inagami T. Identification of an essential signalling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. J Biol Chem. 1996; 271: 14169–14175.
39.
Enslen H, Tokumitsu H, Stork PJ, Davis RJ, Soderling TR. Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade. Proc Natl Acad Sci U S A. 1996; 93: 10803–10808.
40.
Pandey P, Avraham S, Kumar S, Nakazawa A, Place A, Ghanem L, Rana A, Kumar V, Majumder PK, Avraham H, et al. Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism. J Biol Chem. 1999; 274: 10140–10144.
41. Lavoie JN, Lambert H, Hickey E, Weber LA, Landry J. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Mol Cell Biol. 1995; 15: 505–516.[Abstract]
42. Wang P, Bitar KN. Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27. Am J Physiol. 1998; 275: G1454–G1462.[Medline] [Order article via Infotrieve]
43. Ibitayo AI, Sladick J, Tuteja S, Louis-Jacques O, Yamada H, Groblewski G, Welsh M, Bitar KN. HSP27 in signal transduction and association with contractile proteins in smooth muscle cells. Am J Physiol. 1999; 277: G445–G454.[Medline] [Order article via Infotrieve]
44.
Rembold CM, Foster DB, Strauss JD, Wingard CJ, Eyk JE. cGMP-mediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation without myosin light chain dephosphorylation in swine carotid artery. J Physiol. 2000; 524: 865–878.
45. Rembold CM, O’Connor M. Caldesmon and heat shock protein 20 phosphorylation in nitroglycerin- and magnesium-induced relaxation of swine carotid artery. Biochim Biophys Acta. 2000; 1500: 257–264.[Medline] [Order article via Infotrieve]
46. Woodrum DA, Brophy CM, Wingard CJ, Beall A, Rasmussen H. Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. Am J Physiol. 1999; 277: H931–H939.[Medline] [Order article via Infotrieve]
47. Fuchs LC, Giulumian AD, Knoepp L, Pipkin W, Dickinson M, Hayles C, Brophy C. Stress causes decrease in vascular relaxation linked with altered phosphorylation of heat shock proteins. Am J Physiol. 2000; 279: R492–R498.
48.
Wilk-Blaszczak MA, Stein B, Xu S, Barbosa MS, Cobb MH, Belardetti F. The mitogen-activated protein kinase p38-2 is necessary for the inhibition of N-type calcium current by bradykinin. J Neurosci. 1998; 18: 112–118.
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