MEK modulates force-fluctuation-induced relengthening of canine tracheal smooth muscle.

Tidal breathing, and especially deep breathing, is known to antagonise bronchoconstriction caused by airway smooth muscle (ASM) contraction; however, this bronchoprotective effect of breathing is impaired in asthma. Force fluctuations applied to contracted ASM in vitro cause it to relengthen, force-fluctuation-induced relengthening (FFIR). Given that breathing generates similar force fluctuations in ASM, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. Thus it is of considerable interest to understand what modulates FFIR, and how ASM might be manipulated to exploit this phenomenon. It was demonstrated previously that p38 mitogen-activated protein kinase (MAPK) signalling regulates FFIR in ASM strips. Here, it was hypothesised that the MAPK kinase (MEK) signalling pathway also modulates FFIR. In order to test this hypothesis, changes in FFIR were measured in ASM treated with the MEK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Increasing concentrations of U0126 caused greater FFIR. U0126 reduced extracellular signal-regulated kinase 1/2 phosphorylation without affecting isotonic shortening or 20-kDa myosin light chain and p38 MAPK phosphorylation. However, increasing concentrations of U0126 progressively blunted phosphorylation of high-molecular-weight caldesmon (h-caldesmon), a downstream target of MEK. Thus changes in FFIR exhibited significant negative correlation with h-caldesmon phosphorylation. The present data demonstrate that FFIR is regulated through MEK signalling, and suggest that the role of MEK is mediated, in part, through caldesmon.

Identification of capsaicin-sensitive rectal mechanoreceptors activated by rectal distension in mice.

Rodents detect visceral pain in response to noxious levels of rectal distension. However, the mechanoreceptors that innervate the rectum and respond to noxious levels of rectal distension have not been identified. Here, we have identified the mechanoreceptors of capsaicin-sensitive rectal afferents and characterized their properties in response to circumferential stretch of the rectal wall. We have also used the lethal spotted (ls/ls) mouse to determine whether rectal mechanoreceptors that respond to capsaicin and stretch may also develop in an aganglionic rectum that is congenitally devoid of enteric ganglia. In wild type (C57BL/6) mice, graded increases in circumferential stretch applied to isolated rectal segments activated a graded increase in firing of slowly-adapting rectal mechanoreceptors. Identical stimuli applied to the aganglionic rectum of ls/ls mice also activated similar graded increases in firing of stretch-sensitive rectal afferents. In both wild type and aganglionic rectal preparations, focal compression of the serosal surface using von Frey hairs identified mechanosensitive "hot spots," that were associated with brief bursts of action potentials. Spritzing capsaicin (10 microM) selectively onto each identified mechanosensitive hot spot activated an all or none discharge of action potentials in 32 of 56 identified hot spots in wild type mice and 24 of 62 mechanosensitive hot spots in the aganglionic rectum of ls/ls mice. Each single unit activated by both capsaicin and circumferential stretch responded to low mechanical thresholds (1-2 g stretch). No high threshold rectal afferents were ever recorded in response to circumferential stretch. Anterograde labeling from recorded rectal afferents revealed two populations of capsaicin-sensitive mechanoreceptor that responded to stretch: one population terminated within myenteric ganglia, the other within the circular and longitudinal smooth muscle layers. In the aganglionic rectum of ls/ls mice, only the i.m. mechanoreceptors were identified. Both myenteric and i.m. mechanoreceptors could be identified by their immunoreactivity to the anti-TRPV1 antibody and the vesicular glutamate transporter, Vglut2. Myenteric mechanoreceptors had a unique morphology, consisting of smooth bulbous nodules that ramified within myenteric ganglia. In summary, the rectum of wild type mice is innervated by at least two populations of capsaicin-sensitive rectal mechanoreceptor, both of which respond to low mechanical thresholds within the innocuous range. These findings suggest that the visceral pain pathway activated by rectal distension is likely to involve low threshold rectal mechanoreceptors that are activated within the normal physiological range.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle.

We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of +/-16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 +/- 27.6% Lref and hysteresivity by 31.8 +/- 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 +/- 5.2% Lref and hysteresivity by 4.14 +/- 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.

ERK inhibition attenuates 5-HT-induced contractions in fetal and adult ovine carotid arteries.

Growth and differentiation-related pathways are much more active in immature than in mature, fully differentiated smooth muscle. Because mitogen-activated protein kinases (MAPK) are intimately involved with growth and differentiation, and the extracellular signal-regulated kinase (ERK) subfamily of MAPKs are involved in some contractile responses, the present studies examined the hypothesis that ERKs play an important and age-dependent role in smooth muscle contraction. The MAPK inhibitors PD098059 and UO126 both inhibited serotonin (5-HT) concentration-response relations more effectively in carotid arteries from term fetal lambs, than in corresponding arteries from mature non-pregnant adult sheep. This inhibition involved significant decreases in both the pD2 (adult: 2-fold; fetus: 4- to 15-fold) and the maximum efficacy (adult: 15-19%; fetus: 34-39%) of 5-HT. Accompanying this age-dependent effect on contraction, quantitative Western blot assays revealed that ERK1 and ERK2 abundances were 39% and 164% greater, respectively, in fetal than in adult carotid arteries. The abundance of the putative ERK target, caldesmon, however, was about 7-fold greater in adult than in fetal arteries. Together, the present results support the view that ERK abundance and activity is upregulated in fetal relative to adult arteries, and that one consequence of this upregulation is that the contribution of ERKs to contraction, at least that initiated by 5-HT2a receptors, is greater in fetal than adult carotid arteries. Whereas the phosphorylation mechanisms through which ERKs augment contraction remain uncertain and controversial, the present results suggest that emphasis should be shifted away from caldesmon and toward other critical contractile proteins, and how these proteins may contribute differently to development of agonist-induced contractile force in immature and mature arteries.

Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.

Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of sphingosine 1-phosphate (Sph-1-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-1-P produced rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) across both human and bovine pulmonary artery and lung microvascular endothelial cells. This substance also reversed barrier dysfunction elicited by the edemagenic agent thrombin. Sph-1-P-mediated barrier enhancement was dependent upon G(ialpha)-receptor coupling to specific members of the endothelial differentiation gene (Edg) family of receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P-enhanced TER occurred in conjunction with Rac GTPase- and p21-associated kinase-dependent endothelial cortical actin assembly with recruitment of the actin filament regulatory protein, cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability.

PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs.

Pulmonary artery smooth muscle cell (PASMC) adhesion, spreading, and migration depend on matrix-stimulated reorganization of focal adhesions. Platelet-derived growth factor (PDGF) activates intracellular signal transduction cascades that also regulate adhesion, spreading, and migration, but the signaling molecules involved in these events are poorly defined. We hypothesized that phosphatidylinositol (PI) 3-kinases and Src tyrosine kinases translate matrix and PDGF-initiated signals into cell motility. In experiments with cultured canine PASMCs, inhibition of PI 3-kinases with wortmannin (0.3 microM) and LY-294002 (50 microM) and of Src kinase with PP1 (30 microM) did not decrease spontaneous (nonstimulated) or PDGF-stimulated (10 ng/ml) adhesion onto collagen. PI 3-kinase and Src kinase activities, however, were necessary for cell spreading: PP1 inhibited cell spreading and Src Tyr-418 phosphorylation in a concentration-dependent manner. Inhibition of PI 3-kinase and Src partially reduced cell migration, while at 10 and 30 microM, PP1 eliminated migration, likely due to inhibition of PDGF receptors. In conclusion, both PI 3-kinases and Src tyrosine kinases are components of pathways that mediate spreading and migration of cultured PASMCs on collagen.

Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle.

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.

p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 Mapk.

Cell migration contributes to many physiological processes and requires dynamic changes in the cytoskeleton. These migration-dependent cytoskeletal changes are partly mediated by p21-activated protein kinases (PAKs). At least four closely related isoforms, PAK1, PAK2, PAK3, and PAK4, exist in mammalian cells. In smooth muscle cells, little is known about the expression, activation, or ability of PAKs to regulate migration. Our study revealed the existence of three PAK isoforms in cultured tracheal smooth muscle cells (TSMCs). Additionally, we constructed adenoviral vectors encoding wild type and a catalytically inactive PAK1 mutant to investigate PAK activation and its role in TSMC migration. Stimulation of TSMCs with platelet-derived growth factor (PDGF) increased the activity of PAK1 over time. Overexpression of mutant PAK1 blocked PDGF-induced chemotactic cell migration. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) in cells overexpressing wild-type PAK1 was similar to vector controls; however, p38 MAPK phosphorylation was severely reduced by overexpression of the PAK1 mutant. Collectively, these results suggest a role for PAK1 in chemotactic TSMC migration that involves catalytic activity and may require signaling to p38 MAPK among other pathways.

Modulatory role of ERK MAPK-caldesmon pathway in PDGF-stimulated migration of cultured pulmonary artery SMCs.

Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) phosphorylate caldesmon in vivo, but the function of caldesmon phosphorylation in smooth muscle physiology is controversial. We hypothesized that ERK MAPKs and caldesmon modulate chemotactic migration of cultured canine pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF; 10 ng/ml) and endothelin-1 (ET-1; 100 nM) transiently activated ERK MAPKs: PDGF produced higher maximal and more potent activation of ERK MAPKs over 5 h. While both PDGF and ET-1 increased caldesmon phosphorylation, only PDGF stimulated migration of cultured cells (13 times over basal migration). At concentrations from 0.01 to 10 nM, ET-1 failed to enhance migration; 100 nM ET-1 produced only a slight increase (1.31 +/- 0.18 times basal migration). ET-1 (100 nM) did not potentiate migration triggered by 0.5 or 3 ng/ml PDGF. The MEK1 inhibitor PD-98059 (50 microM) abolished the PDGF-stimulated phosphorylation of ERK MAPKs and caldesmon and reduced cell migration by 50%. We conclude that while ERK MAPK activity is not required to initiate migration, an ERK MAPK-caldesmon pathway may modulate later events necessary for PDGF-stimulated migration of cultured PASMCs.

Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle.

In canine colon, M2/M3 muscarinic receptors are coupled to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. We tested the hypothesis that this coupling is mediated by enzymes of the phosphatidylinositol (PI) 3-kinase family. RT-PCR and Western blotting demonstrated expression of two isoforms, PI 3-kinase-alpha and PI 3-kinase-gamma. Muscarinic stimulation of intact muscle strips (10 microM ACh) activated PI 3-kinase-gamma, ERK and p38 MAP kinases, and MAP kinase-activated protein kinase-2, whereas PI 3-kinase-alpha activation was not detected. Wortmannin (25 microM) abolished the activation of PI 3-kinase-gamma, ERK, and p38 MAP kinases. MAP kinase inhibition was a PI 3-kinase-gamma-specific effect, since wortmannin did not inhibit recombinant activated murine ERK2 MAP kinase, protein kinase C, Raf-1, or MAP kinase kinase. In cultured muscle cells, newborn calf serum (3%) activated PI 3-kinase-alpha and PI 3-kinase-gamma isoforms, ERK and p38 MAP kinases, and stimulated chemotactic cell migration. Using wortmannin and LY-294002 to inhibit PI 3-kinase activity and PD-098059 and SB-203580 to inhibit ERK and p38 MAP kinases, we established that these enzymes are functionally important for regulation of chemotactic migration of colonic myocytes.

Mitogen-activated protein kinases regulate cytokine gene expression in human airway myocytes.

The signal transduction pathways regulating smooth-muscle gene expression and production of cytokines in response to proinflammatory mediators are undefined. Cultured human bronchial smooth-muscle cells were treated for 20 h with a cytokine cocktail containing interleukin (IL)-1beta, tumor necrosis factor-alpha, and interferon-gamma. A complementary DNA expression array containing 588 genes was used to follow cytokine-stimulated gene expression. The expression and secretion of the cytokines IL-1beta, IL-6, and IL-8 significantly increased after 20 h of stimulation as measured by relative reverse transcriptase/ polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting techniques. Expression of IL-6 and IL-8 was sensitive to SB203580, the specific inhibitor of p38 mitogen-activated protein (MAP) kinase and PD98059, an inhibitor of MAP kinase kinase. Expression of IL-1beta was sensitive only to PD98059. Together, these results demonstrate that the p38 and extracellular signal-regulated protein kinase MAP kinase pathways are required for proinflammatory mediator- induced cytokine expression in airway myocytes. The generation of chemokines and cytokines in airway smooth muscle also provides evidence that smooth-muscle cells have the ability to contribute to the inflammatory response.

Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway.

Mitogen-activated protein (MAP) kinases signal to proteins that could modify smooth muscle contraction. Caldesmon is a substrate for extracellular signal-related kinases (ERK) and p38 MAP kinases in vitro and has been suggested to modulate actin-myosin interaction and contraction. Heat shock protein 27 (HSP27) is downstream of p38 MAP kinases presumably participating in the sustained phase of muscle contraction. We tested the role of caldesmon and HSP27 phosphorylation in the contractile response of vascular smooth muscle by using inhibitors of both MAP kinase pathways. In intact smooth muscle, PD-098059 abolished endothelin-1 (ET-1)-stimulated phosphorylation of ERK MAP kinases and caldesmon, but p38 MAP kinase activation and contractile response remained unaffected. SB-203580 reduced muscle contraction and inhibited p38 MAP kinase and HSP27 phosphorylation but had no effect on ERK MAP kinase and caldesmon phosphorylation. In permeabilized muscle fibers, SB-203580 and a polyclonal anti-HSP27 antibody attenuated ET-1-dependent contraction, whereas PD-098059 had no effect. These results suggest that ERK MAP kinases phosphorylate caldesmon in vivo but that activation of this pathway is unnecessary for force development. The generation of maximal force may be modulated by the p38 MAP kinase/HSP27 pathway.

Coupling of M(2) muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle.

Coupling of M(2) and M(3) muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M(3) receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser(789)), a putative downstream target of MAP kinases. Alkylation of M(3) receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 microM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M(2) receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M(2) receptor activation in smooth muscle.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle.

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 microM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 microM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M(2) antagonist AF-DX 116 (1 microM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 +/- 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M(2) receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

Maturation attenuates the effects of cGMP on contraction, [Ca2+]i and Ca2+ sensitivity in ovine basilar arteries.

The present study explores the hypothesis that age-related variations in cerebrovascular responses to vasodilators reflect corresponding age-dependent differences in the mechanisms coupling changes in cytosolic cGMP to vasorelaxation. The experiments focused on cGMP's ability to decrease either [Ca2+]i or myofilament Ca2+ sensitivity, because both effects can contribute to cGMP-induced vasodilation. Use of the cGMP analog 8-pCPT-cGMP minimized problems associated with limited cell permeation or cGMP hydrolysis. In fetal basilars contracted with 10 microM serotonin, the EC30 for 8-pCPT-cGMP-induced relaxation was 6 microM. In fura-2 loaded fetal basilars, pretreatment with 6 microM 8-pCPT-cGMP significantly depressed the sensitivity of [Ca2+]i to 5HT, and also myofilament sensitivity to calcium, but only in fetal arteries. In fetal basilar arteries contracted with 120 mM potassium, the EC30 for 8-pCPT-cGMP-induced relaxation was 25 microM. In fura-2 loaded ovine arteries, pretreatment with 25 microM 8-pCPT-cGMP had no effect on the ability of graded concentrations of potassium to elevate [Ca2+]i but reduced potassium's ability to induce contraction and attenuated myofilament calcium sensitivity; these latter effects were significant only in fetal arteries. In alpha-toxin permeabilized preparations, 25 microM 8-pCPT-cGMP significantly depressed both basal- and agonist-stimulated myofilament calcium sensitivity, only in fetal but not in adult basilars. Together, these results demonstrate that: (1) sensitivity to cGMP is greater in fetal than adult sheep arteries independent of method of contraction; (2) cGMP can reduce [Ca2+]i but only in agonist-contracted and not in potassium-contracted arteries; (3) and cGMP attenuates myofilament calcium sensitivity regardless of method of contraction. Overall, the data demonstrate that variations in the ability of cGMP to produce vasodilatation reflect age-, artery-, and agonist-dependent differences in the combination of mechanisms mediating responses to cGMP.

A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration.

Smooth muscle cells are exposed to growth factors and cytokines that contribute to pathological states including airway hyperresponsiveness, atherosclerosis, angiogenesis, smooth muscle hypertrophy, and hyperplasia. A common feature of several of these conditions is migration of smooth muscle beyond the initial boundary of the organ. Signal transduction pathways activated by extracellular signals that instigate migration are mostly undefined in smooth muscles. We measured migration of cultured tracheal myocytes in response to platelet-derived growth factor, interleukin-1beta, and transforming growth factor-beta. Cellular migration was blocked by SB203580, an inhibitor of p38(MAPK). Time course experiments demonstrated increased phosphorylation of p38(MAPK). Activation of p38(MAPK) resulted in the phosphorylation of HSP27 (heat shock protein 27), which may modulate F-actin polymerization. Inhibition of p38(MAPK) activity inhibited phosphorylation of HSP27. Adenovirus-mediated expression of activated mutant MAPK kinase 6b(E), an upstream activator for p38(MAPK), increased cell migration, whereas overexpression of p38alpha MAPK dominant negative mutant and an HSP27 phosphorylation mutant blocked cell migration completely. The results indicate that activation of the p38(MAPK) pathway by growth factors and proinflammatory cytokines regulates smooth muscle cell migration and may contribute to pathological states involving smooth muscle dysfunction.

p38 mitogen-activated protein kinase expression and activation in smooth muscle.

There is relatively little known about expression and activation of p38 mitogen-activated protein kinases (MAPKs) through G protein-linked, seven-transmembrane-spanning (STM) receptors in mammalian smooth muscle. To investigate the role of p38 MAPK in smooth muscle, we cloned and sequenced the p38 MAPK expressed in canine smooth muscles. A full-length clone of the canine p38 MAPK expressed in colonic smooth muscle was obtained by RT-PCR. The deduced amino acid sequence revealed 99% identity to the human p38 MAPK and differed from the human enzyme in only two conservative substitutions. The deduced molecular mass of the canine p38 MAPK is 41.2 kDa, with a calculated isoelectric point of 5.41. Canine p38 MAPK was found to be expressed in colonic, tracheal, and vascular smooth muscles and underwent increased tyrosine phosphorylation in response to motor neurotransmitters, acetylcholine (ACh) and neurokinin A (NKA), in colonic smooth muscle. There was an eightfold increase in p38 MAPK phosphorylation after a 10-min incubation with ACh and a threefold increase with NKA. We also identified a p38 immunoreactive kinase activity isolated from colonic smooth muscle homogenate by Mono Q chromatography. Partially purified p38 MAPK and activated recombinant p38 MAPK (Mpk2) phosphorylated both the known p38 MAPK substrate ATF2, as well as porcine stomach h-caldesmon in vitro. The results suggest that elements of the "stress-response" pathway may be coupled to transcriptional control as well as to cytoskeletal and possibly contractile protein phosphorylation in mammalian smooth muscle.

Endothelin-1 activates MAP kinases and c-Jun in pulmonary artery smooth muscle.

We tested the coupling of endothelin receptors to mitogen-activated protein kinases (MAPK) and nuclear factor c-Jun in intact canine pulmonary artery smooth muscle. Muscle rings denuded of endothelium were stimulated with 10(-7)M ET-1 and frozen during contraction. An <> kinase assay with myelin basic protein as substrate revealed protein kinase activities at 98, 75, 55, 50, 44 and 40 kDa. Erk1 and Erk2 MAPK were activated by ET-1 to 5.4+/-0.97 and 4.03+/-1. 54 times basal activity at 10 min. Using phospho-specific antibodies, we found increased threonine/tyrosine phosphorylation of p38 and JNK1 MAPK to 2.04+/-0.47 and 2.56+/-0.72 times basal. ET-1 increased the phosphorylation level of nuclear factor c-Jun with a time-course closely matching the activation of JNK1 and p38 MAPK. Therefore, endothelin receptors initiate intracellular signals leading to activation of Erk, p38 and JNK1 MAPK pathways and ultimately to nuclear targets. The activation of JNK1 MAPK seems closely related to the phosphorylation of nuclear transcription factor c-Jun.

Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle.

The 27-kDa heat shock protein (HSP27) is expressed in a variety of tissues in the absence of stress and is thought to regulate actin filament dynamics, possibly by a phosphorylation/dephosphorylation mechanism. HSP27 has also been suggested to be involved in contraction of intestinal smooth muscle. We have investigated phosphorylation of HSP27 in airway smooth muscle in response to the muscarinic agonist carbachol. Carbachol increased 32P incorporation into canine tracheal HSP27 and induced a shift in the distribution of charge isoforms on two-dimensional gels to more acidic, phosphorylated forms. The canine HSP27 amino acid sequence includes three serine residues corresponding to sites in human HSP27 known to be phosphorylated by mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2. To determine whether muscarinic receptors are coupled to a "stress response" pathway in smooth muscle culminating in phosphorylation of HSP27, we assayed MAPKAP kinase-2 activity and tyrosine phosphorylation of p38 mitogen-activated protein (MAP) kinase, the enzyme thought to activate MAPKAP kinase-2. Recombinant canine HSP27 expressed in Escherichia coli was a substrate for MAPKAP kinase-2 in vitro as well as a substrate for endogenous smooth muscle HSP27 kinase, which was activated by carbachol. Carbachol also increased tyrosine phosphorylation of p38 MAP kinase. SB-203580, an inhibitor of p38 MAP kinases, reduced activation of endogenous HSP27 kinase activity and blocked the shift in HSP27 charge isoforms to acidic forms. We suggest that HSP27 in airway smooth muscle, in addition to being a stress response protein, is phosphorylated by a receptor-initiated signaling cascade involving muscarinic receptors, tyrosine phosphorylation of p38 MAP kinase, and activation of MAPKAP kinase-2.