Coupling of M2 muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle

2000 ◽  
Vol 278 (3) ◽  
pp. G429-G437 ◽  
Author(s):  
Amy K. Cook ◽  
Michael Carty ◽  
Cherie A. Singer ◽  
Ilia A. Yamboliev ◽  
William T. Gerthoffer
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Muscarinic Receptors ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Subsequent Treatment ◽  
P38 Map Kinase

Coupling of M2 and M3 muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M3 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 (Ser789), a putative downstream target of MAP kinases. Alkylation of M3 receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 μM 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 M2 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 M2receptor activation in smooth muscle.

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

2000 ◽  
Vol 278 (6) ◽  
pp. H1899-H1907 ◽  
Author(s):  
Ilia A. Yamboliev ◽  
Jason C. Hedges ◽  
Jack L.-M. Mutnick ◽  
Leonard P. Adam ◽  
William T. Gerthoffer
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Map Kinase ◽  
Map Kinases ◽  
Contractile Response ◽  
P38 Map Kinase ◽  
Hsp27 Phosphorylation ◽  
Sb 203580

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.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

2000 ◽  
Vol 278 (4) ◽  
pp. C718-C726 ◽  
Author(s):  
Jason C. Hedges ◽  
Brian C. Oxhorn ◽  
Michael Carty ◽  
Leonard P. Adam ◽  
Ilia A. Yamboliev ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Map Kinases ◽  
Isometric Force ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Tracheal Smooth Muscle ◽  
Muscarinic Agonists

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 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM 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 M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 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.

Activation of MAP kinases in airway smooth muscle

1997 ◽  
Vol 272 (2) ◽  
pp. L244-L252 ◽  
Author(s):  
W. T. Gerthoffer ◽  
I. A. Yamboliev ◽  
J. Pohl ◽  
R. Haynes ◽  
S. Dang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Airway Smooth Muscle ◽  
Map Kinases ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Mitogen Activated Protein ◽  
Intact Muscle ◽  
Triton X

To test the hypothesis that mitogen-activated protein (MAP) kinases are activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulated canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin basic protein kinase activities corresponding to ERK1 and ERK2 immunoreactive proteins were activated twofold above the basal level within 5 min by 1 microM carbachol. MAP kinase activity assayed in crude homogenates using a synthetic peptide substrate (APRTPGGRR) also increased twofold above basal in muscles stimulated with 1 microM carbachol. Two protein kinases separated by Mono-Q chromatography were identified on Western blots as ERK1 and ERK2 MAP kinases. Carbachol stimulation increased caldesmon phosphorylation in intact muscle, and purified caldesmon was a substrate for activated murine ERK2 MAP kinase. Activated ERK2 MAP kinase added to Triton X-100-permeabilized fibers potentiated Ca2+-induced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, which is consistent with coupling of MAP kinases to phosphorylation of caldesmon in vivo.

scholarly journals Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190

2015 ◽  
Vol 309 (7) ◽  
pp. C491-C500 ◽  
Author(s):  
Samantha Gardner ◽  
Sean M. Gross ◽  
Larry L. David ◽  
John E. Klimek ◽  
Peter Rotwein
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Cell Fusion ◽  
Muscle Cell ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
P38 Map Kinase ◽  
Myoblast Differentiation ◽  
Igf I ◽  
Muscle Biology

The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38α/β is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached ∼90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis.

PTHrP stimulated by the calcium-sensing receptor requires MAP kinase activation

2003 ◽  
Vol 284 (2) ◽  
pp. E435-E442 ◽  
Author(s):  
R. John MacLeod ◽  
Naibedya Chattopadhyay ◽  
Edward M. Brown
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Hormone Secretion ◽  
P38 Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Humoral Hypercalcemia Of Malignancy ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Hek Cells

Increases in extracellular calcium concentration ([Ca2+]o) stimulate from normal and malignant cells secretion of parathroid hormone-related protein (PTHrP), a major mediator of humoral hypercalcemia of malignancy. Because the calcium-sensing receptor (CaR) is a determinant of calcium-regulated hormone secretion, we examined whether HEK cells stably transfected with human CaR secreted PTHrP in response to CaR stimulation. Increases in [Ca2+]o or neomycin and Gd3+ all substantially increased PTHrP secretion in CaR-HEK cells but had no effect on nontransfected cells. CaR activation likewise increased PTHrP transcripts. PD-098059 and U-0126, inhibitors of the mitogen-activated protein kinase kinase MEK1/2, abolished CaR-stimulated secretion but had no effect on basal secretion. An inhibitor of p38 MAP kinase, SB-203580, also attenuated CaR-stimulated secretion. Western analysis revealed that CaR activation caused a robust increase in MEK1/2 and p38 MAP kinase phosphorylation. A Src family kinase inhibitor, PP2, blocked both basal and CaR-stimulated secretion. We conclude that CaR specifically mediates the effect of increasing [Ca2+]o on PTHrP synthesis and secretion and that activated MEK1/2 and p38 MAP kinases are determinants of the CaR's stimulation of PTHrP secretion.

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

1997 ◽  
Vol 273 (5) ◽  
pp. L930-L940 ◽  
Author(s):  
Janice K. Larsen ◽  
Ilia A. Yamboliev ◽  
Lee A. Weber ◽  
William T. Gerthoffer
Keyword(s):  
Smooth Muscle ◽  
Stress Response ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Airway Smooth Muscle ◽  
Muscarinic Receptors ◽  
P38 Map Kinase ◽  
Mitogen Activated Protein

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 increased32P 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.

scholarly journals Monocyte-derived Prostaglandin E2 inhibits antigen-specific cutaneous immunity during ageing

2020 ◽  
Author(s):  
Emma S Chambers ◽  
Milica Vukmanovic-Stejic ◽  
Barbara B Shih ◽  
Hugh Trahair ◽  
Priya Subramanian ◽  
...  
Keyword(s):  
Older Adults ◽  
Prostaglandin E2 ◽  
Map Kinase ◽  
Kinase Inhibitor ◽  
Tissue Injury ◽  
P38 Map Kinase ◽  
Prostaglandin E ◽  
Varicella Zoster ◽  
Cutaneous Immunity

AbstractAgeing results in a decline in immune function. We showed previously that healthy older humans (>65 years old) have reduced antigen-specific cutaneous immunity to varicella zoster virus (VZV) antigen challenge. This was associated with p38 MAP kinase driven inflammation that was induced by mild tissue injury caused by the injection of the antigen itself. Here we show that non-specific injury induced by injection of air or saline into the skin of older adults recruits CCR2+CD14+ monocytes by CCL2 produced by senescent fibroblasts. These monocytes reduced TRM proliferation via secretion of prostaglandin E2 (PGE2). Pre-treatment with a p38-MAPK inhibitor (Losmapimod) in older adults in vivo significantly decreased CCL2 expression, recruitment of monocyte into the skin, COX2 expression and PGE2 production. This enhanced the VZV response in the skin. Therefore, local inflammation arising from interaction between senescent cells and monocytes leads to immune decline in the skin during ageing, a process that can be reversed.SummaryInflammation resulting from tissue injury blocks antigen-specific cutaneous immunity during ageing. Monocytes recruited to the skin inhibit TRM function through COX2-derived prostaglandin E2 production. Blocking inflammation and resulting prostaglandin E2 production with a p38-MAP kinase inhibitor significantly enhances cutaneous antigen-specific responses.

Spontaneous shift in transcriptional profile of explanted glomeruli via activation of the MAP kinase family

2000 ◽  
Vol 279 (5) ◽  
pp. F954-F959 ◽  
Author(s):  
Yoshihisa Ishikawa ◽  
Tsuneo Konta ◽  
Masanori Kitamura
Keyword(s):  
Gene Expression ◽  
Map Kinase ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
P38 Map Kinase ◽  
Transcriptional Profile ◽  
Activator Protein 1 ◽  
Monocyte Chemoattractant Protein 1 ◽  
Mitogen Activated Protein

To understand how isolation and explantation of glomeruli affect the function of resident cells, the present study investigated the transcriptional profile of explanted normal glomeruli. We found that ex vivo incubation of glomeruli spontaneously expressed monocyte chemoattractant protein-1 (MCP-1) and stromelysin, the genes regulated by activator protein-1 (AP-1). The expression was suppressed by heparin and quercetin, the drugs with anti-AP-1 activities. The gene expression was preceded by 1) induction of AP-1 components c- fos and c- jun and 2) phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein (MAP) kinase, and c-Jun NH2-terminal kinase (JNK), the upstream inducers/activators of AP-1. Suppression of ERK by PD098059 abrogated induction of c- fos and c- jun, and the p38 MAP kinase inhibitor SB203580 attenuated c- fos expression. Furthermore, treatment with either PD098059, SB203580, or the JNK-AP-1 inhibitor curcumin diminished the expression of MCP-1 and stromelysin. The transcriptional profile of glomerular cells thus alters dramatically after explantation of glomeruli. It is, at least in part, due to activation of multiple MAP kinases that lead to induction of AP-1-dependent gene expression.

scholarly journals Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists

2002 ◽  
Vol 283 (6) ◽  
pp. H2282-H2287 ◽  
Author(s):  
Michael P. Massett ◽  
Zoltan Ungvari ◽  
Anna Csiszar ◽  
Gabor Kaley ◽  
Akos Koller
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Map Kinase ◽  
Map Kinases ◽  
P38 Map Kinase ◽  
Intraluminal Pressure ◽  
Myogenic Tone ◽  
Ang Ii ◽  
Wall Tension ◽  
Sb 203580

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinases have been implicated in the modulation of agonist-induced contractions of large vessels. However, their role in pressure- and agonist-induced constrictions of skeletal muscle arterioles, which have a major role in regulating peripheral resistance, is not clearly elucidated. Thus constrictions of isolated rat gracilis muscle arterioles (∼80 μm in diameter) to increases in intraluminal pressure and to norepinephrine (NE) or angiotensin II (ANG II) were assessed in the absence or presence of chelerythrine, PD-98058, and SB-203580 (inhibitors of PKC, p42/44 and p38 MAP kinase pathways, respectively). Arteriolar constriction to NE and ANG II were significantly reduced by chelerythrine (by ∼90%) and unaffected by SB-203580, whereas PD-98058 decreased only ANG II-induced constrictions (by ∼60%). Pressure-induced increases in wall tension (from 0.1 to 0.7 N/m) resulted in significant arteriolar constrictions (50% maximum) that were abolished by chelerythrine without altering smooth muscle intracellular Ca2+ concentration ([Ca2+]i) (fura 2 microfluorimetry). PD-98058 and SB-203580 significantly decreased the magnitude of myogenic tone (by 20% and 60%, respectively) and reduced the sensitivity of the myogenic mechanism to wall tension, causing a significant rightward shift in the wall tension-myogenic tone relationship without affecting smooth muscle [Ca2+ i]. MAP kinases were demonstrated with Western blotting. Thus in skeletal muscle arterioles 1) PKC is involved in both myogenic and agonist-induced constrictions , 2) PD-98058-sensitive p42/44 MAP kinases modulate both wall tension-dependent and ANG II-induced constrictions, whereas 3) a SB-203580-sensitive p38 MAP kinase pathway seems to be specifically involved in the mechanotransduction of wall tension.

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