Ilia A. Yamboliev

A Role for p38MAPK/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-1β, and transforming growth factor-β. Cellular migration was blocked by SB203580, an inhibitor of p38MAPK. Time course experiments demonstrated increased phosphorylation of p38MAPK. Activation of p38MAPK resulted in the phosphorylation of HSP27 (heat shockprotein 27), which may modulate F-actin polymerization. Inhibition of p38MAPK activity inhibited phosphorylation of HSP27. Adenovirus-mediated expression of activated mutant MAPK kinase 6b(E), an upstream activator for p38MAPK, increased cell migration, whereas overexpression of p38α MAPK dominant negative mutant and an HSP27 phosphorylation mutant blocked cell migration completely. The results indicate that activation of the p38MAPK pathway by growth factors and proinflammatory cytokines regulates smooth muscle cell migration and may contribute to pathological states involving smooth muscle dysfunction.

Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle.

1. Phosphorylation of caldesmon was assayed in canine colonic circular smooth muscle strips labelled with 32P and stimulated with 10 microM acetylcholine. Caldesmon was isolated by two‐dimensional non‐equilibrium pH gel electrophoresis. Stimulation with acetylcholine increased caldesmon phosphorylation significantly from a basal level of 0.6 +/‐ 0.07 to 1.1 +/‐ 0.15 mol P1 (mol caldesmon)‐1 after 2 min. 2. MAP kinase activities were measured in SDS extracts of muscle by a gel reconstitution method using myelin basic protein. Myelin basic protein kinase activities were observed at 38, 44, 50 and 57 kDa by the gel reconstitution method. Endogenous caldesmon kinase activities were also identified by the gel reconstitution method at 38, 44 and 50 kDa. The 38 and 44 kDa kinases comigrated with proteins labelled by anti‐ERK1 MAP kinase antibodies on Western blots. Both 38 and 44 kDa MBP kinase activities increased significantly during contractions induced by 10 microM acetylcholine, 0.1 microM neurokinin A and 70 mM potassium. 3. Phorbol dibutyrate (0.1 microM) potentiated activation of MAP kinases and contraction of depolarized muscles while producing a decrease in fura‐2 fluorescence ratio. This suggests that protein kinase C activation is coupled to MAP kinase activity in colonic smooth muscle. 4. MAP kinases isolated form muscle homogenates by Mono Q chromatography were assayed using the specific MAP kinase substrate peptide APRTPGGRR. Stimulation of muscles for 2 min with 10 microM acetylcholine activated both ERK1 and ERK2 MAP kinase activities 2‐fold. 5. To determine the effects of caldesmon phosphorylation by MAP kinase on the cross‐bridge cycle, actin sliding velocity was measured with an in vitro motility assay. Unphosphorylated turkey gizzard caldesmon (3 microM) significantly reduced mean sliding velocity. Phosphorylation of caldesmon with sea star ERK1 MAP kinase reversed the inhibitory effect of caldesmon on sliding velocity. The results are consistent with a protein kinase cascade being activated by contractile agonists in gastrointestinal smooth muscle which activates ERK MAP kinases leading to phosphorylation of caldesmon. Phosphorylation of caldesmon in vivo may reverse inhibitory influences of caldesmon on cross‐bridge cycling.

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

1 Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈30 min completely inhibited expressed guinea‐pig ClC‐3 currents (IgpClC‐3), while intracellular dialysis with antigen‐preabsorbed anti‐ClC‐3 Ab failed to affect IgpClC‐3. 2 Anti‐ClC‐3 Ab was used as a selective probe to examine the relationship between endogenous ClC‐3 expression and native volume‐sensitive outwardly rectifying anion channels (VSOACs) in guinea‐pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti‐ClC‐3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen‐preabsorbed anti‐ClC‐3 Ab. 3 It is suggested that endogenous ClC‐3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti‐ClC‐3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC‐3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

ClC‐3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery

1 Cl− channels have been implicated in essential cellular functions including volume regulation, progression of cell cycle, cell proliferation and contraction, but the physiological functions of the ClC‐3 channel are controversial. We tested the hypothesis that the ClC‐3 gene (ClCn‐3) is upregulated in hypertensive pulmonary arteries of monocrotaline‐treated rats, and upregulated ClC‐3 channel aids viability of pulmonary artery smooth muscle cells (PASMCs). 2 Experimental pulmonary hypertension was induced in rats by a single subcutaneous administration of monocrotaline (60 mg kg−1). Injected animals developed characteristic features of pulmonary hypertension including medial hypertrophy of pulmonary arteries and right ventricular hypertrophy. 3 Reverse transcriptase–polymerase chain reaction (RT–PCR), immunohistochemistry and Western immunoblot analysis indicated that histopathological alterations were associated with upregulation of the ClC‐3 mRNA and protein expression in both smooth muscle cells of hypertensive pulmonary arteries and in cardiac myocytes. 4 RT–PCR analysis of mRNA, extracted from canine cultured PASMCs, indicated that incubation with the inflammatory mediators endothelin‐1 (ET‐1), platelet‐derived growth factor (PDGF), interleukin‐1beta (IL‐1β) and tumor necrosis factor alpha (TNFα), but not transforming growth factor beta (TGFβ), upregulated ClC‐3 mRNA. 5 Adenovirus‐mediated delivery and overexpression of ClC‐3 in canine PASMCs improved cell viability against increasing concentrations of hydrogen peroxide (H2O2, range 50–250 μM). 6 In conclusion, upregulation of ClC‐3 in rat hypertensive lung and heart is a novel observation. Our functional data suggest that upregulation of ClC‐3 is an adaptive response of inflamed pulmonary artery, which enhances the viability of PASMCs against reactive oxygen species.

Hypotonic Activation of Short ClC3 Isoform Is Modulated by Direct Interaction between Its Cytosolic C-terminal Tail and Subcortical Actin Filaments

Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.

PDGF and IL-1β upregulate cofilin and LIMK2 in canine cultured pulmonary artery smooth muscle cells

Actin cytoskeleton reorganization is regulated by various actin-binding proteins. Cofilin is the principal filament-depolymerizing protein, whose activity is reduced upon phosphorylation by LIMK. Thus, LIMK and cofilin comprise a signal transduction module regulating actin turnover and myogenic tone in healthy vasculature. Novel functions of smooth muscle cells (SMCs) in the hypertensive pulmonary artery, such as increased motility and proliferation, are supported by the actin cytoskeleton. We therefore hypothesized that bioactive peptides that affect these SMC functions may also result in an upregulation of LIMK and cofilin expression. Semiquantitative RT-PCR and immunoblotting indicated that LIMK2 and cofilin mRNA and protein expression is upregulated in canine pulmonary artery SMCs (PASMCs) exposed to PDGF or IL-1β (10 ng/ml). Inhibition of ERK MAPKs (U-0126, 10 µM) or p38 MAPK (PD-169316, 10 µM), but not PI3Ks (LY-294002, 50 µM), reduced LIMK2 and cofilin gene expression stimulated by PDGF or IL-1β. Inhibition of ROCK (Y-27632, 10 µM) reduced only the IL-1β-stimulated LIMK2 and cofilin expression. These novel observations in PASMCs indicate that LIMK2 and cofilin expression can be induced by PDGF or IL-1β. This parallel upregulation of LIMK2 and cofilin may have potentially broad functional significance for the progress of pulmonary artery disease.

Storage and secretion of β-NAD, ATP and dopamine in NGF-differentiated rat pheochromocytoma PC12 cells

In nerve–smooth muscle preparations β‐nicotinamide adenine dinucleotide (β‐NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. β‐NAD is released, along with noradrenaline and adenosine 5′‐triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and β‐NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve–smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single‐cell model to examine mechanisms of concomitant neurosecretion. Using high‐performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5′‐diphosphate, adenosine 5′‐monophosphate, adenosine, β‐NAD and its immediate metabolites ADP‐ribose and cyclic ADP‐ribose in superfused nerve growth factor‐differentiated rat pheochromocytoma PC12 cells. β‐NAD, DA and ATP were released constitutively and upon stimulation with high‐K+ solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high‐K+‐evoked release of β‐NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that β‐NAD, ATP and DA are stored in both small synaptic‐like vesicles and large dense‐core‐like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP‐25 and sites not associated with SNAP‐25.

Distinct Effects of Contraction Agonists on the Phosphorylation State of Cofilin in Pulmonary Artery Smooth Muscle

We hypothesized that agonist-induced contraction correlates with the phospho-cofilin/cofilin (P-CF/CF) ratio in pulmonary artery (PA) rings and cultured smooth muscle cells (PASMCs). PA rings were used for isometric contractions and along with PASMCs for assay of P-CF/CF by isoelectric focusing and immunoblotting. The P-CF/CF measured 22.5% in PA and differentiated PASMCs, but only 14.8% in undifferentiated PASMCs. With comparable contraction responses in PA, endothelin-1 (100 nM) and norepinephrine (1 μM) induced a 2-fold increase of P-CF/CF, while angiotensin II (1 μM) induced none. All agonists activated Rho-kinase and LIMK2, and activation was eliminated by inhibition of Rho-kinase. Microcystin LF (20 nM) potentiated the angiotensin II, but not the 5-hydroxytryptamine (1 μM)-mediated increase of P-CF/CF. In conclusion, all tested agonists activate the Rho-kinase-LIMK pathway and increase P-CF/CF. Angiotensin II activates PP2A and counteracts the LIMK-mediated CF phosphorylation. CF phosphorylation stabilizes peripheral actin structures and may contribute to the maximal contraction of PA.

Novel localization of CD38 in perivascular sympathetic nerve terminals

Using high performance liquid chromatography fraction analysis we have recently established that numerous smooth muscle preparations, including the canine mesenteric artery and vein, release beta-nicotinamide adenine dinucleotide upon short-pulse electrical field stimulation in tetrodotoxin- and omega-conotoxin GVIA-sensitive manners [ Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder. J Biol Chem 279:48893-48903.]. The beta-nicotinamide adenine dinucleotide metabolites ADP-ribose and cyclic ADP-ribose are also present in the tissue superfusates. CD38 is a multifunctional enzyme involved in the degradation of beta-nicotinamide adenine dinucleotide to ADP-ribose and cyclic ADP-ribose. Western immunoblot analysis revealed that CD38 is expressed in both artery and vein. Confocal laser scanning microscopy established colocalization of CD38 with tyrosine hydroxylase, synaptotagmin and synaptic vesicle protein in both blood vessels. High performance liquid chromatography with fluorescence detection demonstrated that whole tissue segments metabolize 1,N(6)-etheno-nicotinamide adenine dinucleotide to 1,N(6)-etheno-ADP-ribose and nicotinamide-guanine dinucleotide to cyclic GDP-ribose, suggesting the presence of both nicotinamide adenine dinucleotide-glycohydrolase and ADP-ribosyl cyclase activities in these blood vessels. Both enzymes appear to be associated with the membrane fraction, and therefore might be attributed to CD38. These data demonstrate a previously uncharacterized localization of CD38 in perivascular autonomic nerve terminals. Therefore, the beta-nicotinamide adenine dinucleotide/CD38 system may provide new mechanisms in autonomic neurovascular control.

Hypotonic Activation of Volume-sensitive Outwardly Rectifying Anion Channels (VSOACs) Requires Coordinated Remodeling of Subcortical and Perinuclear Actin Filaments

Cell volume regulation requires activation of volume-sensitive outwardly rectifying anion channels (VSOACs). The actin cytoskeleton may participate in the activation of VSOACs but the roles of the two major actin pools remain undefined. We hypothesized that structural reorganization of both subcortical and perinuclear actin filaments (F-actin) contributes to the hypotonic activation of VSOACs. Hypotonic stress of pulmonary artery smooth muscle cells (PASMCs) was associated with reorganization of both peripheral and perinuclear F-actin, and with activation of VSOACs. Preincubation with cytochalasin D caused prominent dissociation of perinuclear, but not of subcortical F-actin. Cytochalasin D failed to induce isotonic activation and delayed the hypotonic activation of VSOACs. F-actin stabilization by phalloidin delayed both the hypotonic stress-induced dissociation of membrane-associated actin filaments and the activation kinetics of VSOACs. PKCε, which was proposed to phosphorylate and inhibit VSOACs, colocalized primarily with F-actin and the net kinase activity remained unchanged during hypotonic cell swelling. In conclusion, normal hypotonic activation of VSOACs requires disruption of peripheral F-actin but intact perinuclear F-actin; interference with this pattern of actin reorganization delays the activation kinetics of VSOACs. The cell swelling-induced peripheral actin dissociation may underlie the observed translocation of PKCε, which leads to a net decrease of PKCε inhibitory activity in submembranous sites. Thus, reorganization of actin and PKCε may establish conditions for mechano- and/or signal transduction-mediated activation of VSOACs.