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cGMP-dependent protein kinase I (cGKI) induces relaxation of smooth muscle via several pathways that include inhibition of intracellular [Ca.sup.2+] signaling and/or involve activation of myosin phosphatase. In the present study, we investigated these mechanisms comparatively in colon and jejunum longitudinal smooth muscle from mice. In simultaneous recordings from colon muscle, 8-bromo-cGMP (8-Br-cGMP) reduced both carbachol-induced tension and carbachol-induced increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). These effects of 8-Br-cGMP were absent in colon from mice carrying a mutated inositol-1,4,5 trisphosphate receptor l-associated G kinase substrate (IRAG) gene or lacking cGKI. However, in jejunum, 8-Br-cGMP reduced carbachol-induced tension but did not change corresponding [[[Ca.sup.2+]].sub.i] signals. This setting was also observed in jejunum from mice carrying a mutated IRAG gene, whereas no response to 8-Br-cGMP was observed in jejunum from mice lacking cGK1. After inhibition of phosphatase activity by calyculin A, 8-Br-cGMP did not relax jejunum but still relaxed colon muscle. In Western blot analysis, 8-BrcGMP reduced the signal for phosphorylated MYPT-1 in carbachol-stimulated jejunum but not in colon. These results suggest that cGMP/cGKI signaling differentially inhibits contraction in the muscles investigated: in jejunum, inhibition is performed without changing [[[Ca.sup.2+]].sub.i] and is dependent on phosphatase activity, whereas in colon, inhibition is mediated by inhibition of [[[Ca.sup.2+]].sub.i] signals. inositol-1,4,5 trisphosphate receptor I-associated G kinase substrate; myosin phosphatase targeting subunit 1; guanosine 3',5'-cyclic monophosphate-dependent protein kinase I; colon; jejunum doi: 10.1152/ajpgi.00095.2009

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation. p21-activated kinase; 5-hydroxytryptamine; smooth muscle cells; cytoskeleton

We have earlier shown that the treatment of A10 vascular smooth muscle cells with S-nitroso-N-acetyl-penicillamine (SNAP); nitric oxide donor (NO) for 24 h decreased the expression of natriuretic peptide receptor C (NPR-C) and adenylyl cyclase signaling. The present study was undertaken to examine the implication of different signaling mechanisms in a NO-induced response. The treatment of A10 vascular smooth muscle cells with SNAP decreased the expression of NPR-C and [G.sub.i][alpha] proteins in a time-dependent manner. The expression of [G.sub.i][alpha] proteins was decreased at 6 h, whereas the expression of NPR-C was attenuated at 2 h. The NPR-C-mediated inhibition of adenylyl cyclase was attenuated (~50%) after 2 h of treatment and was completely abolished after 6 h of treatment. The decreased expression of NPR-C and NPR-C-mediated attenuation of adenylyl cyclase after 2 h of treatment was reversed to control levels by PD-98059, a MEK inhibitor. SNAP also modulated the ERK1/2 phosphorylation in a time-dependent manner; an increase was observed up to 2 h, and, thereafter, the ERK1/2 phosphorylation was decreased. On the other hand, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823 inhibitor of soluble guanylyl cyclase and protein kinase G, respectively, and Mn(III)tetrakis(4-benzoic acid)porphyrin, a scavenger of peroxynitrite, were unable to restore the SNAP-induced decreased expression of NPR-C protein and increased ERK1/2 phosphorylation to control levels. However, the decreased levels of phosphorylated ERK1/2 and [G.sub.i][alpha] proteins were restored to control levels by 8-bromo-cAMP. These results indicate that a temporal relationship follows between a NO-induced decreased expression of NPR-C and [G.sub.i][alpha] proteins. The decreased expression of NPR-C is mediated through cGMP-independent but MAPK-dependent pathway, whereas NO-induced decreased levels of cAMP may contribute to the decreased activation of MAPK and thereby decreased the expression of [G.sub.i][alpha] proteins. G proteins; mitogen-activated protein kinase

Phenotypic changes in airway smooth muscle occur with airway inflammation and asthma. These changes may be induced by alterations in the extracellular matrix that initiate signaling pathways mediated by integrin receptors. We hypothesized that integrin-linked kinase (ILK), a multidomain protein kinase that binds to the cytoplasmic tail of [beta]-integrins, may be an important mediator of signaling pathways that regulate the growth and differentiation state of airway smooth muscle. We disrupted signaling pathways mediated by ILK in intact differentiated tracheal muscle tissues by depleting ILK protein using ILK antisense. The depletion of ILK protein increased the expression of the smooth muscle differentiation marker genes myosin heavy chain (SmMHC), SM22[alpha], and calponin and increased the expression of SmMHC protein. Conversely, the overexpression of ILK protein reduced the mRNA levels of SmMHC, SM22[alpha], and calponin and SmMHC protein. Analysis by chromatin immunoprecipitation showed that the binding of the transcriptional regulator serum response factor (SRF) to the promoters of SmMHC, SM22[alpha], and calponin genes was increased in ILK-depleted tissues and decreased in tissues overexpressing ILK. ILK depletion also increased the amount of SRF that localized within the nucleus. ILK depletion and overexpression, respectively, decreased and increased the activation of its downstream substrate protein kinase B (PKB/Akt). The pharmacological inhibition of Akt activity also increased SRF binding to the promoters of smooth muscle-specific genes and increased expression of smooth muscle proteins, suggesting that ILK may exert its effects by regulating the activity of Akt. We conclude that ILK is a critical regulator of airway smooth muscle differentiation. ILK may mediate signals from integrin receptors that control airway smooth muscle differentiation in response to alterations in the extracellular matrix. smooth muscle myosin heavy chain; serum response factor

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of allergic asthma through animal models that have shown that IL-13 is both necessary and sufficient to cause airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is a primary effector of AHR, and IL-13 increases the responsiveness of ASM, by increasing [Ca.sup.2+] release intracellularly, to bronchoconstrictors such as histamine. The mechanisms and signaling pathways mediating this effect are incompletely understood. We have investigated the pathways through which IL-13 regulates the [Ca.sup.2+] response to histamine in primary human ASM cell cultures. Functional IL-13 receptors were demonstrated by IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mitogen-activated protein kinases (MAPKs). IL-13 increased [Ca.sup.2+] responses to histamine. The augmentation of [Ca.sup.2+] signaling was not affected by inhibition of STAT6 or p38 MAPK signaling but was prevented by concurrent inhibition of c-jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) MAPKs. This inhibition did not affect the IL-13-induced increase in histamine receptors. We conclude that IL-13 induces potentiation of [Ca.sup.2+] responses to contractile agonists by affecting mechanisms downstream of receptors. JNK and ERK MAPKs modulate these mechanisms. lung; allergy; inflammation; cytokines; mitogen-activated protein kinase

Tonic physiological activity of RhoA/Rho kinase contributes to the maintenance of penile flaccidity through its involvement in the [Ca.sup.2+] sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho kinase is also involved in the modulation of [Ca.sup.2+] entry induced by [[alpha].sup.1]-adrenoceptor stimulation of penile arteries. Rat penile arteries were mounted in microvascular myographs for simultaneous measurements of intracellular [Ca.sup.2+] ([Ca.sup.2+]i) and force. The Rho-kinase inhibitor Y-27632 markedly reduced norepinephrine-mediated electrically induced contractions and the increases in both [Ca.sup.2+]i and tension elicited by the [[alpha].sub.1]-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro-31-8220 reduced tension without altering the Phe-induced increase in [Ca.sup.2+]i. In the presence of nifedipine, Y-27632 still inhibited the non-L-type [Ca.sup.2+] signal and blunted Phe contraction. Y-27632 did not impair the capacitative [Ca.sup.2+] entry evoked by store depletion with cyclopiazonic acid but largely reduced the [Ba.sup.2+] influx stimulated by Phe in fura-2 AM-loaded arteries. The addition of Y-27632 to arteries depolarized with high KC1 markedly reduced tension without changing [Ca2+]i. In [alpha]-toxin-permeabilized penile arteries stimulated with threshold [Ca.sup.2+] concentrations, Y-27632 inhibited the sensitization induced by either guanosine 5'-O-(3-thiotriphosphate) (GTP3,S) or Phe in the presence of GTP[gamma]S. However, Y-27632 failed to alter contractions induced by a maximal concentration of free [Ca.sup.2+] These results suggest that Rho kinase, besides its contribution to the [Ca.sup.2+] sensitization of the contractile proteins, is also involved in the regulation of [Ca.sup.2+] entry through a nonselective cation channel activated by [alpha]-adenoceptor stimulation in rat penile arteries. penile arteries; calcium entry; nonselective cation channels; calcium sensitization

Caffeine has been shown to increase the [Ca.sup.2+] release frequency ([Ca.sup.2+] sparks) from the sarcoplasmic reticulum (SR) through ryanodine-sensitive stores and relax gastric fundus smooth muscle. Increased [Ca.sup.2+] store refilling increases the frequency of [Ca.sup.2+] release events and store refilling is enhanced by CaM kinase II (CaMKII) phosphorylation of phospholamban (PLB). These findings suggest that transient, localized [Ca.sup.2+] release events from the SR may activate CaMKII and contribute to relaxation by enhancing store refilling due to PLB Thr17 phosphorylation. To investigate this possibility, we examined the effects of caffeine on CaMKII, muscle tone, and PLB phosphorylation in murine gastric fundus smooth muscle. Caffeine (1 mM) hyperpolarized and relaxed murine gastric fundus smooth muscle and activated CaMKII. Ryanodine, tetracaine, or cyclopiazonic acid each prevented CaMKII activation and significantly inhibited caffeine-induced relaxation. The large-conductance [Ca.sup.2+]-activated [K.sup.+] channel blocker iberiotoxin, but not apamin, partially inhibited caffeine-induced relaxation. Caffeine-induced CaMKII activation increased PLB Thr17, but not PLB Ser16 phosphorylation. 3-Isobutyl-1-methylxanthine increased PLB Ser16 phosphorylation, but not PLB Thr17 phosphorylation. The CaMKII inhibitor KN-93 inhibited caffeine-induced relaxation and PLB Thr17 phosphorylation. These results show that caffeine-induced CaMKII activation and PLB phosphorylation play a role in the relaxation of gastric fundus smooth muscles. [Ca.sup.2+]/CaM-dependent protein kinase II

Hyperinsulinemia is a major risk factor for the development of vascular disease. We have reported that insulin increases the motility of vascular smooth muscle cells via a hydrogen peroxide-mediated mechanism and that nitric oxide (NO) attenuates insulin-induced motility via a cGMP-mediated mechanism. Events downstream of cGMP elevation have not yet been investigated. The aim of our study was to test the hypothesis that antimotogenic effects of NO and cGMP in cultured rat aortic smooth muscle cells are mediated via PKG, followed by reduction of cytoplasmic [Ca.sup.2+] levels and increased protein tyrosine phosphatase-proline, glutamate, serine, and threonine activity, leading to suppression of agonist-induced elevation of hydrogen peroxide levels and cell motility. Treatment of primary cultures with adenovirus expressing PKG-1[alpha] mimicked NO-induced inhibition of insulin-elicited hydrogen peroxide elevation and cell motility, whereas treatment with the pharmacological PKG inhibitor Rp-8-bromo-3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS) rescued the stimulatory effects of insulin that were suppressed by NO donor. Treatment of cells with insulin failed to increase cytoplasmic [Ca.sup.2+] levels, whereas NO donor decreased cytoplasmic [Ca.sup.2+] levels in the presence or absence of insulin. Treatment of cells with the [Ca.sup.2+] chelator BAPTA mimicked the effects of PKG and the NO donor and increased the activity of PTP-PEST. Finally, treatment with a dominant negative allele of PTP-PEST reversed the inhibitory effect of BAPTA on cell motility and hydrogen peroxide elevation. We conclude that NO-induced inhibition of cell motility occurs via PKG-mediated reduction of basal cytoplasmic [Ca.sup.2+] levels, followed by increased PTP-PEST activity, leading to decreased hydrogen peroxide levels and reduced cell motility. calcium chelator; protein tyrosine phosphatase-proline; glutamate; serine; and threonine; insulin; hydrogen peroxide

In Triton-skinned phasic ileal smooth muscle, constitutively active recombinant p21-activated kinase (PAK3) has been shown to induce [Ca.sup.2+]-independent contraction, which is accompanied by phosphorylation of caldesmon and desmin (Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, and Mak AS. J Biol Chem 273: 23433-23439, 1998). In the present study, we investigated whether PAK has a broad impact on smooth muscle in general by testing the hypothesis that PAK induces [Ca.sup.2+]-independent contractions and/or [Ca.sup.2+] sensitization in tonic airway smooth muscle and that the process is mediated via phosphorylation of caldesmon. In the absence of [Ca.sup.2+] (pCa > 9), constitutively active glutathione-S-transferase-murine PAK3 (GST-mPAK3) caused force generation of Triton-skinned canine tracheal smooth muscle (TSM) fibers to ~40% of the maximal force generated by [Ca.sup.2+] at pCa 4.4. In addition, GST-mPAK3 enhanced [Ca.sup.2+] sensitivity of contraction by increasing force generation by 80% at intermediate [Ca.sup.2+] concentrations (pCa 6.2), whereas it had no effect at pCa 4.4. Catalytically inactive GST-mPAK[3.sup.K297R] had no effect on force production. Using antibody against one of the PAK-phosphorylated sites ([Ser.sup.657]) on caldesmon, we showed that a basal level of phosphorylation of caldesmon occurs at this site in skinned TSM and that PAK-induced contraction was accompanied by a significant increase in the level of phosphorylation. Western blot analyses show that PAK1 is the predominant PAK isoform expressed in murine, rat, canine, and porcine TSM. We conclude that PAK causes [Ca.sup.2+]-independent contractions and produces [Ca.sup.2+] sensitization of skinned phasic and tonic smooth muscle, which involves an incremental increase in caldesmon phosphorylation. p21-activated kinase; monomeric G proteins; calcium sensitization; asthma; tonic smooth muscle

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-[epsilon] is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-[epsilon] would decrease acute HPV in adult C57BL6xSV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% [O.sub.2]) was compared with responses to angiotensin II (ANG II) and KCl. PKC-[epsilon] -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-L-arginine augmented HPV in PKC-[epsilon] +/+ but not -/- mice. Inhibition of [Ca.sup.2+]-gated [K.sup.+] channels ([K.sub.Ca]) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated [K.sup.+] channel ([K.sub.v]) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased [K.sub.v] channel expression could contribute to blunted HPV in PKC-[epsilon] -/- mice. Therefore, expression of the [O.sub.2]-sensitive [K.sub.v] channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Ky3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-[epsilon] blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or [K.sub.Ca] channel activity. Instead, the absence of PKC-[epsilon] allows increased expression of [K.sub.v] channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV. murine knockout; isolated, perfused lung; pulmonary artery; smooth muscle cells; potassium channels

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on [Thr.sup.421]/[Ser.sup.424], [Thr.sup.389], and [Thr.sup.229] that paralleled p70S6 kinase activation. p70S6 kinase([Thr.sup.421]/[Ser.sup.424]) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase (Thr.sup.389) and p70S6 kinase([Thr.sup.229]) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt([Ser.sup.308]), Akt([Ser.sup.473]), and mammalian target of rapamycin([Ser.sup.2448]) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells. insulin-like growth factor-I; proliferation; protein kinase C; protein kinase B; Akt; extracellular signal-regulated kinase 1/2; and p38 mitogen-activated protein kinase

This study examined the expression of inositol 1,4,5-trisphosphate (I[P.sub.3]) receptor (I[P.sub.3]R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate I[P.sub.3]Rs and inhibit I[P.sub.3]-dependent [Ca.sup.2+] release, which mediates the initial phase of agonist-induced contraction. PKG-I[alpha] and PKG-I[beta] were expressed in gastric smooth muscle cells, together with I[P.sub.3]-R-associated cG-kinase substrate, a protein that couples PKG-I[beta] to I[P.sub.3]R-I. I[P.sub.3]R-I and I[P.sub.3]R-III were also expressed, but only I[P.sub.3]R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-I[alpha] in vitro showed that PKA phosphorylated the same site as PKG (presumably [S.sup.1755]) and an additional PKA-specific site ([S.sup.1589]). In intact muscle cells, agents that activated PKG or both PKG and PKA induced I[P.sub.3]R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H, 8H, 1H,-2,7b,11a-trizadizo-benzo9-(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced I[P.sub.3]R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to I[P.sub.3]R-I in intact muscle cells. The pattern of I[P.sub.3]R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-I[alpha]. Phosphorylation of I[P.sub.3]R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited I[P.sub.3]-induced [Ca.sup.2+] release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that I[P.sub.3]R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of I[P.sub.3]-dependent [Ca.sup.2+] release. relaxation, gastric muscle, calcium release

We investigated the effects of thrombin on the induction of heat shock proteins (HSP) 70 and 27, and the mechanism behind the induction in aortic smooth muscle A10 cells. Thrombin increased the level of HSP27 but had little effect on the level of HSP70. Thrombin stimulated the accumulation of HSP27 dose dependently between 0.01 and 1 U/ml and cycloheximide reduced the accumulation. Thrombin stimulated an increase in the level of HSP27 mRNA and actinomycin D suppressed the thrombin-increased mRNA level. Thrombin induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The HSP27 accumulation by thrombin was reduced by SB-203580 and PD-169316 but not by SB-202474. SB-203580 and PD-169316 suppressed the thrombin-induced phosphorylation of p38 MAPK. SB-203580 reduced the thrombin-increased level of HSP27 mRNA. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by thrombin. Dissociation was inhibited by SB-203580. Thrombin induced the phosphorylation of HSP27 and the phosphorylation was suppressed by SB-203580. These results indicate that thrombin stimulates not only the dissociation of HSP27 but also the induction of HSP27 via p38 MAPK activation in aortic smooth muscle cells. serine protease; A20 cell; intracellular signaling

Telokin is an acidic protein with a sequence identical to the COOH-terminal domain of myosin light chain kinase (MLCK) produced by an alternate pro-rooter of the MLCK gene. Although it is abundantly expressed in smooth muscle, its physiological function is not understood. In the present study, we attempted to clarify the function of telokin by analyzing its spatial and temporal localization in living single smooth muscle cells. Primary cultured smooth muscle cells were transfected with green fluorescent protein (GFP)-tagged telokin. The telokin-GFP localized mostly diffusely in cytosol. Stimulation with both sodium nitroprusside (SNP) and 8-bromo-cyclic GMP induced translocation of GFP-tagged telokin to near plasma membrane in living single smooth muscle cells. The translocation was slow, and it took more than 10 min at room temperature. Mutation of the phosphorylation sites of telokin (S13A, S19A, and S13A/S19A) significantly attenuated SNP-induced translocation. Both KT-5823 (cGMP-dependent protein kinase inhibitor) and PD-98059 (mitogen-activated protein kinase inhibitor) diminished the telokin-GFP translocation. These results suggest that telokin changes its intracellular localization because of phosphorylation at Ser13 and/or Ser19 via the cGMP-signaling pathway. green fluorescent protein; myosin; myosin light chain kinase; guanosine 3',5'-cyclic monophosphate-dependent protein kinase; phosphorylation

In this study, we examined the role of insulin in the control of vascular smooth muscle cell (VSMC) migration in the normal vasculature. Platelet-derived growth factor (PDGF) increased VSMC migration, which was inhibited by pretreatment with insulin in a dose-dependent manner. Insulin also caused a 60% decrease in PDGF-stimulated mitogen-activated protein kinase (MAPK) phosphorylation and activation. Insulin inhibition of MAPK was accompanied by a rapid induction of MAPK phosphatase (MKP-1), which inactivates MAPKs by dephosphorylation. Pretreatment with inhibitors of the nitric oxide (NO)/cGMP pathway, blocked insulin-induced MKP-1 expression and restored PDGF-stimulated MAPK activation and migration. In contrast, adenoviral infection of VSMCs with MKP-1 or cGMP-dependent protein kinase I[alpha] (cGK I[alpha]), the downstream effector of cGMP signaling, blocked the activation of MAPK and prevented PDGF-directed VSMC migration. Expression of antisense MKP-1 RNA prevented insulin's inhibitory effect and restored PDGF-directed VSMC migration and MAPK phosphorylation. We conclude that insulin inhibition of VSMC migration may be mediated in part by NO/ cGMP/cGK I[alpha] induction of MKP-1 and consequent inactivation of MAPKs. nitric oxide; guanosine 3',5'-cyclic monophosphate; cGMP-dependent protein kinase I[alpha]; platelet-derived growth factor; hypertension

We examined the role of mitogen-activated protein kinase ([p.sup.44/42] MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a [pp60.sup.c-src] inhibitor), and antibodies to [pp60.sup.c-src] and [p.sup.120] ras GTPase-activating protein ([p.sup.120] rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of [p.sup.120] rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of [p.sup.44/42] MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that [pp60.sup.c-src] and [p.sup.44/42] MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles. tonic smooth muscle; tyrosine kinase; tyrosine phosphorylation; [p.sup.120] ras GTPase-activating protein; [p.sup.190] rho GTPase-activating protein

We tested the hypothesis that mechanical plasticity of airway smooth muscle may be mediated in part by the p38 mitogen-activated protein (MAP) kinase pathway. Bovine tracheal smooth muscle (TSM) strips were mounted in a muscle bath and set to their optimal length, where the active force was maximal ([F.sub.o]). Each strip was then contracted isotonically (at 0.32 [F.sub.o]) with ACh (maintained at [10.sup.-4] M) and allowed to shorten for 180 min, by which time shortening was completed and the static equilibrium length was established. To simulate the action of breathing, we then superimposed on this steady distending force a sinusoidal force fluctuation with zero mean, at a frequency of 0.2 Hz, and measured incremental changes in muscle length. We found that TSM strips incubated in 10 [micro]M SB-203580-HCl, an inhibitor of the p38 MAP kinase pathway, demonstrated a greater degree of fluctuation-driven lengthening than did control strips, and upon removal of the force fluctuations they remained at a greater length. We also found that the force fluctuations themselves activated the p38 MAP kinase pathway. These findings are consistent with the hypothesis that inhibition of the p38 MAP kinase pathway destabilizes muscle length during physiological loading. mitogen-activated protein; contraction; plasticity; perturbed myosin binding

We have investigated the role of heat shock protein 27 (HSP27) phosphorylation and the association of HSP27 with contractile proteins actin, myosin, and tropomyosin. Smooth muscle cells were labeled with [[sup.32]P]orthophosphate. C2-ceramide (0.1 [micro]M), an activator of protein kinase C (PKC), induced a sustained increase in HSP27 phosphorylation that was inhibited by calphostin C. C2-ceramide-induced (0.1 [micro]M) sustained colonic smooth muscle cell contraction was accompanied by significant increases in the association of HSP27 with tropomyosin and in the association of HSP27 with actin. The significant increases occurred at 30 s after stimulation and were sustained at 4 min. Contraction was also associated with strong colocalization of HSP27 with tropomyosin and with actin as observed after immunofluorescent labeling of tropomyosin, actin, and HSP27 followed by confocal microscopy. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that phosphorylation of HSP27 could affect myosin association with actin. In conclusion 1) HSP27 phosphorylation appears to be necessary for reorganization of HSP27 inside the cell and seems to be directly correlated with the PKC signal transduction pathway, and 2) agonist-induced phosphorylation of HSP27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin. heat shock protein; tropomyosin; protein kinase C; mitogen-activated protein kinase

We examined effects of 17[beta]-estradiol ([E.sub.2]) on human vascular smooth muscle cell (VSMC) proliferation under normal (5 mmol/l) and high (25 mmol/l) glucose concentrations. Platelet-derived growth factor (PDGF) BB (20 ng/ml)-induced increases in DNA synthesis and proliferation were greater in high than normal glucose concentrations; the difference in DNA synthesis was abolished by a protein kinase C (PKC)-[beta] inhibitor, LY-379196 (30 nmol/l). Western blotting showed that PKC-[[beta].sub.1] protein increased in cells exposed to high glucose, whereas PKC-[alpha] protein and total PKC activity remained unchanged, compared with normal glucose cultures. In normal glucose, [E.sub.2] (1-100 nmol/l) inhibited PDGF-induced DNA synthesis by 18-37% and cell proliferation by 16-22% in a concentration-dependent manner. The effects of [E.sub.2] were blocked by the estrogen receptor (ER) antagonist ICI-182780, indicating ER dependence. In high glucose, the inhibitory effect of [E.sub.2] on VSMC proliferation was abolished but was restored in the presence of the PKC-[beta] inhibitor LY-379196. Thus high glucose enhances human VSMC proliferation and attenuates the antiproliferative effect of [E.sub.2] in VSMC via activation of PKC-[beta]. high glucose; estrogen; proliferation; smooth muscle cells; protein kinase C-[beta]

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-[beta]-D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced [Ca.sup.2+] release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels. protein kinase A; phosphodiesterase; cyclic nucleotides; relaxation

ERK MAP kinases regulate smooth muscle contraction in ovine uterine artery: effect of pregnancy. Am J Physiol Heart Circ Physiol 282: H292-H300, 2002.--The present study investigated the potential role of extracellular signal-regulated kinase (ERK) in uterine artery contraction and tested the hypothesis that pregnancy upregulated ERK-mediated function in the uterine artery. Isometric tension in response to phenylephrine (PE), serotonin (5-HT), phorbol 12,13-dibutyrate (PDBu), and KCl was measured in the ring preparation of uterine arteries obtained from nonpregnant and near-term (140 days gestation) pregnant sheep. Inhibiting ERK activation with PD-98059 did not change the KCl-evoked contraction but significantly inhibited the contraction to 5-HT in both nonpregnant and pregnant uterine arteries. PD-98059 did not affect PE-induced contraction in the uterine arteries of nonpregnant sheep but significantly decreased it in the uterine arteries of pregnant sheep. In accordance, PE stimulated activation of ERK in uterine arteries of pregnant sheep, which was blocked by PD-98059. PD-98059-mediated inhibition of the PE-induced contraction was associated with a decrease in both intracellular [Ca.sup.2+] concentration and [Ca.sup.2+] sensitivity of contractile proteins in the uterine arteries of pregnant sheep. PDBu-mediated contraction was significantly less in pregnant than in nonpregnant uterine arteries. PD-98059 had no effect on PDBu-induced contraction in nonpregnant but significantly increased it in pregnant uterine arteries. In addition, PD-98059 significantly enhanced PDBu-stimulated protein kinase C activity. The results indicate that ERK plays an important role in the regulation of uterine artery contractility, and its effect is agonist dependent. More importantly, pregnancy selectively enhances the role of ERK in [[alpha].sub.1]-adrenoceptor-mediated contractions and its effect in suppressing protein kinase C-mediated contraction in the uterine artery. mitogen-activated protein kinase; PD-98059; calcium; protein kinase C; [[alpha].sub.1]-adrenoceptor; extracellular signal-regulated kinase

Hayabuchi, Y., N. B. Standen, and N. W. Davies. Angiotensin II inhibits and alters kinetics of voltage-gated [K.sup.+] channels of rat arterial smooth muscle. Am J Physiol Heart Circ Physiol 281: H2480-H2489, 2001.--The vasoconstrictor angiotensin II (ANG II) inhibits several types of [K.sup+] channels. We examined the inhibitory mechanism of ANG II on voltage-gated [K.sup.+] ([K.sub.v) currents (I.sub.Kv]) recorded from isolated rat arterial smooth muscle using patch-clamp techniques. Application of 100 nM ANG II accelerated the activation of [I.sub.Kv] but also caused inactivation. These effects were abolished by the A[T.sub.1] receptor antagonist losartan. The protein kinase A (PKA) inhibitor Rp-cyclic 3',5'-hydrogen phosphothioate adenosine (100 [micro]M) and an analog of diacylglycerol, 1,2dioctanyoyl-rac-glycerol (2 [micro]M), caused a significant reduction of [I.sub.Kv] Furthermore, the combination of 5 [micro]M PKA inhibitor peptide 5-24 (PKA-IP) and 100 [micro]M protein kinase C (PKC) inhibitor peptide 19-27 (PKC-IP) prevented the inhibition by ANG II, although neither alone was effective. The ANG II effect seen in the presence of PKA-IP remained during addition of the [Ca.sup.2+]-dependent PKC inhibitor Go6976 (1 [micro]M) but was abolished in the presence of 40 [micro]M PKC-[epsilon] translocation inhibitor peptide. These results demonstrate that ANG II inhibits Ky channels through both activation of PKC-[epsilon] and inhibition of PKA. potassium channel; activation; inactivation; protein kinase A; protein kinase C Received 22 May 2001; Accepted in Final Form 7 August 2001

Keef, Kathleen D., Joseph R. Hume, and Juming Zhong. Regulation of cardiac and smooth muscle [Ca.sup.2+] channels ([Ca.sub.v]1.2a,b) by protein kinases. Am J Physiol Cell Physiol 281: C1743-C1756, 2001.--High voltage-activated [Ca.sup.2+] channels of the [Ca.sub.v]1.2 class (L-type) are crucial for excitation-contraction coupling in both cardiac and smooth muscle. These channels are regulated by a variety of second messenger pathways that ultimately serve to modulate the level of contractile force in the tissue. The specific focus of this review is on the most recent advances in our understanding of how cardiac [Ca.sub.v]1.2a and smooth muscle [Ca.sub.v]1.2b channels are regulated by different kinases, including cGMP-dependent protein kinase, cAMP-dependent protein kinase, and protein kinase C. This review also discusses recent evidence regarding the regulation of these channels by protein tyrosine kinase, calmodulin-dependent kinase, purified G protein subunits, and identification of possible amino acid residues of the channel responsible for kinase regulation. cGMP-dependent protein kinase; cAMP-dependent protein kinase; protein kinase C; G proteins

Smooth and skeletal muscle pyruvate kinase and its differential coupling to creatine kinase are discussed. It appears that ability to form diazymatic complexes with creatine kinase is in skeletal muscle pyruvate kinase (SKPK).

The role of phosphorylation of G protein-coupled receptors (GPCR) by G protein-coupled receptor kinase (GRK) in airway smooth muscle physiology was investigated in mice. To this end, the airway pressure time index and heart rate responses to intravenous injection of the cholinergic agonist methacholine in genetically engineered mice that lack one copy of GRK2 were examined. Results show that GRK3 may be involved in attenuating the cholinergic response of airway smooth muscle and in controlling the chronotropic element of the baroreceptor reflex.

The role of endothelin and ceramide in the differential activation of phosphoinositide 3-kinase in colonic smooth muscle cells has been investigated. Endothelin was found to induce a sustained rise in phosphoinositide 3-kinase activity at both 30 seconds and four minutes of stimulation. Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein, on the other hand, significantly restricted both C(sub 2) ceramide-induced and endothelin-induced phosphoinositide 3-kinase activation and contraction.

Changes in the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin were analyzed to investigate whether a mechanosensitive transduction signaling pathway is present in tracheal smooth muscles. The FAK and paxillin were found to be involved in the integrin-mediated mechanotransduction process in tracheal smooth muscles. Their phosphorylation was also observed to be higher when the muscles are stimulated isometrically at a long muscle length than at a short length.

Natriuretic peptide secretion and its regulation by the heart are discussed. The peptides are secreted by the heart, actually by atrial cardiomyocytes, normally, but in cardiac hypertropy also by ventricular myocytes. They are important hormones in blood pressure control and excretion of water. Atrial stretch through mechano-sensitive ion channel is one of the major triggers of release of the peptides. Intracellular calcium homeostasis controlled by several ion channels is thought to be a key element in regulation.

The role of extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAP) or MEK activation in the contraction of the ferret aorta smooth muscle contraction using the specific MEK inhibitor PD 098059 was examined. Caldesmon phosphorylation at the MAP kinase site was determined to illustrate the relationship between MAP kinase activation and Ca2+ -independent smooth muscle contraction. The findings suggest the critical role of caldesmon phosphorylation in MAP kinase activation and smooth muscle contraction.

Studies were performed to investigate the existence of swelling-activated or volume-sensitive Cl- currents, also identified as the CIC-3 channel in cardiac myocytes, in the smooth muscle cells of the canine colon. The morphologic and molecular properties of the CIC-3 channel were described. The findings confirmed CIC-3 gene expression in the tonic and phasic smooth muscles of the dog and mouse gastro-intestinal (GI) tracts. They also suggest the involvement of CIC-3 channels in altering extracellular osmolarity and radical stretch in the GI smooth muscle cells.

A study was conducted to characterize the signaling pathways influencing the growth effects of insulin-like growth factor-I in human intestinal smooth muscle cells. Primary cultures of muscle cells were passaged after confluence by washing them three times with phosphate-buffered saline. Results indicated that intestinal muscle cells support distinct phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways to mediate growth.

Airway ring segments from guinea pigs, mice and human fetuses were constricted with the concentration of acetylcholine producing 50-75% maximum contraction. Guinea pig airways contracted 29+/-5%, mouse airways contracted 23+/-6% and human fetal airways contracted 30+/-8%. Findings showed that amiloride-induced airway contraction in guinea pigs and mice closely resemble the response in isolated human airways and that the process may involve the Na+/H+ antiporter and protein kinase C.

The role of protein kinase C (PKC) in beta-hexosaminidase (Hex)-induced proliferation of arterial smooth muscle cells (ASMCs) was analyzed to determine the mechanisms of ASM hypertrophy/hyperplasia. Analysis of Hex-induced mitogenesis in bovine ASMCs indicated the presence of post-PKC activation mechanisms that mediate Hex activity. Furthermore, inhibition of PKC activity attenuated Hex-induced mitogenesis in ASMCs.

The role of mitogen-activated protein kinase/ extracellular signal-regulated kinase (ERK) kinase-1 (MEK1) in ERK-activation was analyzed in bovine tracheal myocytes cotransfected with epitope-tagged ERK2. The activation of the cotransfected ERK2 in bovine tracheal myocytes by platelet-derived growth factors (PDGF) was attenuated by the transient transfection of the inactive form of MEK1. On the other hand, the transient transfection of bovine tracheal myocytes with the constitutive active form of MEK1 led to PDGF-induced ERK activation and DNA synthesis.

The activity of mitogen-activated protein (MAP) kinases were analyzed in isolated canine tracheal smooth muscles by gel renaturation assay and mono-Q chromatography of tissue homogenates. Gel renaturation assay and mono-Q chromatography of canine tracheal smooth muscle homogenates indicated the presence of significant ERK1 and ERK2 MAP kinases that were activated by muscarinic stimulation. Carbarchol stimulation of canine tracheal smooth muscles also activated MAP kinases.

The KCa channels of smooth muscle cells (SMCs) can be activated by iloprosts and opened by (cAMP) cyclicadenosine monophosphate-dependent protein kinase (PKA). The PKA-induced phosphorylation of the KCa channels of SMCs mediates iloprost-activation of the channel which increases the levels of intracellular calcium ions. The activating effect of iloprost was simulated by utilizing 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphothioate. Rp-8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate inhibited the activity of iloprost.

The K+ adenosine triphosphate (ATP) potassium channels mediate membrane depolarization in smooth muscle cells. Elevated concentration of K+ ATP channel activity depends on the presence of endogenous vasodilators such as adenosine and calcitonin gene-related peptides. The ion channel activity is also regulated by the vasoconstrictor activation of protein kinase A and the vasoconstrictor inhibition of protein kinase C.

I. INTRODUCTION In this review we hope to describe current thinking on the mechanisms of smooth muscle contraction. Discussion is confined, with a few exceptions, to studies performed in contractile [...]

Studies on human vascular smooth muscle cells (SMCs) show that growth factor-induced mitogenesis is linked with nuclear translocation of mitogen-activated protein kinase (MAPK), and prolonged activation and tyrosine phosphorylation. MAPK activation and tyrosine phosphorylation peaks 3 to 10 minutes after agonists exposure to human vascular SMCs. Correlation is absent between mitogenicity and the amount of MAPK activation, but it occurs with the duration of MAPK activation.

Large-conductance, [Ca.sup.2+]-activated [K.sup.+] ([BK.sub.Ca]) channels in smooth muscle cells are unique because they integrate changes in both intracellular [Ca.sup.2+] and membrane potential. Protein kinases such as cAMP-dependent protein kinase, cGMP-dependent protein kinase and protein kinase C can affect tissue function by `tuning' the apparent [Ca.sup.2+-] and/or voltage-sensitivity of the [BK.sub.Ca] channel to physiological changes in both [Ca.sup.2+] concentrations and membrane potential. However, despite the central importance of kinase-mediated modulation of [BK.sub.Ca] channels in different smooth muscle tissues, many key issues, including the sites and mechanisms of actions of protein kinases, remain unresolved. In this article, the role of protein kinases in the regulation of [BK.sub.Ca] channels is discussed.

We examined the effects of Rho kinase on contraction and intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in guinea pig trachealis by measuring isometric force and the fura 2 signal [340- to 380-nm fluorescence ratio (F340/F380)]. A Rho kinase inhibitor, Y-27632 (1-1,000 [micro]M), inhibited methacholine (MCh)-induced contraction, with a reduction in F340/ F380 in a concentration-dependent manner. The values of EC50 for contraction and F340/F380 induced by i [micro]M MCh with Y-27632 were 27.3 [+ or -] 5.1 and 524.1 [+ or -] 31.0 [micro]M, respectively. With 0.1 [micro]M MCh, the values for these parameters were decreased to 1.0 [+ or -] 0.1 and 98.2 [+ or -] 6.2 [micro]M, respectively. Tension-F340/F380 curves for MCh indicated that Y-27632 caused an ~50% inhibition of MCh-induced contraction, without a reduction in F340/F380. These effects of Y-27632 were not inhibited by a protein kinase C inhibitor, GF-109203X. Our results indicate that inhibition of Rho kinase attenuates both [Ca.sup.2+] sensitization and [[[Ca.sup.2+]].sub.i]. methacholine; calcium sensitivity; fura 2; protein kinase C; small G proteins

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block [Ca.sup.2+] mobilization and Na.sup.+]-[K.sup.+]-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [[[Ca.sup.2+]].sub.i] resembled that induced by the [Ca.sup.2+] channel blocker nifedipine and were reversed specifically by the [Ca.sup.2+] channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of [Ca.sup.2+] influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined. smooth muscle tone; smooth muscle cells; phorbol esters; calcium influx; calcium channel activator

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 [+ or -] 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 [micro]M) 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. endothelin-1; PD-98059; extracellular signal-regulated kinase; mitogen-activated protein kinase; platelet-derived growth factor; smooth muscle cells

Importance of PKC and PI3Ks in ethanol-induced contraction of cerebral arterial smooth muscle. Am J Physiol Heart Circ Physiol 280: H2144-H2152, 2001.--We investigated the relationships of two potential intracellular signaling pathways, protein kinase C (PKC) and phosphatidylinositol 3-kinases (PI3Ks), to ethanol-induced contractions in cerebral arteries. Ethanol (20-200 mM) induces concentration-dependent constriction in isolated canine basilar arteries that is inhibited in a concentration-dependent manner by pretreatment of these vessels with [10.sup.-9] -[10.sup.-3] M Go-6976 (an antagonist selective for PKC-[Alpha] and PKC-[Beta]I), [10.sup.-10] -[10.sup.-4] M bisindolylmaleimide I (a specific antagonist of PKC), and [10.sup.-10] -[10.sup.-4] M wortmannin or [10.sup.-8] -[10.sup.-2] M LY-294002 (selective antagonists of PI3Ks). Ethanol-induced increases in intracellular [Ca.sup.2+] concentration (from ~100 to ~500 nM) in canine basilar smooth muscle cells are also suppressed markedly (~20-70%) in the presence of a similar concentration range of Go-6976, bisindolymaleimide I, wortmannin, or LY-294002. This study suggests that activation of PKC isoforms and PI3Ks appears to be an important signaling pathway in ethanol-induced vasoconstriction of cerebral blood vessels. canine basilar arteries; cerebrovasospasm; ethanol; stroke; protein kinase C; phosphatidylinositol 3-kinases

ERK activation and mitogenesis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 280: L1019-L1029, 2001.--Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. [PGE.sub.2] and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma. asthma; antisense extracellular signal-regulated kinase; p90 ribosomal S6 kinase; c-Fos; cyclin D1

Activation of class IA PI3K stimulates DNA synthesis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 280: L1009-L1018, 2001.--The precise mechanisms that regulate increases in airway smooth muscle (ASM) mass in asthma are unknown. This study determined whether class IA phosphatidylinositol 3-kinase (PI3K) is sufficient to stimulate DNA synthesis and characterized the PI3K isoforms expressed in human ASM cells. ASM cells express class IA, II, and III PI3K but not class IB. Because thrombin induces ASM cell proliferation, we investigated whether thrombin can stimulate class IA PI3K. Transient transfection of ASM cells with hemagglutinin-tagged p85 PI3K followed by immunostaining revealed that in quiescent cells, p85 was expressed diffusely in the cytoplasm and after stimulation with thrombin p85 translocated to the cell membrane. Microinjection of ASM cells with a dominant negative class IA PI3K inhibited thrombin-induced DNA synthesis by 30% and epidermal growth factor (EGF)- or serum-induced DNA synthesis by 13 and 28%, respectively (P [is less than] 0.05 by [chi square] analysis). In parallel experiments, transfection or microinjection of cells with constitutively active PI3K markedly increased DNA synthesis in transfected cells 10.5-fold and in microinjected cells 12.7-fold (P [is less than] 0.05 by [chi square] analysis) compared with cells transfected or microinjected with control plasmid. Interestingly, constitutively active PI3K augmented EGF-induced DNA synthesis but had little effect on that induced by serum or thrombin in ASM cells. Collectively, these data suggest that class IA PI3K is activated by thrombin and is sufficient to induce ASM cell growth. phosphatidylinositol 3-kinase; airway remodeling; asthma; signaling

Rapacon-Baker, Michelle, Fan Zhang, Michael L. Pucci, Hui Guan, and Alberto Nasjletti. Expression of myogenic constrictor tone in the aorta of hypertensive rats. Am J Physiol Regulatory Integrative Comp Physiol 280: R968-R975, 2001.--We investigated the effect of intraluminal pressure or stretch on the development of tone in the descending thoracic aorta from rats with aortic coarctation-induced hypertension of 7-14 days duration. Increments of pressure [is greater than] 100 mmHg decreased the diameter of thoracic aortas from hypertensive but not from normotensive rats. The pressure-induced constriction was not demonstrable in vessels superfused with calcium-free buffer. Stretched rings of aorta from hypertensive rats exhibited a calcium-dependent constrictor tone accompanied by elevated calcium influx that varied in relation to the degree of stretch. Blockers of L-type calcium channels and inhibitors of protein kinase C reduced both basal tone and calcium influx in aortic rings of hypertensive rats. Hence, the thoracic aorta of hypertensive rats expresses a pressure- and stretch-activated constrictor mechanism that relies on increased calcium influx through L-type calcium channels via a protein kinase C-regulated pathway. The expression of such a constrictor mechanism is suggestive of acquired myogenic behavior. myogenic tone; protein kinase C; calcium influx; aortic smooth muscle; hypertension

Cao, W., Q. Chen, U. D. Sohn, N. Kim, M. T. Kirber, K. M. Harnett, J. Behar, and P. Biancani. [Ca.sup.2+]-induced contraction of cat esophageal circular smooth muscle cells. Am J Physiol Cell Physiol 280: C980-C992, 2001.--ACh-induced contraction of esophageal circular muscle (ESO) depends on [Ca.sup.2+] influx and activation of protein kinase C[Epsilon] (PKC[Epsilon]). PKC[Epsilon], however, is known to be [Ca.sup.2+] independent. To determine where [Ca.sup.2+] is needed in this PKC[Epsilon]-mediated contractile pathway, we examined successire steps in [Ca.sup.2+]-induced contraction of ESO muscle cells permeabilized by saponin. [Ca.sup.2+] (0.2-1.0 [micro]M) produced a concentration-dependent contraction that was antagonized by antibodies against PKC[Epsilon] (but not by PKC[Beta]II or PKC[Gamma] antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDP[Beta]s. Addition of 1 [micro]M [Ca.sup.2+] to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). [Ca.sup.2+]-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by [AACOCF.sub.3] (cytosolic phospholipase [A.sub.2] inhibitor). These data suggest that [Ca.sup.2+] may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKC[Epsilon], which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTP[Gamma]S augmented [Ca.sup.2+]-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular [Ca.sup.2+] and G protein activation), producing DAG and AA, and activating PKC[Epsilon]-dependent mechanisms to cause contraction. calcium; smooth muscle; protein kinase C; phospholipase C; phospholipase D; myosin phosphorylation