Tissue length modulates "stimulated actin polymerization," force augmentation, and the rate of swine carotid arterial contraction

"Stimulated actin polymerization" has been proposed to be involved in force augmentation, in which prior submaximal activation of vascular smooth muscle increases the force of a subsequent maximal contraction by ～15%. In this study, we altered stimulated actin polymerization by adjusting tissue length and then measured the effect on force augmentation. At optimal tissue length (1.0 L(o)), force augmentation was observed and was associated with increased prior stimulated actin polymerization, as evidenced by increased prior Y118 paxillin phosphorylation without changes in prior S3 cofilin or cross-bridge phosphorylation. Tissue length, per se, regulated Y118 paxillin, but not S3 cofilin, phosphorylation. At short tissue length (0.6 L(o)), force augmentation was observed and was associated with increased prior stimulated actin polymerization, as evidenced by reduced prior S3 cofilin phosphorylation without changes in Y118 paxillin or cross-bridge phosphorylation. At long tissue length (1.4 L(o)), force augmentation was not observed, and there were no prior changes in Y118 paxillin, S3 cofilin, or cross-bridge phosphorylation. There were no significant differences in the cross-bridge phosphorylation transients before and after the force augmentation protocol at all three lengths tested. Tissues contracted faster at longer tissue lengths; contractile rate correlated with prior Y118 paxillin phosphorylation. Total stress, per se, predicted Y118 paxillin phosphorylation. These data suggest that force augmentation is regulated by stimulated actin polymerization and that stimulated actin polymerization is regulated by total arterial stress. We suggest that K(+) depolarization first leads to cross-bridge phosphorylation and contraction, and the contraction-induced increase in mechanical strain increases Y118 paxillin phosphorylation, leading to stimulated actin polymerization, which further increases force, i.e., force augmentation and, possibly, latch.     

Force suppression and the crossbridge cycle in swine carotid artery

Cyclic nucleotides can relax arterial smooth muscle without reductions in crossbridge phosphorylation, a process termed force suppression. There are two potential mechanisms for force suppression: 1) phosphorylated crossbridges binding to thin filaments could be inhibited or 2) the attachment of thin filaments to anchoring structures could be disrupted. These mechanisms were evaluated by comparing histamine-stimulated swine arterial smooth muscle with and without forskolin-induced force suppression and with and without latrunculin-A-induced actin filament disruption. At matched force, force suppression was associated with higher crossbridge phosphorylation and shortening velocity at low loads when compared with tissues without force suppression. Shortening velocity at high loads, noise temperature, hysteresivity, and stiffness did not differ with and without force suppression. These data suggest that crossbridge phosphorylation regulates the crossbridge cycle during force suppression. Actin disruption with latrunculin-A was associated with higher crossbridge phosphorylation when compared with tissues without actin disruption. Shortening velocity, noise temperature, hysteresivity, and stiffness did not differ with and without actin disruption. These data suggest that actin disruption interferes with regulation of crossbridge cycling by crossbridge phosphorylation. Stiffness was linearly dependent on stress, suggesting that the force per attached crossbridge was not altered with force suppression or actin disruption. These data suggest a difference in the mechanical characteristics observed during force suppression and actin disruption, implying that force suppression does not mechanistically involve actin disruption. These data are most consistent with a model where force suppression involves the inhibition of phosphorylated crossbridge binding to thin filaments. force suppression; heat shock protein 20; vascular smooth muscle     

The Small GTPase RhoA Regulates the Contraction of Smooth Muscle Tissues by Catalyzing the Assembly of Cytoskeletal Signaling Complexes at Membrane Adhesion Sites

The activation of the small GTPase RhoA is necessary for ACh-induced actin polymerization and airway smooth muscle (ASM) contraction, but the mechanism by which it regulates these events is unknown. Actin polymerization in ASM is catalyzed by the actin filament nucleation activator, N-WASp and the polymerization catalyst, Arp2/3 complex. Activation of the small GTPase cdc42, a specific N-WASp activator, is also required for actin polymerization and tension generation. We assessed the mechanism by which RhoA regulates actin dynamics and smooth muscle contraction by expressing the dominant negative mutants RhoA T19N and cdc42 T17N, and non-phosphorylatable paxillin Y118/31F and paxillin ΔLD4 deletion mutants in SM tissues. Their effects were evaluated in muscle tissue extracts and freshly dissociated SM cells. Protein interactions and cellular localization were analyzed using proximity ligation assays (PLA), immunofluorescence, and GTPase and kinase assays. RhoA inhibition prevented ACh-induced cdc42 activation, N-WASp activation and the interaction of N-WASp with the Arp2/3 complex at the cell membrane. ACh induced paxillin phosphorylation and its association with the cdc42 GEFS, DOCK180 and α/βPIX. Paxillin tyrosine phosphorylation and its association with βPIX were RhoA-dependent, and were required for cdc42 activation. The ACh-induced recruitment of paxillin and FAK to the cell membrane was dependent on RhoA. We conclude that RhoA regulates the contraction of ASM by catalyzing the assembly and activation of cytoskeletal signaling modules at membrane adhesomes that initiate signaling cascades that regulate actin polymerization and tension development in response to contractile agonist stimulation. Our results suggest that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to agonist -induced smooth muscle contraction.     

Role of Rho in Ca(2+)-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle

We investigatedwhether Rho activation is required for Ca²⁺-insensitivepaxillin phosphorylation, myosin light chain (MLC) phosphorylation, andcontraction in tracheal muscle. Tyrosine-phosphorylated proteins havebeen implicated in the Ca²⁺-insensitive contractileactivation of smooth muscle tissues. The contractile activation oftracheal smooth muscle increases tyrosine phosphorylation of thecytoskeletal proteins paxillin and focal adhesion kinase. Paxillin isimplicated in integrin-mediated signal transduction pathways thatregulate cytoskeletal organization and cell motility. In fibroblastsand other nonmuscle cells, paxillin tyrosine phosphorylation depends onthe activation of Rho and is inhibited by cytochalasin, an inhibitor ofactin polymerization. In permeabilized muscle strips, we found that AChinduced Ca²⁺-insensitive contraction, MLC phosphorylation,and paxillin tyrosine phosphorylation. Ca²⁺-insensitivecontraction and MLC phosphorylation induced by ACh were inhibited by C3transferase, an inhibitor of Rho activation; however, C3 transferasedid not inhibit paxillin tyrosine phosphorylation. Ca²⁺-insensitive paxillin tyrosine phosphorylation was alsonot inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D,or by the inhibition of MLC phosphorylation. We conclude that, intracheal smooth muscle, Rho mediates Ca²⁺-insensitivecontraction and MLC phosphorylation but that Rho is not required forCa²⁺-insensitive paxillin tyrosine phosphorylation.Paxillin phosphorylation also does not require actomyosin activation,nor is it inhibited by the actin filament capping agent cytochalasin D.

     

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

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

Relationship between paxillin and myosin phosphorylation during muscarinic stimulation of smooth muscle

The tyrosinephosphorylation of paxillin increases in association with forcedevelopment during tracheal smooth muscle contraction, suggesting thatpaxillin plays a role in the contractile activation of smooth muscle[Z. L. Wang, F. M. Pavalko, and S. J. Gunst. Am.J. Physiol. 271 (CellPhysiol. 40): C1594-C1602, 1996]. We compared the Ca²⁺ sensitivity ofthe tyrosine phosphorylation of paxillin and myosin light chain (MLC)phosphorylation in tracheal muscle and evaluated whether MLCphosphorylation is necessary to induce paxillin phosphorylation. Ca²⁺-depleted muscle strips werestimulated with10⁷-10⁴M acetylcholine (ACh) in 0, 0.05, 0.1, or 0.5 mM extracellular Ca²⁺. In the absence ofextracellular Ca²⁺,10⁴ M ACh induced a maximalincrease in paxillin phosphorylation without increasing MLCphosphorylation or force. Increases in extracellularCa²⁺ concentration did not furtherincrease paxillin phosphorylation. However, during stimulation with10⁶ M ACh, paxillinphosphorylation increased with increases in extracellular Ca²⁺ concentration. We concludethat the tyrosine phosphorylation of paxillin can be stimulated bysignaling pathways that do not depend onCa²⁺ mobilization and that theactivation of contractile proteins is not required to elicit paxillinphosphorylation.

     

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or beta-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in beta-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.     

Paxillin is a novel cellular target for converging Helicobacter pylori-induced cellular signaling

Paxillin is involved in the regulation of Helicobacter pylori-mediated gastric epithelial cell motility. We investigated the signaling pathways regulating H. pylori-induced paxillin phosphorylation and the effect of the H. pylori virulence factors cag pathogenicity island (PAI) and outer inflammatory protein (OipA) on actin stress fiber formation, cell phenotype, and IL-8 production. Gastric cell infection with live H. pylori induced site-specific phosphorylation of paxillin tyrosine (Y) 31 and Y118 in a time- and concentration-dependent manner. Activated paxillin localized in the cytoplasm at the tips of H. pylori-induced actin stress fibers. Isogenic oipA mutants significantly reduced paxillin phosphorylation at Y31 and Y118 and reduced actin stress fiber formation. In contrast, cag PAI mutants only inhibited paxillin Y118 phosphorylation. Silencing of epidermal growth factor receptor (EGFR), focal adhesion kinase (FAK), or protein kinase B (Akt) expression by small-interfering RNAs or inhibiting kinase activity of EGFR, Src, or phosphatidylinositol 3-kinase (PI3K) markedly reduced H. pylori-induced paxillin phosphorylation and morphologic alterations. Reduced FAK expression or lack of Src kinase activity suppressed H. pylori-induced IL-8 production. Compared with infection with the wild type, infection with the cag PAI mutant and oipA mutant reduced IL-8 production by nearly 80 and 50%. OipA-induced IL-8 production was FAK- and Src-dependent, although a FAK/Src-independent pathway for IL-8 production also exists, and the cag PAI may be mainly involved in this pathway. We propose paxillin as a novel cellular target for converging H. pylori-induced EGFR, FAK/Src, and PI3K/Akt signaling to regulate cytoskeletal reorganization and IL-8 production in part, thus contributing to the H. pylori-induced diseases.     

Depletion of focal adhesion kinase by antisense depresses contractile activation of smooth muscle

Focal adhesion kinase (FAK)undergoes tyrosine phosphorylation in response to the contractilestimulation of tracheal smooth muscle. We hypothesized that FAK mayplay an important role in signaling pathways that regulate smoothmuscle contraction. FAK antisense or FAK sense was introduced intomuscle strips by reversible permeabilization, and strips were incubatedwith antisense or sense for 7 days. Antisense decreased FAK expressioncompared with that in untreated and sense-treated tissues, but it didnot affect the expression of vinculin or myosin light chain kinase. Increases in force, intracellular free Ca²⁺, and myosinlight chain phosphorylation in response to stimulation with ACh or KClwere depressed in FAK-depleted tissues, but FAK depletion did notaffect the activation of permeabilized tracheal muscle strips withCa²⁺. The tyrosine phosphorylation of paxillin, a substratefor FAK, was also significantly reduced in FAK-depleted strips. Weconclude that FAK is a necessary component of the signaling pathwaysthat regulate smooth muscle contraction and that FAK plays a role in regulating intracellular free Ca²⁺ and myosin light chain phosphorylation.

     

Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery

Increases in cyclic nucleotide levels induce smooth muscle relaxation by deactivation [reductions in myosin regulatory light chain (MRLC) phosphorylation (e.g., by reduced [Ca²⁺])] or force suppression (reduction in force without reduction in MRLC phosphorylation). Ser¹⁶-heat shock protein 20 (HSP20) phosphorylation is the proposed mediator of force suppression. We evaluated three potential hypotheses whereby Ser¹⁶-HSP20 phosphorylation could regulate smooth muscle force: 1) a threshold level of HSP20 phosphorylation could inactivate a thin filament as a whole, 2) phosphorylation of a single HSP20 could fully inactivate a small region of a thin filament, or 3) HSP20 phosphorylation could weakly inhibit myosin binding at either the thin- or thick-filament level. We tested these hypotheses by analyzing the dependence of force on Ser¹⁶-HSP20 phosphorylation in swine carotid media. First, we determined that swine HSP20 has a second phosphorylation site at Ser¹⁵⁷. Ser¹⁵⁷-HSP20 phosphorylation values were high and did not change during contractile activation or forskolin-induced relaxation. Forskolin significantly increased Ser¹⁶-HSP20 phosphorylation. The relationship between Ser¹⁶-HSP20 phosphorylation and force remained linear and was shifted downward in partially activated muscles relaxed with forskolin. Neither forskolin nor nitroglycerin induced actin depolymerization as detected using the F/G-actin ratio method in smooth muscle homogenates. These results suggest that force suppression does not occur in accordance with the first hypothesis (inactivation of a thin filament as a whole). Our data are more consistent with the second and third hypotheses that force suppression is mediated by full or partial inhibition of local myosin binding at the thin- or thick-filament level. cAMP; cGMP; nitric oxide; vascular smooth muscle     

Load-dependent kinetics of force production by smooth muscle myosin measured with optical tweezers

Muscle contraction is driven by the cyclical interaction of myosin with actin, coupled with ATP hydrolysis. Myosin attaches to actin, forming a crossbridge that produces force and movement as it tilts or rocks into subsequent bound states before finally detaching. It has been hypothesized that the kinetics of one or more of these mechanical transitions are dependent on load, allowing muscle to shorten quickly under low load, but to sustain tension economically, with slowly cycling crossbridges under high load conditions. The idea that muscle biochemistry depends on mechanical output is termed the 'Fenn effect'. However, the molecular details of how load affects the kinetics of a single crossbridge are unknown. Here, we describe a new technique based on optical tweezers to rapidly apply force to a single smooth muscle myosin crossbridge. The crossbridge produced movement in two phases that contribute 4 nm + 2 nm of displacement. Duration of the first phase depended in an exponential manner on the amplitude of applied load. Duration of the second phase was much less affected by load, but was significantly shorter at high ATP concentration. The effect of load on the lifetime of the bound crossbridge is to prolong binding when load is high, but to accelerate release when load is low or negative.     

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca²⁺ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction. tissue transfection; plasmids; cytoskeleton; talin; immunofluorescence     

Role of Cyclic Nucleotide-Dependent Actin Cytoskeletal Dynamics: [Ca2+]i and Force Suppression in Forskolin-Pretreated Porcine Coronary Arteries

Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca²⁺]_i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca²⁺]_i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.     

Central role of paxillin phosphorylation in regulation of LFA-1 integrins activity and lymphocyte migration

Coordinated changes of actin cytoskeleton and cell adhesion accompany maturation of lymphoid cells, their migration through lymphoid organs and to sites of inflammation, as well as metastasis of transformed cells. Here we discuss the central role of the actin-regulating adaptor protein, paxillin, during lymphocyte transition from a polarized, motile cell phenotype with partially active LFA-1 integrins to a round and immobile one with fully active LFA-1. In Baf3 murine pro-B lymphocytes, the former phenotype is induced by IL-3 that stimulates a FAK-mediated phosphorylation of paxillin at tyrosines (Y) 31 and 118 and a consequent Rac1 activation. Rearrangements of actin cytoskeleton that lead to the cell''s acquisition of a spherical shape and LFA-1 activation are achieved upon activation of PKC-δ that binds and directly phosphorylates paxillin at threonine (T) 538 with consequent RhoA activation. This is accompanied by dephosphorylation of paxillin Y31/118 and by Rac1 inactivation. We propose a model of signaling cascades that reflects the interplay between the IL-3- and PKC-δ-mediated pathways.Key words: lymphocytes, paxillin, actin, Rac1, RhoA, LFA-1, PKC-δ     

Vasodilator-stimulated Phosphoprotein (VASP) Regulates Actin Polymerization and Contraction in Airway Smooth Muscle by a Vinculin-dependent Mechanism

Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser¹⁵⁷ phosphorylation by different kinases. Inhibition of VASP Ser¹⁵⁷ phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser¹⁵⁷ mediates its localization at the membrane, but that VASP Ser¹⁵⁷ phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM.     

Paxillin phosphorylation controls invadopodia/podosomes spatiotemporal organization

In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.     

Effects of chronic portal hypertension on agonist-induced actin polymerization in small mesenteric arteries

The ability of arterial smooth muscle to respond to vasoconstrictor stimuli is reduced in chronic portal hypertension (PHT). Additional evidence supports the existence of a postreceptor defect in vascular smooth muscle excitation contraction coupling. However, the nature of this defect is unclear. Recent studies have shown that vasoconstrictor stimuli induce actin polymerization in smooth muscle and that the associated increase in F-actin is necessary for force development. In the present study we have tested the hypothesis that impaired actin polymerization contributes to reduced vasoconstrictor function in small mesenteric arteries derived from rats with chronic prehepatic PHT. In vitro studies were conducted on small mesenteric artery vessel rings isolated from normal and PHT rats. Isometric tension responses to incremental concentrations of phenylephrine were significantly reduced in PHT arteries. The ability to polymerize actin in portal hypertensive mesenteric arteries stimulated by phenylephrine was attenuated compared with control. Inhibition of cAMP-dependent protein kinase (PKA) restored agonist-induced actin polymerization of arteries from PHT rats to normal levels. Depolymerization of actin in arteries from normal rats reduced maximal contractile force but not myosin phosphorylation, suggesting a key role for the dynamic regulation of actin polymerization in the maintenance of vascular smooth muscle contraction. We conclude that reductions in agonist-induced maximal force development of PHT vascular smooth muscle is due, in part, to impaired actin polymerization, and prolonged PKA activation may underlie these changes.     

The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.     

Myosin light chain phosphorylation and evidence for latchbridge formation in norepinephrine stimulated canine veins

The role of 20000 dalton myosin light chain phosphorylation in mediating venous smooth muscle contraction was studied in isolated preparations of canine jugular and femoral vein. One min 10(-5) M norepinephrine-induced contraction was accompanied by significant increases in phosphorylation (jugular - 21 to 46%; femoral - 19 to 54%) which were reversed within 10 min after agonist washout. During 40 min stimulation, phosphorylation and isometric force redevelopment rates declined to near basal levels while force was maintained. These findings implicate light chain phosphorylation as a prerequisite for initial tension development by crossbridge cycling in venous smooth muscle. However, long term tension can be maintained through a process similar to the latchbridge state in tracheal and arterial smooth muscle.