Receptor-independent activation of Rho-kinase-mediated calcium sensitisation in smooth muscle.
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ABSTRACT:
SUBMITTER: Ayman S
PROVIDER: S-EPMC1573988 | biostudies-other | 2003 Aug
REPOSITORIES: biostudies-other
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Project description:Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A(2)beta (iPLA(2)beta) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA(2)beta mRNA, protein, and iPLA(2) activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA(2)beta protein up-regulation. Inhibiting iPLA(2)beta activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA(2)beta in iPLA(2)beta-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA(2)beta inhibition. Moreover, increases in iPLA(2) activity and iPLA(2)beta protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA(2)beta, but not iPLA(2)gamma, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA(2)beta up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.
| S-EPMC2838285 | biostudies-literature
Project description:Efficient mechanotransduction in vascular smooth muscle cells (VSMCs) is intimately coupled to physical coupling of the cell to extracellular matrix proteins (ECM) by integrins. Integrin adhesion receptors are essential for normal vascular function and defective integrin signaling is associated with cardiovascular disease. However, less is known about the mechanism of integrin activation in VSMCs in relation to vasoregulation. Our laboratory previously demonstrated that the vasoconstrictor Angiotensin II increases VSMC stiffness in concert with enhanced adhesion to fibronectin (FN), indicating an important role for adhesion in contraction. However, the mechanism of this coordination remains to be clarified. In this study, intracellular Ca2+ ([Ca2+]i) was hypothesized to link integrin activation through inside-out signaling pathways leading to enhanced adhesion in response to AII. By using atomic force microscopy (AFM) with an anti-?5 antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an ?5 integrin antibody. This indicated that increases in cell adhesion to FN induced by AII were occurring through an integrin activation process, as increased membrane integrin expression/receptor density would have been accompanied by increased adhesion to the anti-?5 antibody. Further studies were performed using either KCl or BAPTA-AM to modulate the level of [Ca2+]i. After KCl, VSMCs showed a rapid transient increase in cell stiffness as well as cell adhesion to FN, and these two events were synchronized with superimposed transient increases in the level of [Ca2+]i, which was measured using the Ca2+ indicator, fluo-4. These relationships were unaffected in VSMCs pretreated with the myosin light chain kinase inhibitor, ML-7. In contrast, unstimulated VSMCs incubated with an intracellular calcium chelator, BAPTA-AM, showed reduced cell adhesion to FN as well the expected decrease in [Ca2+]i. These data suggest that in VSMCs, integrin activation is linked to signaling events tied to levels of [Ca2+]i while being less dependent on events at the level of contractile protein activation. These findings provide additional evidence to support a role for adhesion in VSMC contraction and suggest that following cell contractile activation, that adhesion may be regulated in tandem with the contractile event.
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Project description:BackgroundThe Ca2+-independent contraction of vascular smooth muscle is a leading cause of cardiovascular and cerebrovascular spasms. In the previous study, we demonstrated the involvement of Src family protein tyrosine kinase Fyn and Rho-kinase in the sphingosylphosphorylcholine (SPC)-induced abnormal and Ca2+-independent contraction of vascular smooth muscle, but the specific mechanism has not been completely clarified.MethodsPaxillin knockdown human coronary artery smooth muscle cells (CASMCs) and smooth muscle-specific paxillin knockout mice were generated by using paxillin shRNA and the tamoxifen-inducible Cre-LoxP system, respectively. CASMCs contraction was observed by time-lapse recording. The vessel contractility was measured by using a myography assay. Fyn, Rho-kinase, and myosin light chain activation were assessed by immunoprecipitation and western blotting. The paxillin expression and actin stress fibers were visualized by histological analysis and immunofluorescent staining.ResultsThe SPC-induced abnormal contraction was inhibited in paxillin knockdown CASMCs and arteries of paxillin knockout mice, indicating that paxillin is involved in this abnormal contraction. Further study showed that paxillin knockdown inhibited the SPC-induced Rho-kinase activation without affecting Fyn activation. In addition, paxillin knockdown significantly inhibited the SPC-induced actin stress fiber formation and myosin light chain phosphorylation. These results suggest that paxillin, as an upstream molecule of Rho-kinase, is involved in the SPC-induced abnormal contraction of vascular smooth muscle.ConclusionsThe present study demonstrated that paxillin participates in the SPC-induced abnormal vascular smooth muscle contraction by regulating Rho-kinase activation. Video Abstract.
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Project description:OBJECTIVE: To investigate whether Tumor Necrosis Factor-alpha (TNF?) is capable of activating Rho kinase pathway which leads to smooth muscle cell proliferation and the intervention function of Rosuvastatin, and clarify the mechanism and intervention manner of anti-atherosclerosis by Rosuvastatin. METHODS: Wistar neonate rat smooth muscle cells were cultured, and the activity of cell proliferation was determined by methyl thiazolyl tetrazolium (MTT). The expression of Rho kinase genes after the stimulation of TNF? was evaluated by RT-PCR. Western blot method was used to measure the protein expression of proliferating cell nuclear antigen (PCNA) after TNF? stimulation and Rosuvastatin intervention in smooth muscle cell. RESULTS: The TNF? stimulation significantly enhanced the expression of Rho kinase and increased the expression of PCNA protein in smooth muscle cells (P < 0.05). These effects were positively correlated with prolonged treatment whereas additional Rosuvastatin administration inhibited the above-mentioned effects (P < 0.05). CONCLUSIONS: The activation of TNF? mediated Rho kinase signaling pathway can significantly promote smooth muscle cell proliferation, and Rosuvastatin can not only inhibit this pathway but also the induced proliferation.
| S-EPMC3418909 | biostudies-other
Project description:Background and purposeThe Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels.Experimental approachWe examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques.Key resultsThe Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTP?S-activated non-selective cation currents.Conclusions and implicationsThe Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus.
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Project description:Sphingosine-1-phosphate (S1P) has been shown to modulate intracellular Ca(2+) through both G protein-coupled receptors and intracellular second messenger pathways. The precise mechanism by which S1P activates store-operated calcium entry (SOCE) in vascular smooth muscle cells (VSMCs) has not been fully characterized. Because sphingolipids and Ca(2+) modulate proliferation and constriction in VSMCs, characterizing the connection between S1P and SOCE may provide novel therapeutic targets for vascular diseases. We found that S1P triggered STIM1 puncta formation and SOCE in VSMCs. S1P-activated SOCE was inhibited by 2-aminoethoxydiphenyl borate (2-APB), diethylstilbestrol (DES), and gadolinium (Gd(3+)). SOCE was observed in VSMCs lacking either S1P(2) or S1P(3) receptors, suggesting that S1P acts via multiple signaling pathways. Indeed, both extracellular and intracellular S1P application increased the total internal reflection fluorescence signal in VSMCs cells transfected with STIM1-yellow fluorescent protein in a 2-APB-sensitive manner. These data, and the fact that 2-APB, DES, and Gd(3+) all inhibited S1P-induced cerebral artery constriction, suggest that SOCE modulates S1P-induced vasoconstriction in vivo. Finally, S1P-induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P(1), and S1P(3) receptor mRNA. These data demonstrate that S1P can act through both receptors and a novel intracellular pathway to activate SOCE. Because S1P-induced SOCE contributes to vessel constriction and is increased in proliferative VSMCs, it is likely that S1P/SOCE signaling in proliferative VSMCs may play a role in vascular dysfunction such as atherosclerosis and diabetes.
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Project description:Leptin has been implicated as a key upstream mediator of pathways associated with coronary vascular dysfunction and disease. The purpose of this investigation was to test the hypothesis that leptin modifies the coronary artery proteome and promotes increases in coronary smooth muscle contraction and proliferation via influences on Rho kinase signaling. Global proteomic assessment of coronary arteries from lean swine cultured with obese concentrations of leptin (30 ng/mL) for 3 days revealed significant alterations in the coronary artery proteome (68 proteins) and identified an association between leptin treatment and calcium signaling/contraction (four proteins) and cellular growth and proliferation (35 proteins). Isometric tension studies demonstrated that both acute (30 min) and chronic (3 days, serum-free media) exposure to obese concentrations of leptin potentiated depolarization-induced contraction of coronary arteries. Inhibition of Rho kinase significantly reduced leptin-mediated increases in coronary artery contractions. The effects of leptin on the functional expression of Rho kinase were time-dependent, as acute treatment increased Rho kinase activity while chronic (3 day) exposure was associated with increases in Rho kinase protein abundance. Proliferation assays following chronic leptin administration (8 day, serum-containing media) demonstrated that leptin augmented coronary vascular smooth muscle proliferation and increased Rho kinase activity. Inhibition of Rho kinase significantly reduced these effects of leptin. Taken together, these findings demonstrate that leptin promotes increases in coronary vasoconstriction and smooth muscle proliferation and indicate that these phenotypic effects are associated with alterations in the coronary artery proteome and dynamic effects on the Rho kinase pathway.
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Project description:Nonvisual opsin (OPN) receptors have recently been implicated in blue light-mediated photorelaxation of smooth muscle in various organs. Since photorelaxation has not yet been demonstrated in airway smooth muscle (ASM) or in human tissues, we questioned whether functional OPN receptors are expressed in mouse and human ASM. mRNA, encoding the OPN 3 receptor, was detected in both human and mouse ASM. To demonstrate the functionality of the OPN receptors, we performed wire myography of ex vivo ASM from mouse and human upper airways. Blue light-mediated relaxation of ACh-preconstricted airways was intensity and wavelength dependent (maximum relaxation at 430-nm blue light) and was inhibited by blockade of the large-conductance calcium-activated potassium channels with iberiotoxin. We further implicated OPN receptors as key mediators in functional photorelaxation by demonstrating increased relaxation in the presence of a G protein receptor kinase 2 inhibitor or an OPN chromophore (9- cis retinal). We corroborated these responses in peripheral airways of murine precision-cut lung slices. This is the first demonstration of photorelaxation in ASM via an OPN receptor-mediated pathway.
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Project description:The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF?) increases expression of CD38 (a membrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca(2+) ([Ca(2+)]i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae and their constituent protein caveolin-1 are important for ASM [Ca(2+)]i regulation, which is further enhanced by TNF?. Whether caveolae and CD38 are functionally linked in mediating TNF? effects is unknown. In this regard, whether the related cavin proteins (cavin-1 and -3) that maintain structure and function of caveolae play a role is also not known. In the present study, we hypothesized that TNF? effects on CD38 expression and function in human ASM involve caveolae. Caveolar fractions from isolated human ASM cells expressed CD38 and its expression was upregulated by exposure to 20ng/ml TNF? (48h). ASM cells expressed cavin-1 and cavin-3, which were also upregulated by TNF?. Knockdown of caveolin-1, cavin-1 or cavin-3 (using siRNA) all significantly reduced CD38 expression and ADP-ribosyl cyclase activity in the presence or absence of TNF?. Furthermore, caveolin-1, cavin-1 and cavin-3 siRNAs reduced [Ca(2+)]i responses to histamine under control conditions, and blunted the enhanced [Ca(2+)]i responses in TNF?-exposed cells. These data demonstrate that CD38 is expressed within caveolae and its function is linked to the caveolar regulatory proteins caveolin-1, cavin-1 and -3. The link between caveolae and CD38 is further enhanced during airway inflammation demonstrating the important role of caveolae in regulation of [Ca(2+)]i and contractility in the airway.
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Project description:Myogenic responses by resistance vessels are a key component of autoregulation in brain, thus playing a crucial role in regulating cerebral blood flow and protecting the blood-brain barrier against potentially detrimental elevations in blood pressure. Although cerebrovascular disease is often accompanied by alterations in myogenic responses, mechanisms that control these changes are poorly understood. Peroxisome proliferator-activated receptor γ has emerged as a regulator of vascular tone. We hypothesized that interference with peroxisome proliferator-activated receptor γ in smooth muscle would augment myogenic responses in cerebral arteries. We studied transgenic mice expressing a dominant-negative mutation in peroxisome proliferator-activated receptor γ selectively in smooth muscle (S-P467L) and nontransgenic littermates. Myogenic tone in middle cerebral arteries from S-P467L was elevated 3-fold when compared with nontransgenic littermates. Rho kinase is thought to play a major role in cerebrovascular disease. The Rho kinase inhibitor, Y-27632, abolished augmented myogenic tone in middle cerebral arteries from S-P467L mice. CN-03, which modifies RhoA making it constitutively active, elevated myogenic tone to ≈60% in both strains, via a Y-27632-dependent mechanism. Large conductance Ca(2+)-activated K(+) channels (BKCa) modulate myogenic tone. Inhibitors of BKCa caused greater constriction in middle cerebral arteries from nontransgenic littermates when compared with S-P467L. Expression of RhoA or Rho kinase-I/II protein was similar in cerebral arteries from S-P467L mice. Overall, the data suggest that peroxisome proliferator-activated receptor γ in smooth muscle normally inhibits Rho kinase and promotes BKCa function, thus influencing myogenic tone in resistance arteries in brain. These findings have implications for mechanisms that underlie large- and small-vessel disease in brain, as well as regulation of cerebral blood flow.
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