Activation Of Vascular Smooth Muscle Research Articles

Lysophosphatidic acid induces integrin activation in vascular smooth muscle and alters arteriolar myogenic vasoconstriction.

In vascular smooth muscle cells (VSMC) increased integrin adhesion to extracellular matrix (ECM) proteins, as well as the production of reactive oxygen species (ROS) are strongly stimulated by lysophosphatidic acid (LPA). We hypothesized that LPA-induced generation of ROS increases integrin adhesion to the ECM. Using atomic force microscopy (AFM) we determined the effects of LPA on integrin adhesion to fibronectin (FN) in VSMC isolated from rat (Sprague–Dawley) skeletal muscle arterioles. In VSMC, exposure to LPA (2 μM) doubled integrin-FN adhesion compared to control cells (P < 0.05). LPA-induced integrin-FN adhesion was reduced by pre-incubation with antibodies against β1 and β3 integrins (50 μg/ml) by 66% (P < 0.05). Inhibition of LPA signaling via blockade of the LPA G-protein coupled receptors LPAR1 and LPAR3 with 10 μM Ki16425 reduced the LPA-enhanced adhesion of VSCM to FN by 40% (P < 0.05). Suppression of ROS with tempol (250 μM) or apocynin (300 μM) also reduced the LPA-induced FN adhesion by 47% (P < 0.05) and 59% (P < 0.05), respectively. Using confocal microscopy, we observed that blockade of LPA signaling, with Ki16425, reduced ROS by 45% (P < 0.05), to levels similar to control VSMC unexposed to LPA. In intact isolated arterioles, LPA (2 μM) exposure augmented the myogenic constriction response to step increases in intraluminal pressure (between 40 and 100 mm Hg) by 71% (P < 0.05). The blockade of LPA signaling, with Ki16425, decreased the LPA-enhanced myogenic constriction by 58% (P < 0.05). Similarly, blockade of LPA-induced ROS release with tempol or gp91 ds-tat decreased the LPA-enhanced myogenic constriction by 56% (P < 0.05) and 55% (P < 0.05), respectively. These results indicate that, in VSMC, LPA-induced integrin activation involves the G-protein coupled receptors LPAR1 and LPAR3, and the production of ROS, and that LPA may play an important role in the control of myogenic behavior in resistance vessels through ROS modulation of integrin activity.

Abstract 297: Naturally Occurring Cullin-3 Mutations Decrease Substrate Ubiquitination and Acts Dominantly by Sequestering Substrate Adaptors for Cul3

Cullin-Ring ubiquitin Ligases (CRL) regulate protein turnover by promoting ubiquitination of substrate proteins targeting them for proteosomal degradation. Impaired Cullin-3 (Cul3) activity in vascular smooth muscle causes increased RhoA protein and Rho kinase signaling leading to impaired vasodilation and augmented contraction. PHAII patients with de novo mutations in Cul3 which cause skipping of exon 9 in Cul3 (Cul3Δ9) have early onset hypertension, but the mechanistic basis remains unclear. We hypothesize that Cul3Δ9 results in reduced RhoA ubiquitination and degradation, increasing RhoA activity. We cloned a mutant Cul3Δ9 cDNA by “splicing by overhang extension” PCR. Endogenous RhoA protein was increased in HEK293T cells expressing Flag-Cul3Δ9 compared with cells expressing wildtype Flag-Cul3 (Cul3WT). There was no defect in neddylation of Cul3Δ9. However, binding of Cul3Δ9 to RBX1, which is necessary for delivering ubiquitin to the substrate was impaired. Consistent with this, ubiquitination of epitope-tagged RhoA by Cul3Δ9 was significantly impaired compared with Cul3WT both in vivo and in vitro. Thus Cul3Δ9 exhibits impaired ubiquitination of Cul3 substrates. Interestingly, co-IP studies revealed that the substrate adaptor proteins Bacurd1, RhoBTB1 and KLHL3 bound more efficiently to Cul3Δ9 than Cul3WT. Moreover, we observed a significant increase in Cul3Δ9:Cul3WT heterodimers compared to Cul3Δ9:Cul3Δ9 homodimers. This is important because dimerization of Cul3 ubiquitin ligase complexes is required for efficient ubiquitination of some substrates. Thus Cul3Δ9 may act dominantly by interfering with Cul3WT by sequestering substrate adaptors from the CRL3 complex or by directly inhibiting the Cul3 dimer. We conclude that Cul3Δ9 exhibits impaired ubiquitin ligase activity and interferes with activity of Cul3WT. In smooth muscle, this would increase the RhoA pool that can be activated in response to contractile agonists. When combined with our previous data, we suggest that Cul3-mediated regulation of RhoA protein turnover controls both vascular function and arterial blood pressure. Patients carrying Cul3Δ9 mutations may exhibit elevated smooth muscle RhoA/Rho kinase activity contributing to their hypertension.

Phospholemman-Dependent Regulation of Na/K-Atpase Modulates Constriction and Relaxation in Aortic Smooth Muscle

The Na/K ATPase (NKA) plays a role in modulating vascular tone through both NO-dependent and independent pathways. Phospholemman (PLM) is a muscle-specific regulator of Na/K ATPase and, in cardiac muscle, links PKA, PKC, and NO-signaling pathways to NKA stimulation. PLM is expressed in vascular smooth muscle (VSM) and we hypothesized that its phosphorylation may modulate the vascular responses to agonists. We tested this using wild-type (WT), PLM knock-out (KO), and mutant PLM knock-in mice (3SA) in which the PKC and PKA phosphorylation sites (S63, S68, S69) are mutated to alanines. Agonist-induced constriction and relaxation were measured in aortic rings in an isometric wire myograph. Vascular NKA activity was assessed by K-induced relaxation (a surrogate measure of Na/K ATPase activation) and PLM phosphorylation by immunoblotting. In WT aortae, PLM phosphorylation (S63 and S68) was significantly increased in response to phenylephrine (PE) and K-induced relaxation was significantly higher in WT than KO mice (85±7% vs 58±4% of PE-induced pre-constriction; p<0.01), suggesting PLM regulates NKA activity in VSM. The dose-response curve for PE-induced constriction was profoundly shifted up and to the left in 3SA aortae compared to WT suggesting PLM phosphorylation normally limits constriction. Ouabain (300μM) completely abolished this difference. Pretreatment with L-NAME (300μM) also potentiated constrictor responses to PE to a greater extent in WT than 3SA vessels. Relaxation induced by the NO donor spermine NONOate was blunted in vessels from 3SA mice: 67±6% vs 84±2% (U46619-induced constriction; p<0.01 cf WT). In summary, isolated aortic rings from mice expressing unphosphorylatable PLM showed markedly elevated constriction in response to PE and attenuated relaxation in response to an NO donor. Thus, PLM phosphorylation regulates the activity of vascular NKA and this contributes to modulation of aortic constriction and relaxation.

CaMKIIδ-dependent Inhibition of cAMP-response Element-binding Protein Activity in Vascular Smooth Muscle

One transcription factor mediator of Ca(2+)-signals is cAMP response element-binding protein (CREB). CREB expression and/or activity negatively correlates with vascular smooth muscle (VSM) cell proliferation and migration. Multifunctional Ca(2+)/calmodulin-dependent protein kinases, including CaMKII, have been demonstrated to regulate CREB activity through both positive and negative phosphorylation events in vitro, but the function of CaMKII as a proximal regulator of CREB in intact cell systems, including VSM, is not clear. In this study, we used gain- and loss-of-function approaches to determine the function of CaMKIIδ in regulating CREB phosphorylation, localization, and activity in VSM. Overexpression of constitutively active CaMKIIδ specifically increased CREB phosphorylation on Ser(142) and silencing CaMKIIδ expression by siRNA or blocking endogenous CaMKII activity with KN93 abolished thrombin- or ionomycin-induced CREB phosphorylation on Ser(142) without affecting Ser(133) phosphorylation. CREB-Ser(142) phosphorylation correlated with transient nucleocytoplasmic translocation of CREB. Thrombin-induced CREB promoter activity, CREB binding to Sik1 and Rgs2 promoters, and Sik1/Rgs2 transcription were enhanced by a kinase-negative CaMKIIδ2 (K43A) mutant and inhibited by a constitutively active (T287D) mutant. Taken together, these studies establish negative regulation of CREB activity by endogenous CaMKIIδ-dependent CREB-Ser(142) phosphorylation and suggest a potential mechanism for CaMKIIδ/CREB signaling in modulating proliferation and migration in VSM cells.

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.

Insulin-like Growth Factor (IGF) Signaling Requires αvβ3-IGF1-IGF Type 1 Receptor (IGF1R) Ternary Complex Formation in Anchorage Independence, and the Complex Formation Does Not Require IGF1R and Src Activation

Integrin αvβ3 plays a role in insulin-like growth factor 1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk) in non-transformed cells in anchorage-dependent conditions. We reported previously that IGF1 directly binds to αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation in these conditions. The integrin-binding defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, whereas it still binds to IGF1R. We studied if IGF1 can induce signaling in anchorage-independent conditions in transformed Chinese hamster ovary cells that express αvβ3 (β3-CHO) cells. Here we describe that IGF1 signals were more clearly detectable in anchorage-independent conditions (polyHEMA-coated plates) than in anchorage-dependent conditions. This suggests that IGF signaling is masked by signals from cell-matrix interaction in anchorage-dependent conditions. IGF signaling required αvβ3 expression, and R36E/R37E was defective in inducing signals in polyHEMA-coated plates. These results suggest that αvβ3-IGF1 interaction, not αvβ3-extracellular matrix interaction, is essential for IGF signaling. Inhibitors of IGF1R, Src, AKT, and ERK1/2 did not suppress αvβ3-IGF-IGF1R ternary complex formation, suggesting that activation of these kinases are not required for ternary complex formation. Also, mutations of the β3 cytoplasmic tail (Y747F and Y759F) that block β3 tyrosine phosphorylation did not affect IGF1R phosphorylation or AKT activation. We propose a model in which IGF1 binding to IGF1R induces recruitment of integrin αvβ3 to the IGF-IGF1R complex and then β3 and IGF1R are phosphorylated. It is likely that αvβ3 should be together with the IGF1-IGF1R complex for triggering IGF signaling.

Physical activity opposes the age‐related increase in skeletal muscle and plasma endothelin‐1 levels and normalizes plasma endothelin‐1 levels in individuals with essential hypertension

Endothelin-1 has potent constrictor and proliferative activity in vascular smooth muscle, and essential hypertension and aging are associated with increased endothelin-1-mediated vasoconstrictor tone. The aim of this study was to investigate the effect of physical activity, hypertension and age on endothelin-1 levels in plasma and skeletal muscle and endothelin receptors in skeletal muscle in human subjects. In study 1, normotensive (46 ± 1 years, n = 11) and hypertensive (47 ± 1 years, n = 10) subjects were studied before and after 8 weeks of aerobic exercise training. In study 2, young (23 ± 1 years, n = 8), older lifelong sedentary (66 ± 2 years, n = 8) and older lifelong endurance-trained (62 ± 2 years, n = 8) subjects were studied in a cross-sectional design. Skeletal muscle and plasma endothelin-1 levels were increased with age and plasma endothelin-1 levels were higher in hypertensive than normotensive individuals. Eight weeks of exercise training normalized plasma endothelin-1 levels in the hypertensive subjects and increased the protein expression of the ET(A) receptor in skeletal muscle of normotensive subjects. Similarly, individuals that had performed lifelong physical activity had similar plasma and muscle endothelin-1 levels as the young controls and had higher ET(A) receptor levels. Our findings suggest that aerobic exercise training opposes the age-related increase in skeletal muscle and plasma endothelin-1 levels and normalizes plasma endothelin-1 levels in individuals with essential hypertension. This effect may explain some of the beneficial effects of training on the cardiovascular system in older and hypertensive subjects.

Abstract 8885: C1q-induced Activation of Wnt Signaling Triggers Hypertensive Arterial Remodeling

Hypertension is the leading risk factor of many cardiovascular diseases, and the structural remodeling of arteries in response to elevated blood pressure (hypertensive arterial remodeling) contributes to the end-organ damage associated with hypertension. The signaling mechanism that initiates hypertensive arterial remodeling remains elusive. Here we show that complement C1q-induced activation of Wnt signaling plays a critical role in arterial remodeling associated with hypertension. In the early phase of hypertension, proliferation of vascular smooth muscle cells (VSMCs) and activation of canonical Wnt signaling in VSMCs were observed, and inhibition of canonical Wnt signaling suppressed hypertension-induced VSMC proliferation. Complement C1q was highly expressed in macrophages recruited to the vessel wall following blood pressure elevation, and depletion of macrophages attenuated hypertension-induced VSMC proliferation and activation of canonical Wnt signaling in VSMCs. Furthermore, both C1s inhibition and C1qa gene deletion suppressed activation of canonical Wnt signaling in VSMCs and VSMC proliferation in the early phase of hypertension, and prevented subsequent structural arterial remodeling in the chronic phase. Our findings collectively suggest that macrophage-derived C1q triggers hypertensive arterial remodeling through activation of canonical Wnt signaling in VSMCs. Inhibition of C1q-induced Wnt signal activation may be a novel therapeutic strategy to reduce cardiovascular complications in patients with hypertension.

Peripheral vascular response to inspiratory breath hold in paediatric homozygous sickle cell disease

New Findings• What is the central question of this study?Autonomic nervous dysfunction is implicated in complications of sickle cell anaemia (SCA). In healthy adults, a deep inspiratory breath hold (IBH) elicits rapid transient SNS- mediated vasoconstriction detectable using Laser Doppler Flux (LDF) assessment of the finger-tip cutaneous micovasculature.• What is the main finding and its importance?We demonstrate significantly increased resting peripheral blood flow and sympathetic activity in African children with SCA compared to sibling controls and increased sympathetic stimulation in response to vasoprovocation with DIG.This study is the first to observe an inverse association between resting peripheral blood flow and haemoglobin oxygen saturation (SpO2). These phenomena may be an adaptive response to the hypoxic exposure in SCA.There is increasing evidence that autonomic dysfunction in adults with homozygous sickle cell (haemoglobin SS) disease is associated with enhanced autonomic nervous system-mediated control of microvascular perfusion. However, it is unclear whether such differences are detectable in children with SS disease. We studied 65 children with SS disease [38 boys; median age 7.2 (interquartile range 5.1–10.6) years] and 20 control children without symptoms of SS disease [8 boys; 8.7 (5.5–10.8) years] and recorded mean arterial blood pressure (ABP) and daytime haemoglobin oxygen saturation (). Cutaneous blood flux at rest (RBF) and during the sympathetically activated vasoconstrictor response to inspiratory breath hold (IBH) were measured in the finger pulp of the non-dominant hand using laser Doppler fluximetry. Local factors mediating flow motion were assessed by power spectral density analysis of the oscillatory components of the laser Doppler signal. The RBF measured across the two study groups was negatively associated with age (r=−0.25, P < 0.0001), ABP (r=−0.27, P= 0.02) and daytime (r=−0.30, P= 0.005). Children with SS disease had a higher RBF (P= 0.005) and enhanced vasoconstrictor response to IBH (P= 0.002) compared with control children. In children with SS disease, higher RBF was associated with an increase in the sympathetic interval (r=−0.28, P= 0.022). The SS disease status, daytime and age explained 22% of the variance in vasoconstrictor response to IBH (P < 0.0001). Our findings suggest that blood flow and blood flow responses in the skin of young African children with SS disease differ from those of healthy control children, with increased resting peripheral blood flow and increased sympathetic stimulation from a young age in SS disease. They further suggest that the laser Doppler flowmetry technique with inspiratory breath hold manoeuvre appears to be robust for use in young children with SS disease, to explore interactions between , ABP and autonomic function with clinical complications, e.g. skin ulceration.

Abstract 665: Endothelial Nitric Oxide Negatively Modulates Phenylephrine-induced Contraction In Matrix Metalloproteinase 2 (mmp-2)-filled Rabbit Aortic Rings

Endothelial nitric oxide negatively modulates Phenylephrine-induced contraction in Matrix metalloproteinase 2 (MMP-2)-filled rabbit aortic rings The present study aimed to evaluate the vascular function of aortic rings after intraluminal injection of matrix metalloproteinase 2 (MMP-2) and contribution of endothelial nitric oxide (NO) in this response. METHODS: thoracic aorta isolated from New Zealand rabbits (3.0 Kg) was isolated and were performed in situ gelatinolytic assay (intraluminal exposure with Krebs solution or MMP-2 1.2 μg/mL, at 37° C for 30 minutes, with measure of fluorescence intensity - FI) or vascular reactivity. Cumulative concentration-effect curves for phenylephrine (PE, 0.1 nM - 10 μM/L) were performed in endothelium-intact (E+) or denuded (E-) aortic rings, in the absence or presence of L-NAME (non-selective inhibitor of NOS, 100 μM) or Hydroxycobalamine (scavenger of intracellular NO), 30 min before the PE curves on E+ rings. The vascular contraction was normalized by grams of tension by dry tissue (g/g) and were analyzed the agonist maximum effect (Emax) and potency (pD2). Data represent mean ± S.E.M and the results were statistically analyzed using Student t test, one-way or two-way ANOVA with Bonferroni pos-hoc (p&lt;0,05). Results: In situ gelatinolytic activity was increased in the vessels filled with MMP-2 (19.19+0.93U,n=5) compared to vehicle (10.75+0.45U,n=5). In the vascular reactivity, E- aortic rings filled with MMP-2 increased Emax of PE (1893,28+122,40g/g,n=9) versus vehicle E- (1485.73+76.82g/g,n=9), but no differences were observed on Emax of E+ aortic rings (vehicle: 1416.13+120.87g/g,n=7; MMP-2: 1893.28+122.40g/g,n=9). The presence of L-NAME induced an increase in Emax of E+ aortic rings filled with vehicle (1817.60+76.37g/g,n=7) or MMP-2 (2340.50+130.43g/g,n=5). Hydroxycobalamine also increased the Emax of E+ aortic rings with vehicle (1889,81+129,17,n=6) or MMP-2 (2480,12+192,57,n=5). No difference of pD2 value was observed in this study. Conclusion: Data suggest this protease cross the aorta layers from the lumen. An increased vasoconstriction in E- vessels suggests a vascular smooth muscle activity of MMP-2. In the presence of L-NAME and Hydroxycobalamine, vessels filled with MMP-2 shows a greater maximum effect suggesting that NO exerts a negative modulation of the PE-induced vasoconstriction on MMP-2 aortic rings.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

The pathogenesis, diagnosis and treatment of Raynaud phenomenon

The past 10 years have seen the publication of results from several multicentre clinical trials in primary and systemic sclerosis (SSc)-related Raynaud phenomenon. The publication of these studies has occurred as a result of new insights into the pathogenesis of Raynaud phenomenon, which are directing new treatment approaches, and increased international collaboration between clinicians and scientists. Although the pathogenesis of Raynaud phenomenon is complex, abnormalities of the blood vessel wall, of neural control mechanisms and of intravascular (circulating) factors are known to interact and contribute. Key players relevant in drug development include nitric oxide, endothelin-1, alpha adrenergic receptor activation, abnormal signal transduction in vascular smooth muscle, oxidative stress and platelet activation. The main advances in diagnosis have been a clearer understanding of autoantibodies and of abnormal nailfold capillary patterns as independent predictors of SSc, and widespread use and increased availability of capillaroscopy. The ultimate aim is to translate the advances made in the pathophysiology and early diagnosis into development of treatments to prevent and reverse digital vascular dysfunction and injury. This Review provides an update of the pathogenesis, diagnosis and treatment of Raynaud phenomenon. Current and future treatment approaches are discussed, and some key unanswered questions are highlighted.

Mechanisms of Hydrogen Sulfide Effects on Contractile Activity of Vascular Smooth Muscle in Rats

Mechanisms of Hydrogen Sulfide Effects on Contractile Activity of Vascular Smooth Muscle in Rats

Vascular Smooth Muscle Activation Improves Aortic Compliance with Respect to Mechanical Loading

During a pressure rise, the aortic enlargement is followed by an arterial compliance reduction. For elevated pressure levels, vascular smooth muscle (VSM) activation produces an arterial compliance improvement. To better understand this observation, we studied the aortic elasticity function during high pressure states with and without VSM activation. Simultaneous aortic pressure and diameter were measured in conscious dogs. Animals were separated into groups corresponding to different types of VSM activation: PHE group (phenylephrine 5 μg/kg/min, n = 7) and RAS group (renin-angiotensin system stimulation via renal vasoconstriction, n = 7). An arterial biphasic elasticity function was adjusted with a mechanical occlusion intervention. Pressure-diameter loops were compared isobarically between VSM activation and mechanical occlusion. In the PHE group, activation increased aortic distension from 2.6 ± 0.7% to 5.4 ± 1.0% (p < 0.01) and compliance from 0.30 ± 0.07 mm2/mmHg to 0.58 ± 0.11 mm2/mmHg (p < 0.01). Similarly, in the RAS group, aortic distension increased from 4.8 ± 1.5% to 7.4 ± 2.4% (p < 0.05) and compliance from 0.48 ± 0.18 mm2/mmHg to 0.70 ± 0.28 mm2/mmHg (p < 0.05). When VSM activation produces an arterial pressure rise concomitant with an aortic wall contraction, arterial compliance and distension increase with respect to an isobaric mechanical occlusion. The assessment of the aortic geometry helps to explaining the cardiovascular system behavior during acute and chronic high-pressure states.

Механізми впливу сірководню на скоротливу активність гладеньких м’язів судин щурів

The effect of endogenous and exogenous hydrogen sulfide (H2S) on contractile activity of vascular smooth muscle (VSM) was studied. The introduction of substrate synthesis H2S L-cysteine and its donor NaHS in vitro caused concentration-dependent relaxation of VSM of aorta and portal vein. Low concentrations of hydrogen sulfide donor (10(-5) mol/L) caused vasoconstriction of both types of the vessels. It was shown that the reaction of relaxation of VSM in response to NaHS is independent from endothelium. It was revealed that VSM of portal vein are more sensitive to the effects of H2S than VSM of aorta. Removing of aorta periadventitial adipose tissue showed no relaxation reply to the hydrogen sulfide donor NaHS in 70% of experiments. Some of the cellular mechanisms of hydrogen sulfide action were established, namely relaxation of aorta is depended on K(ATP) channel activation. This is manifested by a lack of relaxation of the aortic VSM due to K(ATP) channel inhibitor glibenclamide.

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.

Effect of hydrogen sulfide on the contractile activity of smooth muscle cells from the rat aorta

In preparations of rat aorta, used as a model of muscular type arteries, the method mehanografii studied the effect of hydrogen sulfide on the reduction of isolated of vascular smooth muscle. Found that hydrogen sulfide in concentrations 1—50 mmol increases the mechanical stress of smooth muscle in high-K + medium. At higher concentrations (300—1 000 mmol) H2S leads to lower amplitude giperkalievoy contraction in high-K + medium. Reduction of smooth muscle cells caused by phenylephrine inhibited the action of hydrogen sulfide in the whole range of concentrations. The causes of differences in data obtained with the results of studies in other laboratories, and possible mechanisms of action of hydrogen sulfide on the contractile activity of vascular smooth muscle.

Methionine Sulfoxide Reductase B1 (MsrB1) Recovers TRPM6 Channel Activity during Oxidative Stress

Mg(2+) is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg(2+) homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg(2+) channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg(2+) transport. Remarkably, TRPM6 combines a Mg(2+)-permeable channel with an alpha-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 alpha-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg(2+)-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H(2)O(2)) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H(2)O(2) (TRPM6, 67 +/- 5% of control; TRPM6 + MsrB1, 81 +/- 5% of control). Cell surface biotinylation assays showed that H(2)O(2) treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met(1755) to Ala in TRPM6 reduced the inhibitory effect of H(2)O(2) on TRPM6 channel activity (TRPM6 M1755A: 84 +/- 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met(1755) and could, thereby, function as a modulator of TRPM6 during oxidative stress.

Renal autoregulation and passive pressure-flow relationships in diabetes and hypertension

We investigated renal hemodynamics in isolated, perfused kidneys from rat models of diabetes and hypertension. Autoregulation and passive vascular responses were measured using stepped pressure ramps in the presence of angiotensin II (pEC50) or papaverine (0.1 mM), respectively. Male diabetic heterozygote m(Ren2)27 rats were compared with three male control groups: nondiabetic, normotensive Sprague-Dawley (SD) rats; nondiabetic, hypertensive heterozygote m(Ren2)27 rats; and diabetic, normotensive SD rats. Kidney function (proteinuria, creatinine clearance) was monitored before induction and at monthly intervals. Vascular function was measured in vitro in rats of induction age (6-8 wk) and at 2 and 4 mo postinduction. Renal flow correlated with age, but not diabetes or the Ren2 gene. Kidney weight-specific and body weight-specific renal flow differed between diabetic and nondiabetic rats because diabetic rats had higher kidney but lower body weights. Kidneys from all groups showed effective autoregulation in the presence of angiotensin II. The autoregulatory pressure threshold of m(Ren2)27 rats was higher, and the autoregulation pressure range was wider, compared with SD rats. When vascular smooth muscle activity was blocked with papaverine, pressure-flow responses differed between groups and with time. The m(Ren2)27 rat groups showed higher renal vascular resistance at lower pressures, suggesting greater vascular stiffness. In contrast, diabetic SD rat kidneys demonstrated reduced vessel stiffness. Flow was impaired in diabetic m(Ren2)27 rats at 4 mo, and this correlated with a decline in creatinine clearance. The results suggest that the characteristic late decline in renal filtration function in diabetes- and hypertension-related renal disease follows changes in renal vascular compliance.

Antiproliferation of cardamonin is involved in mTOR on aortic smooth muscle cells in high fructose-induced insulin resistance rats

The objective of this study was to determine possible effects and potential mechanisms of cardamonin on improving insulin resistance and vascular proliferative lesions in the rat's model system. Fed with 60% fructose-enriched diet for 12 weeks, male Sprague–Dawley (SD) rats developed insulin resistance and hyperinsulinemia. They also showed excessive proliferation of the vascular smooth muscle cells (VSMCs) and activation of the mammalian target of rapamycin (mTOR)/translation control proteins p70 ribosomal S6 kinase (P70S6K1)/eukaryotic initiation factor 4E binding protein 1 (4E-BP1) signaling in the rat thoracic aorta. From weeks 9–12, cardamonin was injected into the peritoneal cavity once daily. Under the detection of microscopy and electron microscopy, cardamonin improved hyperinsulinemia and inhibited proliferation of VSMCs in the thoracic aorta of rats in a dose-dependent manner. By the Real-Time RT-PCR, mRNA expression of mTOR, P70S6K1 and 4E-BP1 was significantly reduced in cardamonin treated rats. Similarly, protein over-expression of mTOR and p-P70S6K1 was obviously inhibited by immunohistochemical analyses. These findings suggest that cardamonin may play a role in ameliorating insulin resistance and smooth muscle hyperplasia of major vessels in fructose-induced rats, possibly via a mechanism that involves the modulation of insulin/mTOR signaling.