Mesenteric Artery Smooth Muscle Cells Research Articles

GW25-e0548 Effects of arsenic trioxide on cell apoptosis and voltage-gated potassium currents of rat mesenteric arterial smooth muscle cells

GW25-e0548 Effects of arsenic trioxide on cell apoptosis and voltage-gated potassium currents of rat mesenteric arterial smooth muscle cells

Vasorelaxant effects of novel Kv 7.4 channel enhancers ML213 and NS15370.

The KCNQ-encoded voltage-gated potassium channel family (Kv 7.1-Kv 7.5) are established regulators of smooth muscle contractility, where Kv 7.4 and Kv 7.5 predominate. Various Kv 7.2-7.5 channel enhancers have been developed that have been shown to cause a vasorelaxation in both rodent and human blood vessels. Recently, two novel Kv 7 channel enhancers have been identified, ML213 and NS15370, that show increased potency, particularly on Kv 7.4 channels. The aim of this study was to characterize the effects of these novel enhancers in different rat blood vessels and compare them with Kv 7 enhancers (S-1, BMS204352, retigabine) described previously. We also sought to determine the binding sites of the new Kv 7 enhancers. Both ML213 and NS15370 relaxed segments of rat thoracic aorta, renal artery and mesenteric artery in a concentration-dependent manner. In the mesenteric artery ML213 and NS15370 displayed EC50 s that were far lower than other Kv 7 enhancers tested. Current-clamp experiments revealed that both novel enhancers, at low concentrations, caused significant hyperpolarization in mesenteric artery smooth muscle cells. In addition, we determined that the stimulatory effect of these enhancers relied on a tryptophan residue located in the S5 domain, which is the same binding site for the other Kv 7 enhancers tested in this study. This study has identified and characterized ML213 and NS15370 as potent vasorelaxants in different blood vessels, thereby highlighting these new compounds as potential therapeutics for various smooth muscle disorders.

Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation

Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca2+-activated K+ channels and inward rectifier K+ channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1513-3) contains supplementary material, which is available to authorized users.

Intravascular pressure stimulates functional KV1.5 surface expression in mesenteric artery smooth muscle cells (1077.4)

Intravascular pressure stimulates arterial depolarization, leading to myocyte voltage‐dependent K+ (KV) channel activation that opposes vasoconstriction. We tested the hypothesis that membrane potential regulates smooth muscle cell KV channel surface expression to modulate Kv channel functionality in mesenteric arteries. Q‐PCR indicated predominant expression of KV1.5 and 2.1 in mesenteric arteries. Contrary to expectations, 70% of total and surface KV1.5 protein was lost within 3 hrs in non‐pressurized arteries. In contrast, KV1.5 or 2.1 mRNA and total and surface KV2.1 protein did not change over the same time course. Bafilomycin, a lysosome inhibitor, and MG‐132, a proteasome inhibitor, both prevented KV1.5 loss. Depolarization (30 mM K+) also inhibited KV1.5 loss and this was blocked by nimodipine, a voltage‐dependent Ca2+ channel blocker. Patch‐clamp studies indicated that KV currents sensitive to DPO‐1, a Kv1.5 inhibitor, were 60% smaller in myocytes isolated from arteries kept at zero pressure than controls. Kv current loss was also blocked by bafilomycin. We also show that physiological intravascular pressure maintains functional Kv1 channel activity in arteries. In summary, data indicate that intravascular pressure inhibits KV1.5 channel degradation to maintain functional surface expression of these proteins in arterial myocytes.Grant Funding Source: NHLBI/NIH

PW180 Expression and Function of Ryanodine Receptors in Mesenteric Artery Smooth Muscle Cells from patients with hypertension

PW180 Expression and Function of Ryanodine Receptors in Mesenteric Artery Smooth Muscle Cells from patients with hypertension

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels

The Kv7 family (Kv7.1-7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.

Abstract 303: A Novel Adipocytokine, Omentin Prevents Vascular Smooth Muscle Cell Migration by Inhibiting O 2 -/p38/HSP27 Pathway

(Background) Omentin, a novel adipocytokine, is mainly expressed in the visceral rather than subcutaneous adipose tissue. Secretion and plasma concentration of omentin decrease in the obese subjects. We previously demonstrated that omentin is anti-inflammatory in vascular smooth muscle cells (SMCs). While vascular remodeling via proliferation, migration and apoptosis of SMCs is also important for hypertension development, it remains to be clarified whether omentin affects those processes. (Methods and Results) Cultured rat mesenteric arterial SMCs were stimulated with platelet-derived growth factor (PDGF)-BB (10 ng/ml) in the absence or presence of omentin (300 ng/ml). Omentin pretreatment (2 h) inhibited PDGF (6 h)-induced SMCs migration (30 % inhibition, n=11, P<0.05) as determined by Boyden camber assay. Omentin pretreatment (2 h) inhibited NADPH oxidase -derived O2- generation as determined by dihydroethidium staining and lucigenin assay (30 % inhibition, n=5, P<0.05) as well as phosphorylation of p38 (20 % inhibition, n=19-23, P<0.01) and heat shock protein (HSP) 27 (20 % inhibition, n=14-17, P<0.01) as determined by Western blotting. Omentin pretreatment (2 h) inhibited PDGF (6 h)-induced lamellipodia formation as determined by rhodamine-phalloidin staining. In ex vivo, omentin pretreatment (6 h) also inhibited fetal bovine serum (FBS) (10%)-induced SMCs out-growth from rat isolated mesenteric artery. (Conclusion) We for the first time demonstrate that omentin prevented SMCs migration by preventing the activation of ROS-dependent p38/HSP27 signals. It is suggested that omentin is anti-hypertensive ‘good adipocytokine’.

Abstract 22: Histone Deacetylase (HDAC) 4 Controls Neointimal Hyperplasia via Stimulating Proliferation and Migration of Vascular Smooth Muscle Cells

Histone deacetylases (HDACs) are transcriptional co-regulators. We have recently demonstrated that a class IIa HDAC, HDAC4 promotes reactive oxygen species (ROS)-dependent vascular smooth muscle inflammation and mediates the development of hypertension in spontaneously hypertensive rats. Pathogenesis of hypertension is in part modulated by vascular structural remodeling via proliferation and migration of vascular smooth muscle cells (SMCs). We thus examined whether HDAC4 controls SMCs proliferation and migration. In rat mesenteric arterial SMCs, small interfering RNA (siRNA) against HDAC4 inhibited platelet-derived growth factor (PDGF)-BB-induced SMCs proliferation as determined by a cell counting (51% inhibition, n=7) or bromodeoxyuridine incorporation assay (95% inhibition, n=6) and migration as determined by Boyden chamber assay (71% inhibition, n=3). Expression and activity of HDAC4 were increased by PDGF-BB (30% increase, n=5 and 170% increase, n=4, respectively). HDAC4 siRNA inhibited phosphorylation of p38 (69% inhibition, n=5) and heat shock protein (HSP) 27 (91% inhibition, n=5) and expression of cyclin D1 (58% inhibition, n=5) as measured by Western blotting. HDAC4 siRNA also inhibited PDGF-BB-induce ROS production as measured fluorometrically using 2’ 7’-dichlorofluorescein diacetate (77% inhibition, n=4) and nicotinamide adenine dinucleotide phosphate oxidase activity as measured by lucigenin assay (61% inhibition, n=4). A Ca 2+ /calmodulin (CaM)-dependent protein kinase (CaMK) II inhibitor, KN93 inhibited PDGF-BB-induced SMCs proliferation (58% inhibition, n=4) and migration (75% inhibition, n=3) as well as phosphorylation of HDAC4 (84% inhibition, n=4). In vivo, a class IIa HDACs inhibitor, MC1568 prevented neointimal hyperplasia in mice carotid ligation model (54% inhibition, n=6). MC1568 also inhibited increased activity of HDAC4 in the neointimal lesions. The present results for the first time demonstrate that HDAC4 controls PDGF-BB-induced SMCs proliferation and migration through activation of p38/HSP27 signals via ROS generation in a CaMKII-dependent manner, which may lead to the neointima hyperplasia in vivo.

Effects of different concentrations of dexmedetomidine on BKCa channels in rat mesenteric arterial smooth muscle cells

Objective To evaluate the effects of different concentrations of dexmedetomidine on large-con-ductance Ca2+-activated K+ (BKca) channels in the rat mesenteric arterial smooth muscle (MASM) cells.Methods Sprague-Dawley rats of both sexes,weighing 180-220 g,were used in this study.Single MASM cell was freshly isolated from mesenteric arteries in two steps.Ten cells were chosen and studied.When holding potential was 40 mV and the concentration of free calcium ions was 10-7 mol/L,inside-out patch-clamp technique was used to record the single BKCa channel current before and after application of different concentrations of dexmedetomidine (0,10-9,10-8,10-7,10-6,10-5 mol/L).Total open probability (NPo),amplitude (Am),mean open time (To) and mean close time (To) of single BKca channel were observed and recorded.Results Compared with the baseline value,dexmedetomidine 10-7,10-6 and 10-5 mol/L increased NPo in a concentration-dependent man-ner,and dexmedetomidine 10-9,10-8,10-7,10-6,10-5mol/L shortened Tc (P ＜0.05 or 0.01).Compared with the value obtained when the concentration of dexmedetomidine was 10-9 mol/L,Tc was significantly shortened when the concentration of dexmedetomidine was 10-8 mol/L (P ＜ 0.05),and no significant change was found in Am and To obtained when different concentrations of dexmedetomidine were applied (P ＞ 0.05).Conclusion Dexmedetomidine 10-7,10-6 and 10-5 mol/L activate BKca channels in rat MASM cells in a concentration-depen-dent manner,which is one of the mechanisms of decrease in blood pressure by dexmedetomidine. Key words: Dexmedetomidine ; Large-conductance Ca2 +-activated potassium channels ; Mesenteric arteries; Muscle, smooth, vascular

In vivo loss of estrogen increases voltage‐gated calcium channel function in mesenteric arterial smooth muscle cells

The presence of estrogen in premenopausal women protects against the development of vascular dysfunctions, such as hypertension. Previous studies from our lab demonstrated a slight (p=0.12) increase in voltage‐gated, L‐type Ca2+ channel (CaL) expression in mesenteric arteries from ovariectomized (OVX) mice. Therefore, in this study we hypothesized that the in vivo loss of estrogen will increase Ca2+ influx in mesenteric arterial smooth muscle cells. Mice (C57BL/6) underwent an ovariectomy or sham surgery at 8 weeks of age. At 12 weeks, the mice were sacrificed, mesenteric arteries dissected, and smooth muscle cells isolated. Lack of gonadal estrogen production was verified by a reduction in uterine weights. No significant difference (p=0.496) in weight gain between mice groups was observed. Ratiometric fluorescent imagining was conducted on isolated cells loaded with the Ca2+ fluorescent dye, fura‐2AM. Cells isolated from the mesenteric arteries of OVX mice (n=23) demonstrated an increased (p<0.001) intracellular Ca2+response to the CaL agonist, FPL64176 (1×10−6), compared to the sham mice (n=15). We plan to conduct whole cell Ca2+ currents on these cells in the future. These results suggest that the loss of in vivo estrogen leads to the enhanced influx of Ca2+into arterial cells which may lead to a chronic elevation of vascular tone in postmenopausal women. Support: NIGMM of the NIH, Grant #P20 GM103429–11

Intravascular pressure stimulates functional Kv1.5 surface expression in mesenteric artery smooth muscle cells

Intravascular pressure stimulates arterial depolarization, leading to myocyte voltage‐dependent K+ (KV) channel activation that opposes the resulting vasoconstriction. We tested the hypothesis that membrane potential regulates KV channel surface expression to modulate mesenteric artery contractility. Q‐PCR indicated predominant expression of KV1.5 and 2.1, with lower levels of 1.2 and 2.2 in mesenteric arteries. In isolated arteries at zero pressure, ~70% of total and surface KV1.5 protein was lost in 3 hrs. Total or surface KV2.1 protein did not change. KV1.5 or 2.1 mRNA were also unchanged. Depolarization (30 mM K+) prevented loss of KV1.5 and this was blocked by nimodipine, a voltage‐dependent Ca2+ channel blocker. Bafilomycin, a lysosome inhibitor, prevented KV1.5 loss, and MG‐132, a proteasome inhibitor, inhibited loss by ~50% in non‐pressurized arteries. The functional significance of KV1.5 surface expression was studied in arteries maintained at low (10 mmHg) or physiological (80 mmHg) pressure (3 hrs), with diameter regulation then measured at 80 mmHg. Vasoconstriction to psora‐4, a KV1 inhibitor, or 4‐AP, a broad KV inhibitor, was larger in arteries maintained at physiological pressure than those at low pressure. Data indicate that intravascular pressure inhibits degradation of KV1.5 channels and thus, maintains functional surface expression of these proteins in arterial myocytes. NHLBI/NIH

ASSA13-14-22 The Changes of Large-Conductance Ca2+-Activated Potassium Channels to AngiotensinII in Human Mesenteric Artery Smooth Muscle Cells During Hypertension

<h3>Objective</h3> Angiotensin II (Ang II) is the most important bioactive peptide of renin angiotensin system, combining with its specific receptor, has significant effect on structure and function of the heart and blood vessels. The continuous increasing tension of resistance vessel is the fundamental pathological features of hypertension (HT), and the ion channels on vascular smooth muscle cells (VSMCs) are involved in the regulation of vascular tension. Activation of large conductance calcium-activated potassium channels (BK_Ca) is one of the important mechanisms involved in vascular smooth muscle relaxation. AngII relaxes mesenteric artery via stimulation of the angiotensinII type 2 receptor (AT₂R) with subsequent opening of BK_Ca. It is necessary to investigate the change of AT₂R under the hypertension condition and BK_Ca change response to AngII. It will expand our understanding of the pathogenesis and development of hypertension. <h3>Methods</h3> Using amphotericin-perforated whole-cell recording technique and Western blot analysis, we will know the relationships among AngII, AT₂R and hypertension. <h3>Results</h3> (1) In the amphotericin-perforated whole-cell patch-clamp configuration, after pretreatment with Valsartan (a specific inhibitor of angiotensinII type 1 receptor, 10 μmol/L), ➀ In the normotension (NT) group, 100 nmol/L AngII can augment BK_Ca whole-cell currents in human mesenteric artery VSMCs, the current density of BK_Ca at the voltage of −60 ∼ +20 mV had no significant change before and after adding 100 nmol/L AngII, but the current density of BK_Ca at the voltage of +30 mV, +40 mV, +50 mV and +60 mV increased significantly after adding 100 nmol/L AngII, from (5.84 ± 1.25) pA/pF, (9.19 ± 1.54) pA/pF, (13.32 ± 1.83) pA/pF and (18.06 ± 2.13) pA/pF to (8.68 ± 1.70) pA/pF, (13.24 ± 2.00) pA/pF, (19.44 ± 2.14) pA/pF and (28.09 ± 2.62) pA/pF (n = 10,<i> P</i> &lt; 0.05). ➁ In the HT group, AngII had no significant effect on BK_Ca. (2) The protein amount of the AT₂R in mesenteric artery among the NT group and the HT group was 1.2046 ± 0.0564 (n = 13), 0.9893 ± 0.0543 (n = 6), the expression level of AT₂R protein of the NT group was greater than the HT group (<i>P</i> &lt; 0.05). <h3>Conclusions</h3> AT₂R is downregulated in hypertension, we speculate that the different protein of AT₂R results in the response difference of BK_Ca channel which mediated by AT₂R.

Endosomal proteolysis regulates calcitonin gene‐related peptide responses in mesenteric arteries

Calcitonin gene-related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR●RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin-converting enzyme-1 (ECE-1). However, it is not known if ECE-1 regulates the resensitization of CGRP-induced responses in functional arterial tissue. CLR, ECE-1a-d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA-SMCs) and mesenteric arteries was analysed by RT-PCR and by immunofluorescence and confocal microscopy. CGRP-induced signalling in cells was examined by measuring cAMP production and ERK activation. CGRP-induced relaxation of arteries was measured by isometric wire myography. ECE-1 was inhibited using the specific inhibitor, SM-19712. RMA-SMCs and arteries contained mRNA for CLR, ECE-1a-d and RAMP1. ECE-1 was present in early endosomes of RMA-SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium-independent relaxation of arteries. ECE-1 inhibition had no effect on initial CGRP-induced responses but reduced cAMP generation in RMA-SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. ECE-1 regulated the resensitization of responses to CGRP in RMA-SMCs and mesenteric arteries. CGRP-induced relaxation did not involve endothelium-derived pathways. This is the first report of ECE-1 regulating CGRP responses in SMCs and arteries. ECE-1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine.

Activation of c-SRC underlies the differential effects of ouabain and digoxin on Ca2+ signaling in arterial smooth muscle cells

Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 μg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 μM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.

Abstract 9701: A Novel Adipocytokine Chemerin Stimulates Proliferation and Hypercontractility of Vascular Smooth Muscle: Implication for Pathogenesis of Obesity-related Hypertension

Chemerin is a recently identified adipose tissue-derived cytokine (adipocytokine) which plays roles in inflammatory responses of immune cells such as dendritic cells and macrophages as well as differentiation and metabolisms of adipocytes. Chemerin acts as a chemoattractant for the immune cells, while it induces differentiation of adipocytes and glucose tolerance. Blood chemerin level increases in patients with obesity, and correlates positively with several key factors of metabolic syndrome such as body mass index and triglyceride level. Since blood chemerin level also correlates positively with systolic blood pressure in the obese subjects, we hypothesized that chemerin might relate to pathogenesis of obesity-related hypertension. To test the hypothesis, we examined the effects of chemerin on proliferation and contractility of vascular smooth muscle which play key roles in the development of hypertension. In cultured rat mesenteric arterial smooth muscle cells (SMCs), chemerin (1-100 ng/ml, 24h) stimulated proliferation in a concentration-dependent manner (n=8, P&lt;0.01). Chemerin (100 ng/ml, 20 min) stimulated phosphorylation of Akt (n=8, P&lt;0.05), and an Akt inhibitor prevented the chemerin-induced SMCs proliferation (n=4). Chemerin produced oxidant radicals in SMCs, and an anti-oxidant drug, N-acetyl-L-cysteine inhibited SMCs proliferation (n=6) and Akt phosphorylation (n=4, P&lt;0.05). In rat isolated mesenteric artery, chemerin (100 ng/ml, 30 min) augmented noradenaline (1 nM-1 μM)-induced concentration-dependent contraction (n=6, P&lt;0.05). The effect was higher in endothelium-intact artery than endothelium-denuded artery (n=7), and was normalized by an endothelin type A receptor blocker, BQ123 (n=13). Chemerin had no influence on noradrenaline-stimulated endothelial nitric oxide production (n=10). The present results for the first time demonstrated that chemerin stimulates proliferation of vascular SMCs via oxidative-stress-dependent Akt activation. It is also demonstrated that chemerin mediates hypercontractility of isolated mesenteric artery likely via endothelial-derived endothelin-1. The results may represent chemerin as a new adipocytokine to understand the link between obesity and hypertension.

ARSENIC TRIOXIDE INHIBITS THE VOLTAGE-GATED K+ CHANNELS IN RAT MESENTERIC ARTERIAL SMOOTH MUSCLE CELLS

ARSENIC TRIOXIDE INHIBITS THE VOLTAGE-GATED K+ CHANNELS IN RAT MESENTERIC ARTERIAL SMOOTH MUSCLE CELLS

Abstract 171: Eukaryotic Elongation Factor 2 Kinase (eEF2K) Mediates Vascular Inflammation and Development of Hypertension in Spontaneously Hypertensive Rats

Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca 2+ /calmodulin-dependent protein kinase that mainly regulates protein translation. We have recently demonstrated that eEF2K protein increases in mesenteric artery from spontaneously hypertensive rats (SHR) compared with Wistar Kyoto rats. Pathogenesis of hypertension is modulated in part by vascular inflammation. We examined whether eEF2K mediates vascular inflammatory responses and development of hypertension. In human umbilical vein endothelial cells (HUVECs) and rat mesenteric arterial smooth muscle cells (SMCs), eEF2K phosphorylation was increased by TNF-α (48% increase in HUVECs and 46% increase in SMCs, n=5, P&lt;0.01). In HUVECs, small interfering RNA (siRNA) against eEF2K inhibited induction of VCAM-1 (62% inhibition, n=4) and e-selectin (29% inhibition, n=6) as well as monocyte adhesion (43% inhibition, n=4) by TNF-α (P&lt;0.01). eEF2K siRNA inhibited phosphorylation of JNK (76% inhibition) and NF-κB (54% inhibition) as well as reactive oxygen species (ROS) production (69% inhibition) by TNF-α (n=4, P&lt;0.01). In SMCs, eEF2K siRNA inhibited VCAM-1 induction (41% inhibition, n=5, P&lt;0.05) and phosphorylation of JNK (65% inhibition, n=6, P&lt;0.01) and NF-κB (83% inhibition, n=4, P&lt;0.05) by TNF-α. In vivo, increased blood pressure (systolic blood pressure, SHR; 199.5 mmHg vs. SHR+NH125; 159.4 mmHg, n=4, P&lt;0.01) and increased eEF2K phosphorylation (68% inhibition, n=4), induction of VCAM-1 (62% inhibition, n=4, P&lt;0.05) and hypertrophy (70% inhibition, n=4) in SHR mesenteric artery was normalized by long-term treatment with NH125 (500 μg/kg/day for 6 weeks). In SHR mesenteric artery, impairment of acetylcholine (ACh)-induced endothelium-dependent relaxation was normalized by NH125 (relaxation induced by 30 nM ACh, SHR; 60.4% vs. SHR+NH125; 90.0%, n=4, P&lt;0.01). The present results for the first time demonstrated in cultured ECs and SMCs that eEF2K mediates TNF-induced inflammatory responses via ROS-dependent mechanism. It is also suggested that eEF2K may mediate development of hypertension in SHR likely via inflammation, hypertrophy and endothelial dysfunction.

Death-Associated Protein Kinase 3 Mediates Vascular Inflammation and Development of Hypertension in Spontaneously Hypertensive Rats

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated serine/threonine kinase that mediates cell death. Our recent study demonstrated that DAPK3 protein increases in the mesenteric artery from spontaneously hypertensive rats compared with Wistar Kyoto rats. Pathogenesis of hypertension is modulated at least in part by vascular inflammation. We examined whether DAPK3 mediates vascular inflammatory responses and development of hypertension. In rat mesenteric arterial smooth muscle cells, small interfering RNA against DAPK3 inhibited vascular cell adhesion molecule 1 expression and monocyte adhesion induced by tumor necrosis factor-α. DAPK3 small interfering RNA inhibited phosphorylation of c-Jun amino-terminal kinase, p38, and Akt, as well as reactive oxygen species (ROS) production induced by tumor necrosis factor-α. In human umbilical vein endothelial cells, expressions of vascular cell adhesion molecule 1, endothelial selectin, and cyclooxygenase 2, as well as ROS production induced by tumor necrosis factor-α, were inhibited by DAPK inhibitor. In vivo, blood pressure, ROS production, inflammatory molecule expression (vascular cell adhesion molecule 1 and endothelial selectin), and hypertrophy in isolated mesenteric artery were elevated in spontaneously hypertensive rats (10 weeks old), which were prevented by long-term treatment with a DAPK inhibitor (500 µg/kg per day for 6 weeks). In isolated mesenteric artery, the increased angiotensin II-induced contraction and the impaired acetylcholine-induced endothelium-dependent relaxation in spontaneously hypertensive rats were reversed by a DAPK inhibitor. The present results for the first time demonstrated in cultured smooth muscle cells and endothelial cells that DAPK3 mediates tumor necrosis factor-induced inflammatory responses via ROS-dependent mechanisms. It is also suggested that DAPK3 mediates the development of hypertension in spontaneously hypertensive rats likely via ROS-dependent inflammation, hypertrophy, and hypercontractility.

HDAC4 mediates development of hypertension via vascular inflammation in spontaneous hypertensive rats

Histone deacetylases (HDACs) are transcriptional corepressors. Our recent study demonstrated that HDAC4 protein specifically increases in mesenteric artery from spontaneous hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). Vascular inflammation is important for pathogenesis of hypertension. We examined whether HDAC4 affects vascular inflammatory responses and promotes hypertension. In vivo, blood pressure, reactive oxygen species (ROS) production, and VCAM-1 expression in isolated mesenteric artery were elevated in young SHR (7 wk old) compared with age-matched WKY, which were prevented by long-term treatment of SHR with an HDACs inhibitor, trichostatin A (TSA; 500 μg·kg(-1)·day(-1) for 3 wk). In isolated mesenteric artery, the increased angiotensin II-induced contraction in SHR was reversed by TSA. The endothelium-dependent relaxation induced by ACh in SHR was augmented by TSA. In cultured rat mesenteric arterial smooth muscle cells (SMCs), expression of HDAC4 mRNA and protein was increased by TNF-α (10 ng/ml). TSA (10 μM, pretreatment for 30 min) inhibited VCAM-1 expression and NF-κB phosphorylation induced by TNF (10 ng/ml, 24 h or 20 min) in SMCs. HDAC4 small interfering RNA inhibited TNF-induced monocyte adhesion, VCAM-1 expression, transcriptional activity of NF-κB, and ROS production in SMCs. The present results demonstrated that proinflammatory effects of HDACs may mediate the further development of hypertension in SHR. It is also suggested in cultured vascular SMCs that TNF-induced HDAC4 mediates vascular inflammation likely via VCAM-1 induction through ROS-dependent NF-κB activation.