A7r5 Rat Aortic Smooth Muscle Research Articles

Evidence for the involvement of TRPV2 channels in the modulation of vascular tone in the mouse aorta

AimsTransient receptor potential vanilloid 2 (TRPV2) channels are expressed in both smooth muscle and endothelial cells and participate in vascular mechanotransduction and sensing of high temperatures and lipids. Nevertheless, the impact of TRPV2 channel activation by agonists on the coordinated and cell-type specific modulation of vasoreactivity is unknown. Main methodsAorta from 2- to 4-months-old male Oncins France 1 mice was dissected and mounted in tissue baths for isometric tension measurements. TRPV2 channel expression was assessed by immunofluorescence and western blot in mice aortas and in cultured A7r5 rat aortic smooth muscle cells. Key findingsTRPV2 channels were expressed in all three mouse aorta layers. Activation of TRPV2 channels with probenecid evoked endothelium-dependent relaxations through a mechanism that involved activation of smooth muscle Kir and Kv channels. In addition, TRPV2 channel inhibition with tranilast increased endothelium-independent relaxations to probenecid and this effect was abrogated by the KATP channel blocker glibenclamide, revealing that smooth muscle TRPV2 channels induce negative feedback on probenecid relaxations mediated via KATP channel inhibition. Exposure to the NO donor sodium nitroprusside increased TRPV2 channel translocation to the plasma membrane in cultured smooth muscle cells and enhanced negative feedback on probenecid relaxations. SignificanceIn conclusion, we present the first evidence that TRPV2 channels may modulate vascular tone through a balance of opposed inputs from the endothelium and the smooth muscle leading to net vasodilation. The fact that TRPV2 channel-induced activity can be amplified by NO emphasizes the pathophysiological relevance of these findings.

Regulation of total LC3 levels by angiotensin II in vascular smooth muscle cells

Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II‐induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co‐chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT‐qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II‐mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co‐administration with either losartan 1 μM (AT1R antagonist) or Y‐27632 10 μM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

Curcumin inhibits proliferation, migration and neointimal formation of vascular smooth muscle via activating miR-22

Context Curcumin has antitumor, antioxidative, anti-inflammatory, and anti-proliferative properties. Objective To investigate the role of miR-22 during curcumin-induced changes in vascular smooth muscle cells (VSMC) and neointima formation in balloon-injured rat abdominal aorta. Materials and methods Sprague-Dawley rats were randomised to the sham-operated (n = 10), operated control (injured, n = 10), and curcumin treatment (n = 10) groups. miR-22 expression was determined by real-time PCR. SP1 was assessed by western blot and real-time PCR. Rat aortic smooth muscle A7r5 cells were used to determine VSMC proliferation and migration, which were measured by the MTS, EdU staining, Transwell, and wound healing assays. Results miR-22 levels declined following arterial balloon injury in vivo (48% at 3d, p < 0.05) and serum stimulation in vitro (45% at 24 h, p < 0.01). Functional studies revealed that miR-22 negatively regulated the proliferation and migration of VSMCs by directly targeting the SP1 transcription factor in VSMCs. Curcumin increased the expression of miR-22 (81%, p < 0.05) and decreased the protein expression of SP1 in VSMCs (25%, p < 0.05). miR-22 inhibition was found to attenuate the effects of curcumin on VSMC functions. Curcumin increased miR-22 (46%, p < 0.01), decreased the SP1 protein (19%, p < 0.05), and inhibited vascular neointimal area (48%, p < 0.01) in vivo. Discussion The miR-22/SP1 pathway is involved in the protective role of curcumin during arterial balloon injury, but the mechanisms remain unclear. Conclusion miR-22 is involved in the inhibitory effects of curcumin on VSMCs’ proliferation, migration and neointima hyperplasia after arterial balloon injury in rats. Curcumin could be used to prevent neointimal hyperplasia after angioplasty.

Pu-Erh Tea Relaxes the Thoracic Aorta of Rats by Reducing Intracellular Calcium.

Previous studies suggested that pu-erh tea aqueous extract could lower blood pressure and ameliorate hypertension symptoms. However, the antihypertension mechanisms of pu-erh tea remain unclear. In this work, the direct effects of pu-erh tea on vessels and cells were investigated by detecting isometric tension and intracellular calcium ([Ca2+]i), respectively. Additionally, to identify the main active components, the aqueous extract of pu-erh was separated by organic solvents to obtain various fractions, and the effects of these fractions on arteries were assessed. The results showed that pu-erh aqueous extract vasodilated rat thoracic aortas preconstricted by phenylephrine or KCl. These vasodilation effects were not significantly affected by the removal of the endothelium or by preincubation with potassium channel blockers (tetraethylammonium, glibenclamide, aminopyridine, or barium chloride). Moreover, pu-erh aqueous extract could reduce the vessel contractibility induced by CaCl2 and phenylephrine under KCl-depolarizing or Ca2+-free buffer conditions, respectively. Furthermore, pu-erh aqueous extract attenuated the KCl-induced increase in [Ca2+]i in cultured rat aortic smooth muscle A7r5 cells. In addition, the chloroform precipitate of pu-erh aqueous extract produced the most potent vasodilation. Theabrownins (the characteristic components of pu-erh tea) accounted for 41.91 ± 1.09 % of the chloroform precipitate and vasodilated arteries in an endothelium-independent manner. In addition, the vasodilation effect of caffeine was verified. In conclusion, theabrownins and caffeine should be the two main active components in pu-erh tea. Pu-erh aqueous extract vasodilated arteries in an endothelium-independent manner, which might partly be attributed to the decrease in extracellular Ca2+ influx. Moreover, our study provided data on the potential mechanism of the hypotensive actions of pu-erh tea, which might improve our understanding of the effect of pu-erh tea on the prevention and treatment of hypertension.

Novel Inflammatory Signaling via Interleukin‐13 Decreases Potassium Channel Expression and Increases Vascular Smooth Muscle Excitability

The transcriptional repressor element‐1 (RE1) silencing transcription factor (REST), also known as neuron‐restrictive silencer factor (NRSF) is a gene silencing transcription factor, which has been characterized as a master regulator of the neuronal phenotype. REST plays a key role in the development of the nervous system and is a critical player in the etiology of neurodegenerative diseases such as Huntington's Disease, though little is known about the role of REST in non‐neuronal cells. We are exploring a potential role of REST as a mediator between inflammation and changes in smooth muscle phenotype. Kv7.5 voltage‐dependent potassium channels, encoded by the KCNQ5 gene, are expressed in smooth muscle cells where they play an important role in determining resting membrane potential. Interleukin‐13 (IL‐13) is an inflammatory cytokine, which is implicated as a primary mediator of the phenotypic changes induced by allergic inflammation in many tissues. In the present study, we hypothesized that IL‐13 can increase nuclear REST and decrease KCNQ5 mRNA expression and Kv7.5 protein in vascular smooth muscle cells, providing a potential link between inflammation, REST and potassium channel‐mediated vascular hyper‐responsiveness. In support of this hypothesis, using A7r5 rat aortic smooth muscle cells, we found that 1) IL‐13 treatment significantly increased nuclear REST, and 2) significantly suppressed the expression of KCNQ5 and Kv7.5 currents, resulting in increased sensitivity of A7r5 cells to arginine vasopressin (AVP). 3) REST overexpression also significantly decreased Kv7.5 currents, and 4) significantly increased the sensitivity of A7r5 cells to AVP. We conclude that inflammation that induces IL‐13 may increase nuclear REST levels and thereby decrease KCNQ5 expression and Kv7.5 currents, ultimately increasing vascular smooth muscle cell excitability and contributing to vascular hyper‐responsiveness.Support or Funding InformationThe project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research &amp; Development Service of the VA Office of Research and Development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GPER Attenuates Angiotensin II‐Induced Oxidative Stress via cAMP‐Mediated Regulation of NOX4

Our previous work shows that the G protein‐coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II‐dependent hypertension, in part by inhibiting oxidative stress. In addition, we find that acute estrogenic signaling via GPER involves activation of the cAMP signaling pathway in vascular smooth muscle cells. Ang II stimulates the production of reactive oxygen species (ROS) via NADPH oxidase (NOX) complex activation, but whether GPER interferes with NOX activation and whether this occurs through cAMP signaling is not known. Therefore, the goal of the current study was to assess the molecular mechanisms by which GPER attenuates Ang II‐induced oxidative stress in vitro. A7r5 rat aortic smooth muscle cells were treated overnight with 100 nM Ang II in the presence of vehicle or 100 nM of the GPER agonist G‐1. ROS was measured via electron spin resonance spectroscopy (e‐scan) and CellRox™ Green fluorescence. NOX activity and expression were measured by NADP/NADPH ratio, real time RT‐PCR, and immunoblotting. Both e‐scan and CellRox experiments showed that Ang II increased oxidative stress, while co‐administration of the GPER agonist G‐1 prevented this increase (4.3 × 105 vs 1.9 × 105 AU/mg protein, P&lt;0.001, n=7–8). Similarly, NOX activity was elevated in response to Ang II but normalized by G‐1 (189% vs. 83% RLU, P&lt;0.0001, n=5). When the production of cAMP was inhibited with the adenylyl cyclase inhibitor SQ22536, G‐1 was unable to block Ang II‐induced NOX activity (153% vs. 142% RLU, P=0.39, n=6). Ang II increased NOX4 mRNA over time, with a maximum increase at 4h (3.5‐fold change, P&lt;0.05, n=3). Ang II also increased NOX4 protein (1.2 vs 2.1 ADU/β‐actin, P&lt;0.01, n=2). In contrast, administration of G‐1 for 4h decreased NOX4 mRNA by 60% (P&lt;0.05, n=3) with no significant impact on protein expression (P=0.91, n=2). Preliminary studies using male wildtype and knockout mice show higher expression of NOX4 in response to genetic deletion of GPER. These experiments indicate that GPER inhibits NOX4 transcription and suppresses NOX activity, thereby limiting the production of ROS by Ang II. The impact of GPER on NOX activity was dependent on the production of cAMP. Taken together with our previous work, this study provides insight into how acute estrogen signaling provides cardiovascular protection during Ang II‐induced hypertension and potentially other diseases that are characterized by increased oxidative stress.Support or Funding InformationThis study was funded by National Institutes of Health grant HL133619 to S.H.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel 1, 4-dihydropyridines for L-type calcium channel as antagonists for cadmium toxicity

The present study, we design and synthesize the novel dihydropyridine derivatives, i.e., 3 (a-e) and 5 (a-e) and evaluated, anticonvulsant activity. Initially due to the lacuna of LCC, we modeled the protein through modeller 9.15v and evaluated through servers. Docking studies were performed with the synthesized compounds and resulted two best compounds, i.e., 5a, 5e showed the best binding energies. The activity of intracellular Ca2+ measurements was performed on two cell lines: A7r5 (rat aortic smooth muscle cells) and SH-SY5Y (human neuroblastoma cells). The 5a and 5e compounds was showing the more specific activity on L-type calcium channels, i.e. A7r5 (IC50 = 0.18 ± 0.02 and 0.25 ± 0.63 μg/ml, respectively) (containing only L-type channels) than SH-SY5Y (i.e. both L-type and T-type channels) (IC50 = 8 ± 0.23 and 10 ± 0.18 μg/ml, respectively) with intracellular calcium mobility similar to amlodipine. Finally, both in silico and in vitro results exploring two derivatives 5a and 5e succeeded to treat cadmium toxicity.

Protein Kinase A-Dependent Phosphorylation Modulates the Affinity of Kv7.5 Potassium Channels to Phosphatidylinositol 4,5-Bisphosphate to Enhance Potassium Conductance

PKA-dependent phosphorylation of Kv7.5 potassium channels was previously found to enhance Kv7.5 currents, but the mechanism of the enhancement by PKA as well as putative PKA phosphorylation sites remained unknown. Activity of all known Kv7 channels critically depends on the presence of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2). In the present study, we tested the hypothesis that PKA-dependent phosphorylation increases the affinity of Kv7.5 potassium channels to PIP2. Application of wortmannin (10 µM), an inhibitor of PI-4 kinase that decreases the level of membrane PIP2, caused time-dependent inhibition of the endogenous Kv7.5 current in A7r5 rat aortic smooth muscle cells. Activation of PKA by co-application of forskolin (activator of adenylyl cyclase, 1 µM) with IBMX (phosphodiesterase inhibitor, 500 µM) significantly attenuated the rate and total amount of wortmannin-induced inhibition of endogenous Kv7.5 current. Co-expression of ci-VSP, a voltage dependent phosphatase that dephosphorylates PIP2, with human Kv7.5 (hKv7.5) in A7r5 cells caused pronounced voltage and time-dependent inhibition of hKv7.5 current at membrane voltages positive to 0 mV. Enhancement of the hKv7.5 current observed in the presence of forskolin (10 µM) and IBMX (500 µM) occurred in parallel with the positive shift of the ci-VSP-induced voltage-dependent inhibition of the Kv7.5 current. In the presence of forskolin and IBMX, ci-VSP induced inhibition of the Kv7.5 current at voltages positive to +60 mV. To identify PKA phosphorylation sites we mutated predicted PKA phosphorylation sites S406 and S412 (located near the putative PIP2 binding domain on the C-terminus of the Kv7.5 channels) to alanine. Currents through both mutant channels Kv7.5 S406A and Kv7.5 S412A were enhanced by co-application of forskolin with IBMX to the same degree as wild type Kv7.5 current. Thus, phosphorylation of S406 and S412 cannot account for PKA-dependent Kv7.5 current enhancement. Overall, experimental results support our hypothesis that enhancement of Kv7.5 currents caused by PKA activation is associated with increased affinity of the Kv7.5 potassium channels to PIP2.

Kv7.5 Potassium Channel Subunits are the Primary Target for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

Kv7 (KCNQ) channels, formed as homo- or hetero-tetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, physiological regulation of Kv7 channel activity is still poorly understood. Here, we measure the effect of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) activation on whole cell K+ currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells, or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or hetero-tetrameric channels in A7r5 cells. Stimulation of Gs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously expressed hetero-tetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were almost entirely insensitive to this regulatory pathway. There results suggest that signal transduction mediated responsiveness of Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5>>Kv7.4/Kv7.5>Kv7.4 and that physiological or pathological alteration of subunit stoichiometry of Kv7 channels would determine their responsiveness to the cAMP/PKA pathway.

Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents.

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

Abstract 405: Functional Characterization of Coronary Artery Disease (CAD) Associated SNPs at the ADAMTS7 Locus

SNPs in the genomic region containing the gene ADAMTS7, a gene that codes for a metalloproteinase involved in vascular smooth muscle cell migration, are significantly associated with CAD in humans. Recently, our lab demonstrated that knock out of Adamts7 in mice is protective against atherosclerosis, suggesting that ADAMTS7 is involved in the pathogenesis of cardiovascular disease. Several of the top CAD-associated SNPs (p&lt;5e-8) identified by CARDIoGRAMplusC4D consortium lie in putative enhancer regions of ADAMTS7 .. We investigated the functionality of CAD associated SNPs that overlap 5 active regulatory elements as predicted by vascular smooth muscle cell (VSMC) ChIP-seq signals, ENCODE histone methylation peaks, and aortic smooth muscle cell DNase hypersensitivity peaks. Several regions containing association SNPs showed enhancer activity in minimal promoter luciferase reporter assays. Region 2 showed an 8.2 (p&lt;0.01) and a 32.4 (p&lt;0.005) fold increase in luciferase activity in HEK293T and A7r5 rat aortic smooth muscle cell lines, respectively, when normalized to empty vector control, while Region 5 demonstrated a 4.9 (p&lt;0.01) and 1.8 (p&lt;0.01) normalized fold increase in 293T and A7r5 cells, respectively. Both of these regions contain binding motifs for TCF21, a transcription factor involved in the development of the human coronary vasculature that is also a GWAS locus for CAD, that is disrupted by genotype of the CAD-associated SNP. We performed electrophoretic mobility shift assays (EMSAs) using purified TCF21 protein and showed altered binding in Region 5 based on disease associated SNP variation. Additionally, EMSAs using human coronary artery smooth muscle cell (hCASMC) nuclear extract detected a band shift demonstrating protein binding at both predicted enhancer regions 2 and 5. However, we were not able to detect a significant difference in protein binding between the major and minor allele, suggesting transcription factors other than TCF21 may be regulating these regions. More work is being done using Capture-C technology to interrogate physical interactions between these regions and the promoter of ADAMTS7 , but our current data suggest that top CAD-associated SNPs alter regulatory activity at the ADAMTS7 locus.

Microscopic study of ultrasound-mediated microbubble destruction effects on vascular smooth muscle cells.

To observe vascular smooth muscle cell morphological changes induced by ultrasound combined with microbubbles by Atomic Force Acoustic Microscopy (AFAM). A7r5 rat aortic smooth muscle cells were divided into groups: control group (without ultrasonic irradiation, no micro bubbles) and US+MB group (45 kHz, 0.4 W/cm(2) ultrasound irradiate for 20 seconds with a SonoVue™ concentration of [(56-140)×10(5)/mL]. Cell micro-morphological changes (such as topographic and acoustic prognosis) were detected, before and after ultrasound destruction by AFAM. In cell morphology, smooth muscle cells were spread o and connected to each another by fibers. At the center of the cell, the nuclear area had a rough surface and was significantly elevated from its surroundings. The cytoskeletal structure of the reticular nucleus and cytoplasm in the morphology of A7r5 cells (20 μ m × 20 μ m) were clear before microbubble intervention. After acoustic exciting, the cell structure details of the acoustic image were improved with better resolution, showing the elasticity of different tissues. In the acoustic image, the nucleus was harder, more flexible and uneven compared with the cytoplasm. Many strong various-sized echo particles were stuck on the rough nuclear membrane's substrate surface. The nuclear membrane did not have a continuous smooth surface; there were many obstructions (pores). After ultrasound-intervention was combined with microbubbles, the dark areas of the A7r5 cell images was increased in various sizes and degrees. The dark areas showed the depth or low altitudes of the lower regions, suggesting regional depressions. However, the location and scope of the acoustic image dark areas were not similar to those found in the topographic images. Therefore, it was likely that the dark areas, both from the topographic and acoustic images, were sound-holes. In addition, some cell nuclei become round in different degrees after irradiation. Atomic force microscopy and acoustic excitation method can noninvasively and completely display a cell's structure, connections and elastic properties at a nano scale in just several minutes. The dark areas, both from the topographic and acoustic images, may be sound-holes; therefore, it would be helpful if these sound-holes were found. These findings provide a relationship between cell apoptosis after ultrasound and microbubble ultrasound irradiation, and the sound-hole effect.

Leukotriene B4 Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells

Background/Aims: Arachidonic acid (AA) and its metabolites are important endogenous lipid messengers. In this study, we test the effect of Leukotriene B₄ (LTB₄), a 5-lipoxygenase metabolite of AA, on L-type calcium channels in A7r5 rat aortic vascular smooth muscle cells. Methods: L-type calcium channel currents were recorded by a patch-clamp technique. The mRNA expression of Ca_V1.2 was determined by Real-time RT-PCR. The protein expression of Ca_V1.2 and p38 activity was determined by Western blot analysis. Results: LTB₄ inhibits L-type channel currents in A7r5 cells in a dose-and time- dependent manner. LTB₄ reduced the mRNA/protein expression of Ca_V1.2 channels in A7r5 cells. BLT1 receptor antagonist LY29311 abrogated the inhibitory effect of LTB₄, while BLT2 receptor antagonist LY255283 had no effect. 5Z-7-oxozeaenol and SB203580, which block TAK1 and p38 kinase respectively, abrogated the LTB₄ inhibitory effect on L-type calcium channels. LTB₄ increased p38 activity in A7r5 cells. Blockage of Src, PI3K, JNK and NF-κB kinase had no effects on LTB₄ inhibition of L-type calcium channel currents in A7r5 cells. Conclusion: We conclude that LTB₄ inhibits L-type calcium channels through BLT1-TAk1-p38 signaling pathway. The LTB₄ inhibitory effect on L-type calcium channels may be involved in its pathological processes such as atherosclerosis.

Low-frequency low energy ultrasound combined with microbubbles induces distinct apoptosis of A7r5 cells.

The present study aimed to investigate whether low frequency low energy ultrasound combined with microbubbles induces apoptotic cell death of A7r5 rat aortic vascular smooth muscle cells, and to identify the possible mechanisms underlying this effect. Ultrasonic waves (45 kHz with 0.3 Wcm2 of intensity for 0, 10, 20 and 30 sec) were used together with different dosages of SonoVue™ microbubbles (0, 14, 28, 42 and 56 µl), respectively. The cell viability and apoptotic rate were determined by trypan blue staining immediately following treatment and flow cytometry 24 h thereafter. The treatment conditions resulting in the lowest amount of necrosis, highest apoptotic rate and lowest microbubble dosage was selected for the US+MB group, which was treated with ultrasound combined with microbubbles. The cell proliferation 24 h following treatment was determined and western blot analysis was applied to examine the expression of apoptosis‑associated proteins, B-cell lymphoma 2 (Bcl‑2) and Bcl-2-associated X (Bax). The harmonic acoustic pressure amplitude was measured to obtain the cavitation intensity. The combination of 20 sec ultrasound irradiation and 14 µl SonoVue™ was selected as the treatment conditions for the US+MB group. The results demonstrated that both ultrasound alone (the US group) and in combination with microbubbles significantly inhibited the proliferation of A7r5 cells compared with that of the control (P<0.01), and the suppression in the US+MB group was significantly greater (P<0.01). The apoptotic rate in A7r5 cells induced by this combination treatment (16.62±0.93%) was significantly higher than that in the control (3.93±0.39%; P<0.01) and US (6.88±1.87%; P<0.01) groups. Treatment with ultrasound combined with microbubbles increased the expression of Bax and decreased the ratio of Bcl‑2/Bax compared with those in the control and US groups. The cavitation induced by ultrasound combined with microbubble treatment was more intense than that by ultrasound alone. The results demonstrated that the cell death and apoptosis of A7r5 cells were associated with ultrasound duration and microbubble dosage. Low frequency ultrasound combined with microbubbles induced apoptosis in A7r5 cells through the upregulation of Bax and the downregulation of the Bcl‑2/Bax ratio, where the cavitation effect may have an important role.

Dexamethasone Upregulates Nox1 Expression in Vascular Smooth Muscle Cells

Background/Aim: It has been demonstrated that dexamethasone-induced hypertension can be prevented by the NADPH oxidase inhibitor apocynin. The effect of dexamethasone on NADPH oxidase, however, is unknown. The present study was conducted to investigate the effect of dexamethasone on the gene expression of Nox1, the major NADPH oxidase isoform in vascular smooth muscle cells. Results: Oral treatment of Wistar-Kyoto rats with dexamethasone (0.03 mg/kg/day) for 12 days led to an upregulation of Nox1 mRNA expression in the aorta. In cultured A7r5 rat aortic smooth muscle cells, dexamethasone increased Nox1 mRNA expression in a concentration- and time-dependent manner. The upregulation of Nox1 mRNA expression was completely prevented by the glucocorticoid receptor antagonist mifepristone. The effect of dexamethasone on Nox1 expression was likely to be indirect as it could be largely blocked by cycloheximide, an inhibitor of protein biosynthesis. Dexamethasone increased Nox1 mRNA stability as well as Nox1 transcription. The dexamethasone-induced Nox1 expression was completely prevented by scriptaid, a pan-inhibitor of histone deacetylases (HDAC), indicating a crucial role for HDAC enzymes. In total, A7r5 cells expressed 8 HDAC isoforms, with HDAC1, 5, 6 and 7 being the most abundant ones. Knockdown of these 4 individual HDAC enzymes did not prevent the effect of dexamethasone on Nox1 expression, although HDAC5 knockdown markedly reduced basal Nox1 expression. Conclusion: Dexamethasone upregulates Nox1 expression in vascular smooth muscle cells. This effect involves the glucocorticoid receptor and HDAC enzymes.

Diallyl sulfide as a potential dietary agent to reduce TNF‐α‐ and histamine‐induced proinflammatory responses in A7r5 cells

Oxidative stress-aggravated chronic inflammatory diseases of the airway are well documented; hence, treatment with antioxidants to ameliorate oxidative stress might be an effective strategy to reduce airway complications. The aim of this study was to investigate the effect and molecular mechanism of diallyl sulfide (DAS), which is a natural organosulfuric compound found in garlic, on the inhibition of tumor necrosis factor-alpha (TNF-α)- or histamine-induced inflammation in rat aortic smooth muscle A7r5 cells. A7r5 cells were coincubated with DAS before exposure to TNF-α or histamine. DAS significantly blocked the accumulation of the nuclear p65 protein in TNF-α-induced A7r5 cells by attenuating the TNF-α receptor complex through the dissociation of the TNF receptor-associated death domain and TNF receptor-associated factor 2. Moreover, DAS inhibited histamine-induced inflammation by decreasing reactive oxygen species (ROS) levels by enhancing the nuclear factor-erythroid 2-related factor 2-related antioxidative enzyme. DAS also inhibited inflammation by suppressing interleukin-1β and TNF-α through the inhibition of ROS-induced PI3K/Akt and the downstream NF-κB and activator protein-1. Our results demonstrate that DAS is a potential phytochemical to inhibit TNF-α- and histamine-induced inflammation, suggesting that DAS might be an effective dietary agent for the prevention of oxidative stress-induced inflammation of the airway.

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.

Protein Kinase C-Dependent Modulation of Heterologously Expressed Homomeric Kv7.4, Kv7.5 and Heteromeric Kv7.4/7.5; Differential Sensitivities to Low Concentrations of Vasopressin and PMA in A7R5 Vascular Smooth Muscle Cells

Kv7.4 and Kv7.5 voltage-activated potassium channels are proposed to contribute to the maintenance of resting membrane voltage in smooth muscle cells. We had previously provided evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels when natively or exogenously expressed in vascular smooth muscle cells. Endogenous Kv7 currents in smooth muscle cells are suppressed upon activation of Gq coupled receptors. It remained to be elucidated if both Kv7.4 and Kv7.5 respond similarly to low concentrations of vasopressin (AVP), an agonist of the V1a Gq-coupled receptor. using patch-clamp techniques, we measured currents through human Kv7.4 and Kv7.5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells and compared their sensitivity to AVP (100 pM and 500 pM) and to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1nM). AVP (100 pM) and PMA suppressed currents through Kv7.4, Kv7.5 and Kv7.5/7.5. Currents were reduced with different voltage dependencies and potencies in the rank order: Kv7.5 > Kv7.4/7.5 > Kv7.4. Both AVP and PMA increased the steepness of Kv7.5 voltage-dependent activation and dramatically decreased Gmax. In contrast, AVP and PMA induced a rightward shift of the Kv7.4 activation curve with only a slight reduction in maximal conductance (Gmax). The modulation of Kv7.4/7.5 activation by AVP and PMA had intermediate biophysical characteristics that were distinct from the modulation of either of the homomeric configurations. These findings reinforce the significance of PKC-dependent regulation of the Kv7 channels and suggest a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation.

Regulation and Roles of Urocortins in the Vascular System

Urocortins (Ucns) are members of the corticotropin-releasing factor (CRF) family of peptides. Ucns would have potent effects on the cardiovascular system via the CRF receptor type 2 (CRF2 receptor). Regulation and roles of each Ucn have been determined in the vascular system. Ucns have more potent vasodilatory effects than CRF. Human umbilical vein endothelial cells (HUVECs) express Ucns1-3 mRNAs, and the receptor, CRF2a receptor mRNA. Ucns1-3 mRNA levels are differentially regulated in HUVECs. Differential regulation of Ucns may suggest differential roles of those in HUVECs. Ucn1 and Ucn2 have strong effects on interleukin (IL)-6 gene expression and secretion in rat aortic smooth muscle A7r5 cells. The increase that we observed in IL-6 levels following Ucn treatment of A7r5 cells suggests that smooth muscle cells may be a source of IL-6 secretion under physiological stress conditions. Ucns are important and unique modulators of vascular smooth muscle cells and act directly or indirectly as autocrine and paracrine factors in the vascular system.

Endothelin receptor antagonist activity of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy)acetamide hydrochloride (PABSA) in rat aortic smooth muscle cells and isolated rat thoracic aorta.

Antagonist activities of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy) acetamide hydrochloride (CAS 188710-94-3, PABSA), a novel endothelin (ET) receptor antagonist, for ETA and ETB receptors were evaluated using rat aortic smooth muscle A7r5 cells and isolated rat thoracic aorta. PABSA concentration-dependently inhibited the ET-1-induced increase in intracellular calcium concentration ([Ca2+]i) mediated via ETA receptors in A7r5 cells with an IC50 of 0.17 nmol/l. PABSA antagonized the ETA receptor-mediated contraction induced by ET-1 in endothelium-denuded rat aorta with a Kb of 0.74 nmol/l. The potency of PABSA in inhibiting ETA receptor-mediated vasocontraction was approximately 40- and 100-fold greater than those of BQ-123, a selective ETA antagonist, and bosentan, a mixed ETA/ETB receptor antagonist, respectively. ETB receptor-mediated endothelium-dependent vasorelaxation induced by ET-3 in the aorta was also antagonized by PABSA, with a Kb of 9.8 nmol/l. In contrast, PABSA affected neither the vasocontraction induced by KCl or norepinephrine nor the vasorelaxation induced by acetylcholine or prostaglandin I2 in the aorta. These results suggest that PABSA is a highly potent and selective ETA receptor antagonist.