Porcine Coronary Smooth Muscle Research Articles

Activation of adenosine A2A but not A2B receptors is involved in uridine adenosine tetraphosphate-induced porcine coronary smooth muscle relaxation

Activation of both adenosine A2A and A2B receptors (A2BR) contributes to coronary vasodilation. We previously demonstrated that uridine adenosine tetraphosphate (Up4A) is a novel vasodilator in the porcine coronary microcirculation, acting mainly on A2AR in smooth muscle cells (SMC). We further investigated whether activation of A2BR is involved in Up4A-mediated coronary SMC relaxation. Both A2AR and A2BR may stimulate H2O2 production leading to activation of KATP channels in SMCs, we also studied the involvement of H2O2 and KATP channels in Up4A-mediated effect. Coronary small arteries dissected from the apex of porcine hearts were mounted on wire myograph for Up4A concentration responses. Up4A-induced coronary SMC relaxation was attenuated by A2AR but not A2BR antagonism or non-selective P2R antagonism, despite greater endogenous A2BR expression vs. A2AR in both coronary small arteries and primary cultured coronary SMCs. Moreover, Up4A-induced coronary SMC relaxation was blunted by H2O2 catabolism. This effect was not altered by KATP channel blockade. Combination of H2O2 catabolism and A2AR antagonism attenuated Up4A-induced coronary SMC relaxation to the similar extent as A2AR antagonism alone. Collectively, Up4A-induced porcine coronary SMC relaxation is mediated by activation of A2AR-H2O2 pathway. This process does not involve A2BR, P2R or KATP channels.

Madagascine Induces Vasodilatation via Activation of AMPK.

Madagascine (3-isopentenyloxyemodin) can be chemically synthesized or purified from several Rhamnus species, and it is found to have more potent biological activities than the parent compound emodin. The aim of this study is to characterize the vasodilatory effect of madagascine on vasoconstriction and sphingosylphosphorylcholine induced vasospasm in ex vivo and reveal the potential mechanisms in vitro. The effects of madagascine on vasoconstriction of rat mesenteric resistance arteries (MRAs) induced by K+, methoxamine, and endothelin-1 were, respectively, studied. The cholesterol-enriched porcine coronary vascular smooth muscle (VSM) strips were used to investigate the effects of madagascine on abnormal constriction induced by sphingosylphosphorylcholine (SPC) which has a pivotal role in vasospasm. The vasodilatory effect was induced by madagascine (0.3–100 μM) in isolated rat MRAs and the vasodilatory effect was blocked by NO synthase inhibitor L-NAME and AMPK inhibitor compound C. Madagascine (10 μM) also significantly relaxed the abnormal constriction in porcine VSM induced by SPC and the effect was abolished by compound C. Madagascine significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) in endothelial cells while decreasing the phosphorylation of myosin phosphatase target subunit 1 (MYPT1) in VSM cells. Madagascine-induced vasodilatation was abrogated using small interfering RNA knockdown of AMPK. In summary, madagascine exerted vasodilatation through activating AMPK, leading to the activation of eNOS in endothelium and inhibition of ROCK/MYPT1 in VSM. This study suggests the potential value of madagascine in amelioration of vasospasm related cardiovascular diseases.

Abstract 18708: Vascular Dysfunction in Hyperhomocysteinemia: In vitro Evidence for Endoplasmic Reticulum Stress-mediated Calcium-activated Potassium Channel Dysfunction

Objectives: KCa channels play an important role in the control of vascular tone. Opening of IKCa and SKCa in endothelial cells underlies the classic EDHF pathway and promotes NO production. Smooth muscle BKCa provides a negative feedback mechanism opposing vasoconstriction and is an effector of NO and EDHF. Previous studies demonstrated homocysteine (Hcy), a risk factor for atherosclerosis, compromises NO and EDHF function, however, whether KCa is involved is poorly studied and the underlying mechanisms remain unknown. We studied the effect of Hcy on vascular KCa with the role of endoplasmic reticulum (ER) stress explored. Methods: In vitro studies were performed in porcine coronary arteries and primary cultured porcine coronary endothelial (PCECs) and smooth muscle cells (PCSMCs). IKCa and SKCa-, and BKCa-mediated relaxations were studied in endothelium-intact and -denuded arteries in a myograph. IKCa and SKCa currents in PCECs and BKCa current in PCSMCs were analyzed by whole-cell patch clamp and channel expressions were examined by western blot. Results: Hcy impairs the role of IKCa and SKCa, and BKCa in vasorelaxation. Relaxant responses to channel activators NS309 and NS1619 were attenuated and EDHF-type response was inhibited. Hcy suppressed IKCa and SKCa currents in PCECs and BKCa currents in PCSMCs. Inhibition of ER stress enhanced KCa currents and improved EDHF-type and channel activators-induced responses. Whole-cell protein levels of IKCa and SKCa remained unchanged in Hcy-exposed PCECs whereas IKCa and SKCa at cell surface were significantly decreased. Hcy lowered protein of β1 but not α subunit of BKCa in PCSMCs. The decrease in cell surface IKCa and SKCa and reduction of BKCa β1 were restored by ER stress inhibition. Further, inhibition of PERK increased BKCa β1 protein and enhanced BKCa current. Conclusion: ER stress mediates Hcy-induced vascular dysfunction through inhibition of KCa. Suppression of cell surface expression underlies ER stress-mediated IKCa and SKCa inhibition. Downregulation of BKCa β1 by PERK-ER stress pathway plays a key role in the loss of BKCa function. This study provides new mechanistic insights into the role of ER stress in vascular dysfunction. Supported by RGC GRF CUHK4774/12M & CUHK14118414, and NSFC 81200123.

Upregulation of intermediate-conductance Ca2+-activated K+ channels (KCNN4) in porcine coronary smooth muscle requires NADPH oxidase 5 (NOX5).

AimsNADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca2+-activated K+ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors.Methods and ResultsDihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration.ConclusionsOur findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.

Ovariectomy increases L-type Ca(2+) channel activity in porcine coronary smooth muscle.

Sex hormone status has been demonstrated to play a role in the regulation of ion channel activity. We previously demonstrated increased L-type Ca channel current (ICa) in the coronary smooth muscle cells (SMCs) of male swine compared with female swine. In male swine, endogenous testosterone increases ICa in SMCs by enhanced expression of the pore-forming α1 subunit Cav1.2. Conversely, the role of sex hormones in female swine has not previously been investigated. Therefore, the purpose of the current study was to determine the effect of ovariectomy (OVX) on L-type Ca channel activity and expression in female Yucatan miniature swine. Sexually mature female swine were obtained from a breeder and either left intact (intact female [IF]; n = 5) or ovariectomized (n = 6). Sensitivity to depolarization-induced contractions was increased by OVX. Accordingly, mean (SEM) ICa was enhanced in the OVX group (-9.5 [0.6] pA/pF) compared with the IF group (-4.5 [0.3] pA/pF), although L-type Ca channel α1 subunit (Cav1.2; α1c) messenger RNA (mRNA) and protein expressions were unchanged.Among the L-type Ca channel β subunits, β1 (188 [31]) and β2a (561 [79]) had higher mRNA expression levels (target/18S) than β3 (9 [1]) and β4 (2 [0.1]). Although β2a, β3, and β4 mRNA and protein expressions were not different between groups, protein expression of the β1 subunit (Cavβ1) was decreased in the OVX group compared with the IF group. Endogenous female hormones inhibit L-type Ca channel activity in coronary SMCs potentially via the up-regulation of Cavβ1 subunit expression.

Mechanism of inhibition of delayed rectifier K+ current by diphenyl phosphine oxide‐1 in porcine coronary smooth muscle (1079.10)

Mechanisms involved in DPO‐1‐induced block of KDR were studied in myocytes isolated from the porcine coronary artery. DPO‐1 inhibited the slowly inactivating KDR in a dose‐dependent manner (IC50 = 212 ± 28 nM). Neither the time constant of activation (+20 mV) nor the time constant of deactivation (‐40 mV) was affected by DPO‐1 (1 μM). However, the fast and slow time constants of inactivation at +20 mV were notably shortened by DPO‐1 (fast = 0.52 ± 0.07 vs. 0.14 ± 0.01 s; slow = 5.8 ± 1.1 vs. 0.6 ± 0.2 s). These results suggest that DPO‐1 preferentially binds to the channel in the open state, producing an open channel block of KDR. As such, if KDR channels were open under resting conditions in vascular smooth muscle, then DPO‐1 should depolarize the membrane potential and cause vasoconstriction. To test this hypothesis and show functional relevance, we determined the effect of DPO‐1 on the membrane potential and coronary blood flow. DPO‐1 depolarized membrane potential from –29 ± 1 to ‐14 ± 3 mV. Further, DPO‐1 reduced coronary artery blood flow at 100 mmHg perfusion pressure from 21 to 14 ml/min. Together, these data indicate that DPO‐1 blocks KDR channels in the open state and that KDR channels in vascular smooth muscle are open under resting conditions. Thus, therapeutic tools that target KDR activity could be used to regulate vascular tone and mechanisms controlling KDR merit further study.Grant Funding Source: Supported by T32HL090610, T32HL079995, AHA 13POST1681001813, HL092245

Ovariectomy increases L-type calcium channel activity in porcine coronary smooth muscle

Menopause, published monthly, provides a forum for new research, applied basic science, and clinical guidelines on all aspects of menopause. The scope and usefulness of the journal extend beyond gynecology, encompassing many varied biomedical areas, including internal medicine, family practice, medical subspecialties such as cardiology and geriatrics, epidemiology, pathology, sociology, psychology, anthropology, and pharmacology. This forum is essential to help integrate these areas, highlight needs for future research, and enhance health care.

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca2+-activated K+ channel (BKCa) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BKCa channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K+ currents (STOC, a component pattern of BKCa currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BKCa currents) via an IP3 receptor- and sarcoplasmic Ca2+ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BKCa channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BKCa channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BKCa channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca2+ sensitivity of the channels.

NADPH oxidase (NOX) derived superoxide (O2.‐) upregulates intermediate‐conductance Ca2+ ‐ activated K+ channel (KCa3.1) expression in vascular smooth muscle cells (VSMCs)

Reactive oxygen species (ROS), predominantly from NOX, mediate VSMC phenotypic modulation known to play a role in hypertension, atherosclerosis, and hypertrophy. We have previously shown that upregulation of KCa3.1 via increased AP‐1 activity is required for phenotypic modulation of porcine coronary smooth muscle cells (PCSMC). However, the role of ROS in KCa3.1 regulation remains unknown. Our objective was to determine if NOX regulates KCa3.1 expression via AP‐1. Basic fibroblast growth factor (bFGF, 25ng/mL) increased O2.−production in PCSMC, while the NOX inhibitor apocynin (Apo, 1mM) completely abolished O2.− production. bFGF increased KCa3.1 mRNA levels 2.5 fold; while decreasing smooth muscle myosin heavy chain mRNA levels 80% (P< 0.05) in PCSMC. Apo completely inhibited KCa3.1 mRNA upregulation indicating that NOX derived O2.−is required for KCa3.1 upregulation. Acute bFGF significantly increased K+ current, which was inhibited ~50% by the KCa3.1 specific channel blocker TRAM‐34 (100nM) indicating that bFGF causes an upregulation in KCa3.1 channel activity. bFGF increased AP‐1 promoter activity 2.5 fold, while Apo completely inhibited the response to bFGF. These results support a novel mechanism by which NOX derived O2.− regulates KCa3.1 expression through AP‐1.

Abstract 5257: Inhibition of Myosin Light Chain Phosphatase by Rho Kinase (ROCK) Modulates Arteriolar Tone and Constriction of Coronary Arterioles

ROCK has been suggested to regulate smooth muscle contraction of conduit artery via multiple pathways including direct myosin light chain (MLC) phosphorylation or inhibition of MLC phosphatase (MLCP) activity. Although recent studies have implicated the involvement of ROCK in pathogenesis of cardiovascular disease, the role and expression of ROCK isoforms (ROCK1 and ROCK2) and their signaling pathway for vasomotor regulation in resistance arterioles remain unclear. Using an isolated and pressurized vessel approach, we found that the resting tone and the endothelin-1 (ET-1)-induced constriction of porcine coronary arterioles (60±4 μ m resting diameter) were preferentially inhibited, in a dose-dependent manner, by a potent ROCK2 inhibitor H-1152 compared to a non-selective ROCK antagonist Y-27632. MLCP inhibitor calyculin A reversed the inhibitory effects of H-1152 and Y-27632, but ROCK inhibition failed to prevent or reverse calyculin A-induced arteriolar constriction. These results indicate that ROCK is involved in the development of basal tone and vasoconstriction to ET-1 and that MLCP is a major target of ROCK. Immunoblotting and immunohistochemical data showed that ROCK2 expression is 2-fold higher than ROCK1 in coronary arterioles with preferential media-layer distribution. Both ROCK1 and ROCK2 were also expressed in cultured porcine coronary arteriolar smooth muscle cells (PCASMC) with ROCK2 as the major isoform. In cultured PCASMC, MLC phosphorylation (ser19) elicited by ET-1, thromboxane analogue (U46619) and 80 mM KCl was sensitive to H-1152, supporting the involvement of ROCK2 in regulating coronary arteriolar tone via MLC phosphorylation. Consistent with our functional data, calyculin A increased MLC phosphorylation (ser19) in PCASMC, but ROCK2 inhibition (by H-1152) failed to reverse such effect. We conclude that ROCK2 is the major isoform mediating the development of basal tone and vasoconstrictor activity in coronary resistance arterioles primarily via regulation of MLCP activity.

Advanced glycation endproducts induce a proliferative response in vascular smooth muscle cells via altered calcium signaling

Advanced glycation endproducts (AGEs) are proteins that accumulate in the plasma of diabetics as a result of increased glucose concentrations and are closely linked with vascular disease. The mechanisms involved are still not clear. The aim of this study was to investigate whether AGE-induced changes in calcium (Ca 2+) homeostasis could contribute to these mechanisms. Cultured porcine coronary artery vascular smooth muscle (VSM) cells were preincubated with glycated albumin for 96 h. The sphingosine 1-phosphate (S1P)-induced intracellular Ca 2+ increase, although not increased in amplitude, was significantly prolonged in cells preincubated with glycated albumin. Intracellular Ca 2+ imaging and electrophysiological recording of ion channel currents following release of caged Ca 2+ indicated that this prolonged Ca 2+ rise occurred predominantly via changes in Ca 2+-induced Ca 2+ release. Preincubation with glycated albumin also resulted in a threefold increase in expression of the receptor for AGE. As a consequence of the prolonged intracellular Ca 2+ rise following preincubation with glycated albumin, the S1P-induced activation of the Ca 2+-dependent phosphatase, calcineurin (CaN) was increased. This resulted in increased S1P-induced activation of the Ca 2+-dependent transcription factor, nuclear factor of activated T cells (NFATc). BrdU incorporation in VSM cells was increased in cells preincubated with glycated albumin and was inhibited by the CaN inhibitor, cyclosporin A. In conclusion, AGE can induce VSM proliferation via a prolonged agonist-induced Ca 2+ increase leading to increased activation of CaN and subsequently NFATc. This mechanism may contribute to pathogenesis of vascular disease in diabetes mellitus.

Local Delivery of the K Ca 3.1 Blocker, TRAM-34, Prevents Acute Angioplasty-Induced Coronary Smooth Muscle Phenotypic Modulation and Limits Stenosis

We previously demonstrated that upregulation of intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of K(Ca)3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific K(Ca)3.1 blocker TRAM-34. Expression of K(Ca)3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. K(Ca)3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented K(Ca)3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Blockade of K(Ca)3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.

NADPH Oxidases: New Regulators of Old Functions

Antioxidants & Redox SignalingVol. 8, No. 9-10 Forum EditorialNADPH Oxidases: New Regulators of Old FunctionsKathy K. GriendlingKathy K. GriendlingSearch for more papers by this authorPublished Online:20 Sep 2006https://doi.org/10.1089/ars.2006.8.1443AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByTwenty years of progress in angiotensin converting enzyme 2 and its link to SARS-CoV-2 disease16 October 2020 | Clinical Science, Vol. 134, No. 19Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats16 April 2020 | PLOS ONE, Vol. 15, No. 4Redox Systems Biology of Nutrition and Oxidative Stress5 April 2019 | The Journal of Nutrition, Vol. 149, No. 4The natural phenolic compounds as modulators of NADPH oxidases in hypertensionPhytomedicine, Vol. 55New Therapies for the Treatment of Renal Fibrosis9 August 2019A Systematic Review on Natural Antioxidant Properties of Resveratrol1 September 2018 | Natural Product Communications, Vol. 13, No. 9Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction24 February 2016 | Basic Research in Cardiology, Vol. 111, No. 2Potential Role of Protein Disulfide Isomerase in Metabolic Syndrome-Derived Platelet HyperactivityOxidative Medicine and Cellular Longevity, Vol. 2016Upregulation of Intermediate-Conductance Ca2+-Activated K+ Channels (KCNN4) in Porcine Coronary Smooth Muscle Requires NADPH Oxidase 5 (NOX5)21 August 2014 | PLoS ONE, Vol. 9, No. 8Potential approaches to reverse or repair renal fibrosis11 February 2014 | Nature Reviews Nephrology, Vol. 10, No. 4Caffeic Acid Phenethyl Ester Inhibits Diesel Exhaust Particle—Induced Inflammation of Human Middle Ear Epithelial Cells via NOX4 Inhibition1 September 2013 | Annals of Otology, Rhinology & Laryngology, Vol. 122, No. 9Environmental Toxicology: Oxidative Stress4 December 2012Molecular and cellular mechanisms of pulmonary fibrosis23 July 2012 | Fibrogenesis & Tissue Repair, Vol. 5, No. 1Cell oxidant stress delivery and cell dysfunction onset in type 2 diabetesBiochimie, Vol. 94, No. 9Diabetic Vascular DiseaseReactive Oxygen Species and Oxidative StressThe Oxidative Function of Diferric TransferrinBiochemistry Research International, Vol. 2012Environmental Toxicology: Oxidative StressMolecular Medicine, Vol. 17, No. 11-12Redox regulation of Nox proteinsRespiratory Physiology & Neurobiology, Vol. 174, No. 3Effect of Early Particulate Air Pollution Exposure on Obesity in MiceArteriosclerosis, Thrombosis, and Vascular Biology, Vol. 30, No. 12NAD(P)H Oxidase Mediates TGF- 1-Induced Activation of Kidney Myofibroblasts19 November 2009 | Journal of the American Society of Nephrology, Vol. 21, No. 1Reactive Oxygen Species, Oxidative Stress, and Hypertension17 August 2010The Role of Oxidative Stress in Endothelial Dysfunction and Vascular InflammationRole of p47 phox in regulating Cdc42GAP, vimentin, and contraction in smooth muscle cellsAmerican Journal of Physiology-Cell Physiology, Vol. 297, No. 6Resveratrol Improves Endothelial FunctionArteriosclerosis, Thrombosis, and Vascular Biology, Vol. 29, No. 8Air pollution and cardiac remodeling: a role for RhoA/Rho-kinaseAmerican Journal of Physiology-Heart and Circulatory Physiology, Vol. 296, No. 5Regulation of NADPH Oxidase in Vascular Endothelium: The Role of Phospholipases, Protein Kinases, and Cytoskeletal Proteins Srikanth Pendyala, Peter V. 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Hypoxia-induced vascular smooth muscle relaxation: increased ATP-sensitive K+ efflux or decreased voltage-sensitive Ca2+ influx?

controversy continues regarding the cellular mechanisms of hypoxic vasodilation. The vascular endothelium may contribute to hypoxic dilations by releasing relaxing factors that act on the smooth muscle ([4][1], [6][2]–[8][3]). In contrast, numerous studies have suggested a role of the vascular

IKCa1 is an immediate early response gene mediating coronary smooth muscle phenotypic modulation

We previously demonstrated functional upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) with the development of early coronary atherosclerosis in vivo and as a prerequisite for platelet-derived growth factor BB (PDGF-BB) induced porcine coronary smooth muscle cell (CSMC) dedifferentiation and migration in vitro. Our objective was to examine the temporal regulation of IKCa1 and smooth muscle marker gene expression in porcine CSMC exposed to coronary balloon injury (CBI) in vivo or PDGF-BB in vitro. Two hours following CBI, laser capture microdissection of media from injured vessels demonstrated higher IKCa1, c-jun, and c-fos, but unaltered amounts of SMMHC mRNA compared to control media. PDGF-BB increased enrichment of H4 acetylation and c-jun to the AP-1 binding site of the IKCa1 promoter ~4-fold, an effect blocked by TRAM-34, a specific IKCa1 channel blocker. PDGF-BB increased IKCa1 and decreased myocardin mRNA at 30 minutes, but did not decrease smooth muscle myosin heavy chain (SMMHC) or smoothelin expression until 10 hours. In conclusion, these data indicate that AP-1 dependent upregulation of IKCa1 is an immediate and requisite factor initiating smooth muscle specific gene suppression, i.e. CSMC phenotypic modulation, during coronary injury. Supported by: NIH HL52490

Resting smooth muscle cells as a model for studying vascular cell activation

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also reacquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimick in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.

Contribution of capacitative and non-capacitative Ca 2+-entry to M 3-receptor-mediated contraction of porcine coronary smooth muscle

We studied the contribution of store-operated or capacitative Ca 2+-entry (SOCE or CCE, respectively) through store-operated Ca 2+ channels (SOCCs) and the contribution of Ca 2+-entry through receptor-operated, non-selective cation channels (ROCCs or NSCCs, respectively), on the M 3-receptor-mediated (270 nM Ach) contractile response of porcine coronary smooth muscle strips by means of the respective inhibitors. In the presence of L-VOCC blockade (1 μM verapamil), LOE 908 (inhibition of NSCCs) decreased the contractile response to 75 ± 5% ( p < 0.01, n = 6), 2-APB (inhibition of SOCCs) and SK&F 96365 (inhibition of SOCCs and of NSCCs) decreased the response to 45 ± 4% ( p < 0.001, n = 10) and to 23 ± 2% ( p < 0.001, n = 5), respectively (control: Ach response in the presence of verapamil alone). In the absence of L-VOCC blockade, LOE 908 reduced the Ach-response to 49 ± 7% ( p < 0.001, n = 8) and SK&F 96365 to 3 ± 2% ( p < 0.001, n = 4) of control, whereas 2-APB transiently increased the response (peak effect: 130 ± 11%; p < 0.05, n = 8). We conclude: (1) the main source of activator Ca 2+ during the M 3-receptor-mediated contractile response is the Ca 2+ influx through L-VOCCs; (2) however, in the presence of L-VOCC blockade, the contractile response is mainly due to Ca 2+-entry through SOCCs; (3) NSCCs may be considerably involved in M 3-receptor-mediated contraction as they may serve to depolarize the membrane potential and, thus, to open L-VOCCs; (4) in primary tissue of vascular smooth muscle, both, SOCE and Ca 2+-entry through NSCCs are activated during M 3-receptor stimulation.

Identification of active siRNAs against IGF-IR of porcine coronary smooth muscle cells in a heterologous cell line

Testing of short interfering RNAs (siRNAs) to knock-down gene expression is complicated in primary differentiated cells due to their limited availability and short in vitro life span. The objective of this study was to bypass these limitations by testing selected siRNAs in a heterologous cell line. A plasmid containing a fragment of porcine IGF-I receptor (pIGF-IR) gene cloned downstream of EGFP sequence was constructed and cotransfected with pIGF-IR/siRNAs in HEK 293T human cell line. The abatement of EGFP fluorescence, measured at mRNA and protein level, was compared with that induced by a EGFP/siRNA. Among the three pIGF-IR/siRNAs tested, one was active against the hybrid reporter gene as EGFP/siRNA, while the other two were inactive. When transfected in primary porcine coronary smooth muscle cells (PC-SMCs) and tested by real-time PCR, immunocytofluorimetry and cell motility assay, pIGF-IR/siRNAs confirmed the results obtained in HEK 293T cells, thus establishing that the search of active siRNAs addressed to a gene of a cell and/or a species of interest can be done in a heterologous cell line.

Involvement of p38-mitogen-activated protein kinase in adenosine receptor-mediated relaxation of coronary artery

The purpose of this study was to explore the involvement of adenosine receptor(s) in porcine coronary artery (PCA) relaxation and to define the role of MAPK signaling pathways. Isometric tensions were recorded in denuded PCA rings. 5'-(N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine receptor agonist, induced a concentration-dependent relaxation (EC(50) = 16.8 nM) of PGF(2alpha) (10 microM)-preconstricted arterial rings. NECA-induced relaxation was completely blocked by 0.1 microM SCH-58261 (A(2A) antagonist) at lower doses (1-40 nM) but not at higher doses (80-1,000 nM). MRS-1706 (1 microM, A(2B) antagonist) was able to shift the NECA concentration-response curve to the right. CGS-21680 (selective A(2A) agonist) induced responses similarly to NECA, whereas N(6)-cyclopentyladenosine (A(1) agonist) and Cl-IB-MECA (A(3) agonist) did not. Furthermore, the effect of NECA was attenuated by the addition of SB-203580 (10 microM, p38 MAPK inhibitor) but not by PD-98059 (10 microM, MEK inhibitor). Interestingly, SB-203580 had no effect on CGS-21680-induced relaxation. Western blot analysis demonstrated that PGF(2alpha) and adenosine agonists stimulated p38 MAPK at a concentration of 40 nM in PCA smooth muscle cells. MRS-1706 (1 microM) significantly reduced NECA-induced p38 MAPK phosphorylation. Addition of NECA and SB-203580 alone or in combination inhibited PGF(2alpha)-induced p38 MAPK. Western blot data were further confirmed by p38 MAPK activity measurement using activating transcription factor-2 assay. Our results suggest that the adenosine receptor subtype involved in causing relaxation of porcine coronary smooth muscle is mainly A(2A) subtype, although A(2B) also may play a role, possibly through p38 MAPK pathway.

Cloning, up-regulation, and mitogenic role of porcine P2Y2 receptor in coronary artery smooth muscle cells.

Previous work has shown up-regulation of a UTP-sensitive P2Y receptor in porcine coronary smooth muscle cells (CSMC) of organ-cultured arteries. However, the molecular identity and functional role of this putative receptor remained undefined. Here we report the cloning of the cDNA for this receptor that encodes an open reading frame for a protein of 373 amino acids with the highest homology to the human P2Y(2) receptor (84%). Heterologous expression of this receptor in 1321N1 cells revealed a novel pharmacology in that UTP and ITP were full agonists and UTP was more potent and efficacious than ATP for increasing intracellular [Ca(2+)] and extracellular signal-regulated kinase phosphorylation. Stimulation of subcultured CSMC with UTP, ITP, or ATP induced a concentration-dependent increase in cellular DNA content, protein synthesis, cell number, and proliferating cell nuclear antigen expression, indicating a mitogenic role for P2Y(2) receptors. This was supported by the finding that the treatment of CSMC with antisense oligonucleotides to the cloned cDNA sequence significantly inhibited UTP- and ATP-induced DNA and protein synthesis. In addition, reverse transcription-polymerase chain reaction analysis showed that P2Y(2) receptor mRNA was dramatically increased in cells of organ-cultured arteries compared with freshly harvested arteries, whereas the P2Y(6) receptor mRNA level was unchanged, and the P2Y(4) receptor mRNA was undetectable. This P2Y(2) subtype-specific up-regulation was confirmed in cells of coronary arteries stented in vivo. In conclusion, we have cloned the porcine P2Y(2) receptor with novel pharmacology and demonstrated that this receptor is up-regulated in CSMC of in vitro organ cultures or in vivo stented coronary arteries to mediate the mitogenic effects of nucleotides.