Constrictor-induced translocation of NFAT3 in human and rat pulmonary artery smooth muscle

2005 ◽  
Vol 289 (6) ◽  
pp. L1061-L1074 ◽  
Author(s):  
Asma Yaghi ◽  
Stephen M. Sims
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Rho Kinase ◽  
Pulmonary Vasculature ◽  
Resting Cells ◽  
Maximal Effect ◽  
Activated T Cells ◽  
Western Blots ◽  
Transient Rise ◽  
Low Amplitude

The transcription factor nuclear factor of activated T cells (NFAT) resides in the cytoplasm in resting cells and upon stimulation is dephosphorylated, translocates to the nucleus, and becomes transcriptionally active. NFAT is commonly activated by stimulation of receptors coupled to Ca2+ mobilization; however, little is known about the regulation of NFAT in pulmonary vascular smooth muscle. The aim of this study was to investigate regulation of NFAT in human and rat intralobar pulmonary artery by two constrictors: phenylephrine (PE) and 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 metabolite formed endogenously in lungs. Immunostaining of smooth muscle cells revealed cytoplasmic localization of NFAT in untreated cells, and PE or 20-HETE induced translocation to the nucleus, with maximal effect at 30 min. Cyclosporin A and FK-506 (both 1 μM) inhibited NFAT translocation, indicating involvement of calcineurin. Moreover, the Rho-kinase blocker Y-27632 prevented translocation. Translocation of NFAT was confirmed by Western blots, with NFAT3 the prominent isoform in pulmonary artery. Constrictors caused calcineurin-sensitive translocation of NFAT to nuclei in intact arteries, demonstrating regulation in native tissue. To investigate a role for Ca2+, cells were loaded with fura-2. Whereas PE caused an acute transient rise of [Ca2+]i, 20-HETE caused a prolonged low amplitude rise of [Ca2+]i. The involvement of Rho-kinase in PE- and 20-HETE-induced NFAT3 translocation in pulmonary artery suggests a level of control not previously recognized in smooth muscle. Constrictors of the pulmonary vasculature not only cause acute responses but also activate NFAT, which may alter gene expression in pulmonary health and disease.

scholarly journals Diversity of voltage-dependent K+ channels in human pulmonary artery smooth muscle cells

2004 ◽  
Vol 287 (1) ◽  
pp. L226-L238 ◽  
Author(s):  
Oleksandr Platoshyn ◽  
Carmelle V. Remillard ◽  
Ivana Fantozzi ◽  
Mehran Mandegar ◽  
Tiffany T. Sison ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Critical Role ◽  
Muscle Cells ◽  
Pulmonary Vasculature ◽  
Wide Range ◽  
Voltage Dependent ◽  
Pulmonary Artery Smooth Muscle ◽  
Α Subunits

Electrical excitability, which plays an important role in excitation-contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study examined the heterogeneous nature of native voltage-dependent K+ channels in human PASMC. Both voltage-gated K+ (KV) currents and Ca2+-activated K+ (KCa) currents were observed and characterized. In cell-attached patches of PASMC bathed in Ca2+-containing solutions, depolarization elicited a wide range of K+ unitary conductances (6–290 pS). When cells were dialyzed with Ca2+-free and K+-containing solutions, depolarization elicited four components of KV currents in PASMC based on the kinetics of current activation and inactivation. Using RT-PCR, we detected transcripts of 1) 22 KV channel α-subunits (KV1.1–1.7, KV1.10, KV2.1, KV3.1, KV3.3–3.4, KV4.1–4.2, KV5.1, KV 6.1–6.3, KV9.1, KV9.3, KV10.1, and KV11.1), 2) three KV channel β-subunits (KVβ1–3), 3) four KCa channel α-subunits ( Slo-α1 and SK2–SK4), and 4) four KCa channel β-subunits (KCaβ1–4). Our results show that human PASMC exhibit a variety of voltage-dependent K+ currents with variable kinetics and conductances, which may result from various unique combinations of α- and β-subunits forming the native channels. Functional expression of these channels plays a critical role in the regulation of membrane potential, cytoplasmic Ca2+, and pulmonary vasomotor tone.

Implication of the ryanodine receptor in TRPV4-induced calcium response in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

2012 ◽  
Vol 303 (9) ◽  
pp. L824-L833 ◽  
Author(s):  
Diana Dahan ◽  
Thomas Ducret ◽  
Jean-François Quignard ◽  
Roger Marthan ◽  
Jean-Pierre Savineau ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Calcium Signaling ◽  
Ryanodine Receptor ◽  
Muscle Cells ◽  
Pulmonary Vasculature ◽  
Release Mechanism ◽  
Patch Clamp Technique ◽  
Calcium Response

There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 μM) increased the intracellular calcium concentration ([Ca2+]i). This effect was significantly reduced by a high concentration of ryanodine (100 μM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 μM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 μM of dantrolene did not attenuate the 4α-PDD-induced [Ca2+]i increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca2+ release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.

scholarly journals Mechanisms of BDNF regulation in asthmatic airway smooth muscle

2016 ◽  
Vol 311 (2) ◽  
pp. L270-L279 ◽  
Author(s):  
Bharathi Aravamudan ◽  
Michael A. Thompson ◽  
Christina M. Pabelick ◽  
Y. S. Prakash
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Transient Receptor Potential ◽  
Rho Kinase ◽  
Receptor Potential ◽  
Camp Response Element Binding ◽  
Transient Receptor Potential Channels ◽  
Bdnf Expression ◽  
Activated T Cells ◽  
Tnf Α

Brain-derived neurotrophic factor (BDNF), a neurotrophin produced by airway smooth muscle (ASM), enhances inflammation effects on airway contractility, supporting the idea that locally produced growth factors influence airway diseases such as asthma. We endeavored to dissect intrinsic mechanisms regulating endogenous, as well as inflammation (TNF-α)-induced BDNF secretion in ASM of nonasthmatic vs. asthmatic humans. We focused on specific Ca2+ regulation- and inflammation-related signaling cascades and quantified BDNF secretion. We find that TNF-α enhances BDNF release by ASM cells, via several mechanisms relevant to asthma, including transient receptor potential channels TRPC3 and TRPC6 (but not TRPC1), ERK 1/2, PI3K, PLC, and PKC cascades, Rho kinase, and transcription factors cAMP response element binding protein and nuclear factor of activated T cells. Basal BDNF expression and secretion are elevated in asthmatic ASM and increase further with TNF-α exposure, involving many of these regulatory mechanisms. We conclude that airway BDNF secretion is regulated at multiple levels, providing a basis for autocrine effects of BDNF under conditions of inflammation and disease, with potential downstream influences on contractility and remodeling.

Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway

2004 ◽  
Vol 287 (4) ◽  
pp. L673-L684 ◽  
Author(s):  
Jean-Marc Hyvelin ◽  
Clare O’Connor ◽  
Paul McLoughlin
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Kinase Inhibitor ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Systemic Circulation ◽  
Pulmonary Vasculature ◽  
Tension Development ◽  
Kinase Pathway

Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+ sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the α1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (≤3 μM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in α-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+ activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+ sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.

8-Isoprostane-induced endothelin-1 production by infant rat pulmonary artery smooth muscle cells is mediated by Rho-kinase

2006 ◽  
Vol 41 (6) ◽  
pp. 942-949 ◽  
Author(s):  
Soojin L. Yi ◽  
Crystal Kantores ◽  
Rosetta Belcastro ◽  
Judy Cabacungan ◽  
A. Keith Tanswell ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Endothelin 1 ◽  
Infant Rat ◽  
Pulmonary Artery Smooth Muscle

scholarly journals Distinct Effects of Contraction Agonists on the Phosphorylation State of Cofilin in Pulmonary Artery Smooth Muscle

2008 ◽  
Vol 2008 ◽  
pp. 1-9 ◽  
Author(s):  
Yan-Ping Dai ◽  
Shaner Bongalon ◽  
Violeta N. Mutafova-Yambolieva ◽  
Ilia A. Yamboliev
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Pulmonary Artery ◽  
Rho Kinase ◽  
Fold Increase ◽  
Isometric Contractions ◽  
Maximal Contraction ◽  
Pulmonary Artery Smooth Muscle ◽  
Cultured Smooth Muscle Cells ◽  
20 Nm

We hypothesized that agonist-induced contraction correlates with the phospho-cofilin/cofilin (P-CF/CF) ratio in pulmonary artery (PA) rings and cultured smooth muscle cells (PASMCs). PA rings were used for isometric contractions and along with PASMCs for assay of P-CF/CF by isoelectric focusing and immunoblotting. The P-CF/CF measured 22.5% in PA and differentiated PASMCs, but only 14.8% in undifferentiated PASMCs. With comparable contraction responses in PA, endothelin-1 (100 nM) and norepinephrine (1 μM) induced a 2-fold increase of P-CF/CF, while angiotensin II (1 μM) induced none. All agonists activated Rho-kinase and LIMK2, and activation was eliminated by inhibition of Rho-kinase. Microcystin LF (20 nM) potentiated the angiotensin II, but not the 5-hydroxytryptamine (1 μM)-mediated increase of P-CF/CF. In conclusion, all tested agonists activate the Rho-kinase-LIMK pathway and increase P-CF/CF. Angiotensin II activates PP2A and counteracts the LIMK-mediated CF phosphorylation. CF phosphorylation stabilizes peripheral actin structures and may contribute to the maximal contraction of PA.

scholarly journals Rho kinase modulates postnatal adaptation of the pulmonary circulation through separate effects on pulmonary artery endothelial and smooth muscle cells

2010 ◽  
Vol 299 (6) ◽  
pp. L872-L878 ◽  
Author(s):  
Cristina M. Alvira ◽  
David J. Sukovich ◽  
Shu-Chen Lyu ◽  
David N. Cornfield
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Barrier Function ◽  
Pulmonary Circulation ◽  
Vascular Tone ◽  
Control Period ◽  
Rho Kinase ◽  
Pulmonary Vascular Disease ◽  
No Production ◽  
Arginine Infusion

At birth, pulmonary vasodilation occurs concomitant with the onset of air-breathing life. Whether and how Rho kinase (ROCK) modulates the perinatal pulmonary vascular tone remains incompletely understood. To more fully characterize the separate and interactive effects of ROCK signaling, we hypothesized that ROCK has discrete effects on both pulmonary artery (PA): 1) endothelial cell (PAEC) nitric oxide (NO) production and contractile state; and 2) smooth muscle cell tone independent of endothelial NO synthase (eNOS) activity. To test these hypotheses, NO production and endothelial barrier function were determined in fetal PAEC under baseline hypoxia and following exposure to normoxia with and without treatment with Y-27632, a specific pharmacological inhibitor of ROCK. In acutely instrumented, late-gestation ovine fetuses, eNOS was inhibited by nitro-l-arginine infusion into the left PA (LPA). Subsequently, fetal lambs were mechanically ventilated (MV) with 100% oxygen in the absence (control period) and presence of Y-27632. In PAEC, treatment with Y-27632 had no effect on cytosolic calcium but did increase normoxia-induced NO production. Moreover, acute normoxia increased PAEC barrier function, an effect that was potentiated by Y-27632. In fetal lambs, MV during the control period had no effect on LPA flow. In contrast, MV after Y-27632 increased LPA flow and fetal arterial Po2 (PaO2) and decreased PA pressure. In conclusion, ROCK activity modulates vascular tone in the perinatal pulmonary circulation via combined effects on PAEC NO production, barrier function, and smooth muscle tone. ROCK inhibition may represent a novel treatment strategy for neonatal pulmonary vascular disease.

scholarly journals PDGF induces SphK1 expression via Egr-1 to promote pulmonary artery smooth muscle cell proliferation

2016 ◽  
Vol 310 (11) ◽  
pp. C983-C992 ◽  
Author(s):  
Justin R. Sysol ◽  
Viswanathan Natarajan ◽  
Roberto F. Machado
Keyword(s):  
Cell Proliferation ◽  
Transcription Factor ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Protein Expression ◽  
Selective Inhibition ◽  
Pulmonary Vasculature ◽  
Sphingosine 1 Phosphate ◽  
Life Threatening ◽  
Pulmonary Artery Smooth Muscle

Pulmonary arterial hypertension (PAH) is a progressive, life-threatening disease for which there is currently no curative treatment available. Pathologic changes in this disease involve remodeling of the pulmonary vasculature, including marked proliferation of pulmonary artery smooth muscle cells (PASMCs). Recently, the bioactive lipid sphingosine-1-phosphate (S1P) and its activating kinase, sphingosine kinase 1 (SphK1), have been shown to be upregulated in PAH and promote PASMC proliferation. The mechanisms regulating the transcriptional upregulation of SphK1 in PASMCs are unknown. In this study, we investigated the role of platelet-derived growth factor (PDGF), a PAH-relevant stimuli associated with enhanced PASMC proliferation, on SphK1 expression regulation. In human PASMCs (hPASMCs), PDGF significantly increased SphK1 mRNA and protein expression and induced cell proliferation. Selective inhibition of SphK1 attenuated PDGF-induced hPASMC proliferation. In silico promoter analysis for SphK1 identified several binding sites for early growth response protein 1 (Egr-1), a PDGF-associated transcription factor. Luciferase assays demonstrated that PDGF activates the SphK1 promoter in hPASMCs, and truncation of the 5′-promoter reduced PDGF-induced SphK1 expression. Stimulation of hPASMCs with PDGF induced Egr-1 protein expression, and direct binding of Egr-1 to the SphK1 promoter was confirmed by chromatin immunoprecipitation analysis. Inhibition of ERK signaling prevented induction of Egr-1 by PDGF. Silencing of Egr-1 attenuated PDGF-induced SphK1 expression and hPASMC proliferation. These studies demonstrate that SphK1 is regulated by PDGF in hPASMCs via the transcription factor Egr-1, promoting cell proliferation. This novel mechanism of SphK1 regulation may be a therapeutic target in pulmonary vascular remodeling in PAH.

KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase

2004 ◽  
Vol 287 (4) ◽  
pp. L852-L858 ◽  
Author(s):  
Luke J. Janssen ◽  
Tracy Tazzeo ◽  
Jianmin Zuo ◽  
Evi Pertens ◽  
Shaf Keshavjee
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Contractile Activity ◽  
Rho Kinase ◽  
Western Blots ◽  
Downstream Effector ◽  
Highly Sensitive ◽  
Voltage Dependent ◽  
Evoked Contractions ◽  
Stimulation Of

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 μM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.

Abstract P182: The Novel Perivascular Adipose Tissue Adipokine, Chemerin, Signals Through G i - and Calcium-Dependent Mechanisms

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
David J Ferland ◽  
Emma S Darios ◽  
Richard R Neubig ◽  
Benita Sjögren ◽  
Nguyen Truong ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Isometric Contraction ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Calcium Flux ◽  
Western Blots ◽  
Perivascular Adipose Tissue ◽  
Erk Mapk

Chemerin is an adipokine associated with inflammation, increased blood pressure, and may be a link between the pathologies of obesity and hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via the chemerin receptor and calcium flux in smooth muscle cells. Known mediators of the amplified arterial responsiveness seen in hypertension (L-type Ca 2+ channels, Src, and Rho kinase) were interrogated by isometric contraction of rat aortic rings in parallel with calcium kinetics of rat aortic smooth muscle cells. Western blots were also used to observe phosphorylation of Erk/MAPK. Chemerin-9 (nonapeptide of the chemerin S 157 isoform) caused a concentration-dependent contraction of isolated aorta (EC 50 100 nM) and elicited a concentration-dependent intracellular calcium response (EC 50 10 nM). Both calcium influx and isometric contraction, respectively, were reduced (units of “% of vehicle response”) by Pertussis toxin (G i inhibitor; 0±3% and 23±9%), verapamil (L-type Ca 2+ channel inhibitor; 38±20% and 23±4%), PP1 (Src inhibitor; 43±23% and 15±4%), and Y27632 (Rho Kinase inhibitor; 58±23% and 22±4%) but U73122 (PLC inhibitor) had little to no effect (71±31% and 71±12%). PD098059 (Erk/MAPK inhibitor) did not inhibit chemerin-9 induced contraction (117±19%) and phosphorylation did not change after chemerin-9 stimulation [1.12±0.14 (44 kDa) and 1.11±0.29 (42 kDa) fold-increase with chemerin-9 contraction compared to vehicle, p>0.05]. The chemerin receptor-selective antagonist CCX832 inhibited chemerin-9-induced calcium flux and aortic contraction and calcium flux (0.1±10.3% and 10±7%). These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through the G i -linked chemerin receptor to activate L-type Ca 2+ channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us one step closer to targeting chemerin as a unique form of therapy.

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