Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells

The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 ± 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (∼3-fold) cell surface levels of TRPM4-GFP protein in <10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (∼3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCα and PKCβ activity but was inhibited by blockade of PKCδ with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCδ. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCδ. We conclude that PKCδ activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.

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Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

AJP Cell Physiology ◽

10.1152/ajpcell.00221.2012 ◽

2013 ◽

Vol 305 (8) ◽

pp. C829-C845 ◽

Cited By ~ 22

Author(s):

Hanaa K. B. Motawea ◽

Selvi C. Jeyaraj ◽

Ali H. Eid ◽

Srabani Mitra ◽

Nicholas T. Unger ◽

...

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Cyclic Amp ◽

Cell Surface ◽

Smooth Muscle Cells ◽

Blood Vessels ◽

Muscle Cells ◽

Small Gtpase ◽

Actin Binding ◽

Surface Translocation

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

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Orai1, a critical component of store-operated Ca2+ entry, is functionally associated with Na+/Ca2+ exchanger and plasma membrane Ca2+ pump in proliferating human arterial myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00283.2009 ◽

2009 ◽

Vol 297 (5) ◽

pp. C1103-C1112 ◽

Cited By ~ 71

Author(s):

Sergey G. Baryshnikov ◽

Maria V. Pulina ◽

Alessandra Zulian ◽

Cristina I. Linde ◽

Vera A. Golovina

Keyword(s):

Cell Proliferation ◽

Smooth Muscle ◽

Plasma Membrane ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Transient Receptor Potential ◽

Muscle Cells ◽

Membrane Microdomains ◽

Trpc Channels ◽

Critical Component

Ca2+ entry through store-operated channels (SOCs) in the plasma membrane plays an important role in regulation of vascular smooth muscle contraction, tone, and cell proliferation. The C-type transient receptor potential (TRPC) channels have been proposed as major candidates for SOCs in vascular smooth muscle. Recently, two families of transmembrane proteins, Orai [also known as Ca2+ release-activated Ca2+ channel modulator (CRACM)] and stromal interacting molecule 1 (STIM1), were shown to be essential for the activation of SOCs mainly in nonexcitable cells. Here, using small interfering RNA, we show that Orai1 plays an essential role in activating store-operated Ca2+ entry (SOCE) in primary cultured proliferating human aortic smooth muscle cells (hASMCs), whereas Orai2 and Orai3 do not contribute to SOCE. Knockdown of Orai1 protein expression significantly attenuated SOCE. Moreover, inhibition of Orai1 downregulated expression of Na+/Ca2+ exchanger type 1 (NCX1) and plasma membrane Ca2+ pump isoform 1 (PMCA1). The rate of cytosolic free Ca2+ concentration decay after Ca2+ transients in Ca2+-free medium was also greatly decreased under these conditions. This reduction of Ca2+ extrusion, presumably via NCX1 and PMCA1, may be a compensation for the reduced SOCE. Immunocytochemical observations indicate that Orai1 and NCX1 are clustered in plasma membrane microdomains. Cell proliferation was attenuated in hASMCs with disrupted Orai1 expression and reduced SOCE. Thus Orai1 appears to be a critical component of SOCE in proliferating vascular smooth muscle cells, and may therefore be a key player during vascular growth and remodeling.

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TRPML1 channels initiate Ca2+ sparks in vascular smooth muscle cells

Science Signaling ◽

10.1126/scisignal.aba1015 ◽

2020 ◽

Vol 13 (637) ◽

pp. eaba1015 ◽

Cited By ~ 3

Author(s):

Pratish Thakore ◽

Harry A. T. Pritchard ◽

Caoimhin S. Griffin ◽

Evan Yamasaki ◽

Bernard T. Drumm ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Transient Receptor Potential ◽

Ex Vivo ◽

Ryanodine Receptors ◽

Muscle Cells ◽

Confocal Imaging

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s (“Ca2+ sparks”) and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1−/−) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1−/− mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.

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TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00283.2001 ◽

2002 ◽

Vol 282 (2) ◽

pp. C347-C359 ◽

Cited By ~ 189

Author(s):

Silke Jung ◽

Rainer Strotmann ◽

Günter Schultz ◽

Tim D. Plant

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Transient Receptor Potential ◽

Growth Factor Receptor ◽

Receptor Potential ◽

Muscle Cells ◽

Northern Hybridization ◽

A7r5 Cells ◽

Current Voltage ◽

Transient Receptor

To investigate the possible role of members of the mammalian transient receptor potential (TRP) channel family (TRPC1–7) in vasoconstrictor-induced Ca2+ entry in vascular smooth muscle cells, we studied [Arg8]-vasopressin (AVP)-activated channels in A7r5 aortic smooth muscle cells. AVP induced an increase in free cytosolic Ca2+ concentration ([Ca2+]i) consisting of Ca2+ release and Ca2+ influx. Whole cell recordings revealed the activation of a nonselective cation current with a doubly rectifying current-voltage relation strikingly similar to those described for some heterologously expressed TRPC isoforms. The current was also stimulated by direct activation of G proteins as well as by activation of the phospholipase Cγ-coupled platelet-derived growth factor receptor. Currents were not activated by store depletion or increased [Ca2+]i. Application of 1-oleoyl-2-acetyl- sn-glycerol stimulated the current independently of protein kinase C, a characteristic property of the TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currents in A7r5 cells were increased by flufenamate. Northern hybridization revealed mRNA coding for TRPC1 and TRPC6. We therefore suggest that TRPC6 is a molecular component of receptor-stimulated Ca2+-permeable cation channels in A7r5 smooth muscle cells.

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Upregulation of Na+/Ca2+ exchanger and TRPC6 contributes to abnormal Ca2+ homeostasis in arterial smooth muscle cells from Milan hypertensive rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00356.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. H624-H633 ◽

Cited By ~ 44

Author(s):

Alessandra Zulian ◽

Sergey G. Baryshnikov ◽

Cristina I. Linde ◽

John M. Hamlyn ◽

Patrizia Ferrari ◽

...

Keyword(s):

Blood Pressure ◽

Smooth Muscle ◽

Plasma Membrane ◽

Smooth Muscle Cells ◽

Mesenteric Artery ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Muscle Cells ◽

Mesenteric Arteries ◽

Membrane Microdomains

The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered Ca2+ signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared Ca2+ signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 ± 2 vs. 113 ± 1 mmHg, P < 0.05). Resting cytosolic free Ca2+ concentration (measured with fura-2) and ATP-induced Ca2+ transients were augmented in freshly dissociated arterial myocytes from MHS rats. Ba2+ entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl- sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the Na+/Ca2+ exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by ∼13-fold), whereas the expression of plasma membrane Ca2+-ATPase was not altered. Ca2+ entry via the reverse mode of NCX1 evoked by the removal of extracellular Na+ induced a rapid increase in cytosolic free Ca2+ concentration that was significantly larger in MHS arterial myocytes. The expression of α1/α2 Na+ pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented Ca2+ signaling. We suggest that the increased Ca2+ signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats.

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TMEM16A channels generate Ca2+-activated Cl− currents in cerebral artery smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00404.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. H1819-H1827 ◽

Cited By ~ 62

Author(s):

Candice Thomas-Gatewood ◽

Zachary P. Neeb ◽

Simon Bulley ◽

Adebowale Adebiyi ◽

John P. Bannister ◽

...

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Smooth Muscle Cells ◽

Cerebral Artery ◽

Transmembrane Protein ◽

Cerebral Arteries ◽

Reversal Potential ◽

Muscle Cells ◽

Electrophysiological Properties ◽

Channel Expression

Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca2+-activated Cl− (ClCa) channels in native cells. The molecular identity of proteins that generate ClCa currents in smooth muscle cells (SMCs) of resistance-size arteries is unclear. Similarly, whether cerebral artery SMCs generate ClCa currents is controversial. Here, using molecular biology and patch-clamp electrophysiology, we examined TMEM16A channel expression and characterized Cl− currents in arterial SMCs of resistance-size rat cerebral arteries. RT-PCR amplified transcripts for TMEM16A but not TMEM16B–TMEM16H, TMEM16J, or TMEM16K family members in isolated pure cerebral artery SMCs. Western blot analysis using an antibody that recognized recombinant (r)TMEM16A channels detected TMEM16A protein in cerebral artery lysates. Arterial surface biotinylation and immunofluorescence indicated that TMEM16A channels are located primarily within the arterial SMC plasma membrane. Whole cell ClCa currents in arterial SMCs displayed properties similar to those generated by rTMEM16A channels, including Ca2+ dependence, current-voltage relationship linearization by an elevation in intracellular Ca2+ concentration, a Nerstian shift in reversal potential induced by reducing the extracellular Cl− concentration, and a negative reversal potential shift when substituting extracellular I− for Cl−. A pore-targeting TMEM16A antibody similarly inhibited both arterial SMC ClCa and rTMEM16A currents. TMEM16A knockdown using small interfering RNA also inhibited arterial SMC ClCa currents. In summary, these data indicate that TMEM16A channels are expressed, insert into the plasma membrane, and generate ClCa currents in cerebral artery SMCs.

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TNF-α dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation

AJP Cell Physiology ◽

10.1152/ajpcell.00499.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. C964-C971 ◽

Cited By ~ 46

Author(s):

Sergey Y. Cheranov ◽

Jonathan H. Jaggar

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Laser Scanning ◽

Channel Blocker ◽

Cerebral Arteries ◽

Muscle Cells ◽

Confocal Imaging ◽

Tnf Α ◽

Intracellular Ca ◽

Current Activation

Expression of TNF-α, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF-α regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF-α regulates the diameter of resistance-sized (∼150-μm diameter) cerebral arteries by modulating local and global intracellular Ca2+ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF-α increased Ca2+ spark and Ca2+ wave frequency but reduced global intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells of intact arteries. TNF-α elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (−40 mV) cells, TNF-α increased the frequency and amplitude of Ca2+ spark-induced, large-conductance, Ca2+-activated K+ (KCa) channel transients ∼1.7- and ∼1.4-fold, respectively. TNF-α-induced transient KCa current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF-α induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase blocker that inhibits Ca2+ sparks, and iberiotoxin, a KCa channel blocker, reduced TNF-α-induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF-α activates NAD(P)H oxidase, resulting in an increase in intracellular H2O2 that stimulates Ca2+ sparks and transient KCa currents, leading to a reduction in global [Ca2+]i, and vasodilation.

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Cigarette smoke extract increases expression and plasma membrane trafficking of α 2C ‐ adrenoceptors in human microvascular smooth muscle cells

The FASEB Journal ◽

10.1096/fasebj.2020.34.s1.05225 ◽

2020 ◽

Vol 34 (S1) ◽

pp. 1-1

Author(s):

Dina Salem Maaliki ◽

Ali H. Eid

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Smooth Muscle Cells ◽

Cigarette Smoke ◽

Membrane Trafficking ◽

Muscle Cells ◽

Cigarette Smoke Extract

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Do TRPC-like currents and G protein-coupled receptors interact to facilitate myogenic tone development?

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00460.2011 ◽

2011 ◽

Vol 301 (4) ◽

pp. H1378-H1388 ◽

Cited By ~ 25

Author(s):

Yana Anfinogenova ◽

Suzanne E. Brett ◽

Michael P. Walsh ◽

Osama F. Harraz ◽

Donald G. Welsh

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Transient Receptor Potential ◽

Cerebral Arteries ◽

Receptor Potential ◽

Muscle Cells ◽

Receptor Activation ◽

Ruthenium Red ◽

Pcr Analysis ◽

Whole Cell

The objective of this study was to determine whether Gq/11-coupled receptor activation can enhance the mechanosensitivity of a canonical transient receptor potential (TRPC)-like current and consequently the myogenic responsiveness of rat anterior cerebral arteries. Initial patch-clamp experiments revealed the presence of a basal cation current in isolated smooth muscle cells that displayed evidence of double rectification, which was blocked by trivalent cations (Gd3+ and La3+). PCR analysis identified the expression of TRPC1, 3, 6 and 7 mRNA and, characteristic of TRPC-like current, the whole-cell conductance was insensitive to a Na+-dependent transport (amiloride), TRP vanilloid (ruthenium red), and chloride channel (DIDS, niflumic acid, and flufenamate) inhibitors. One notable exception was tamoxifen, which elicited a dual effect, blocking or activating the TRPC-like current at 1 and 10 μM, respectively. This TRPC-like current was augmented by constrictor agonists (uridine 5′-triphosphate and U46619) or hyposmotic challenge (303 to 223 mOsm/l), a mechanical stimulus. Although each stimulus was effective alone, smooth muscle cells pretreated with agonist did not augment the whole-cell response to hyposmotic challenge. Consistent with these electrophysiological recordings, functional experiments revealed that neither UTP nor U46619 enhanced the sensitivity of intact cerebral arteries to hyposmotic challenge or elevated intravascular pressure. In summary, this study found no evidence that Gq/11-coupled receptor activation augments the mechanosensitivity of a TRPC-like current and consequently the myogenic responsiveness of anterior cerebral arteries.

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