Journal of the American Society of Hypertension 3(2) (2009) 84–95

Review Article

Vascular smooth muscle cell signaling mechanisms for contractionto angiotensin II and endothelin-1

Brandi M. Wynne, MS*, Chin-Wei Chiao, PhD, and R. Clinton Webb, PhDDepartment of Physiology, Medical College of Georgia, Augusta, Georgia, USA

Manuscript received June 28, 2008 and accepted September 25, 2008

Abstract

Vasoactive peptides, such as endothelin-1 and angiotensin II, are recognized by specific receptor proteins located in the cellmembrane of target cells. After receptor recognition, the specificity of the cellular response is achieved by G-protein couplingof ligand binding to the regulation of intracellular effectors. These intracellular effectors will be the subject of this brief re-view on contractile activity initiated by endothelin-1 and angiotensin II. Activation of receptors by endothelin-1 and angio-tensin II in smooth muscle cells results in phospholipase C activation leading to the generation of the second messengersinositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates intracellular Ca2þ release from the sarcoplasmic retic-ulum and DAG causes protein kinase C activation. Additionally, different Ca2þ entry channels, such as voltage-operated, re-ceptor-operated, and store-operated Ca2þ channels, as well as Ca2þ-permeable nonselective cation channels, are involved inthe elevation of intracellular Ca2þ concentration. The elevation in intracellular Ca2þ is transient and initiates contractile ac-tivity by a Ca2þ-calmodulin interaction, stimulating myosin light chain (MLC) phosphorylation. When the Ca2þ concentra-tion begins to decline, Ca2þ sensitization of the contractile proteins is signaled by the RhoA/Rho-kinase pathway to inhibitthe dephosphorylation of MLC phosphatase (MLCP), thereby maintaining force generation. Removal of Ca2þ from the cy-tosol and stimulation of MLCP initiates the process of smooth muscle relaxation. In pathologic conditions such as hyperten-sion, alterations in these cellular signaling components can lead to an overstimulated state causing maintainedvasoconstriction and blood pressure elevation. J Am Soc Hypertens 2009;3(2):84–95. � 2009 American Society ofHypertension. All rights reserved.Keywords: Calcium channel; Rho-kinase; vasoactive peptides; phosphoinositides.

Introduction

In the vasculature, the small arteries and arteriolesregulate the majority of blood flow resistance in the circu-lation. Circulating neurotransmitters, hormones, endothe-lium-derived factors, and even shear stress itself playsa role in modulating smooth muscle tone, and consequently,lumen diameter.1–3 The final product is the control of bloodpressure (BP) and organ blood flow.

This study was supported by the National Institutes of Health(Grant Nos. HL71138 and HL74167).

Conflict of interest: none.*Corresponding author: Brandi M. Wynne, MS, Department of

Physiology, Medical College of Georgia, Augusta, Georgia 30912.Tel: 706-721-3547; fax: 706-721-7299.

E-mail: bwynne@students.mcg.edu

1933-1711/09/$ – see front matter � 2009 American Society of Hyperdoi:10.1016/j.jash.2008.09.002

Arteries are composed of three layers: 1) the tunica ad-ventitia, 2) tunica media, and 3) tunica intima. Althoughthe outer and inner layers are composed mainly of connec-tive tissue and endothelial cells, respectively, the tunica me-dia is composed of smooth muscle cells.3 All smoothmuscle cells, regardless of the stimulus, produce force orcontraction through cross-bridge cycling between actinand myosin filaments. Although vascular smooth musclecells are capable of dynamic changes in gene expressionto either contractile or differentiation proteins, the contrac-tile phenotype in vascular smooth muscle predominates.4

The contractile response of vascular smooth muscle is theproduct of myosin light chain kinase (MLCK) and myosinlight chain phosphatase (MLCP) activation. In smooth mus-cle, this process is initiated by a calcium (Ca2þ)-mediatedchange in the thick (myosin) filaments2 (Figure 1).

In this review, we will discuss the molecular mechanismsby which two common vasoactive peptides: angiotensin IIand endothelin-1 produce smooth muscle contraction.

tension. All rights reserved.

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Because intracellular Ca2þ is fundamental to the contractileprocess, we will also provide a brief review of the cellularmechanisms that regulate this cation.

Angiotensin II Signaling

Angiotensin II, which is the predominate bioactive pep-tide in the renin-angiotensin system (RAS), promotes themaintenance of systemic pressure through various mecha-nisms in the cardiovascular and renal systems.5 Althoughangiotensin II is crucial to salt and water homeostasis,this peptide is also implicated in several cardiovascularconditions, such as hypertension, atherosclerosis and heartfailure, and more specifically in vascular smooth musclecontraction; vascular remodeling including the inductionof hypertrophy and hyperplasia. Canonical angiotensin IIsignaling occurs through a membrane bound heterotrimericG-protein coupled receptor (GPCR). To date, there are twoGPCRs known to mediate angiotensin II function: the AT1

and AT2 receptors.5,6

The AT1 receptor is expressed in a variety of tissues, in-cluding the kidney, heart, adrenal gland, brain, lung, andadipose; however, the focus will be on AT1 receptor activa-tion in arterial smooth muscle. The AT2 receptor is highlyexpressed in fetal tissues, then declines quickly after birth.In adults, the AT2 receptor has a varied tissue distribution aswell, and is detectable in the kidney and adrenals, pancreas,ovary, brain, heart, and vasculature. In vessels, the AT2 re-ceptor is not localized on the smooth muscle, but is mainlyexpressed in the adventitia. After injury, levels of this re-ceptor have been observed to increase, which may explaincurrent dogma suggesting that the AT2 receptor is involvedin cellular growth. In contrast to the vasoconstrictive effectsof the AT1, the AT2 receptor has been shown to modulatevasodilation.7 Less is known about AT2 receptor mecha-nisms than the AT1 receptor; however, it is known that itcouples to Gi and activates tyrosine and serine/threonine

Figure 1. Overview of major contributors to vascular smooth muscle cmediated change in the thick filaments, or myosin. With myosin light cof interacting. ATP hydrolysis is the source for force generation in smyosin head. Relaxation occurs via the action of myosin light chain phing it. ATP, adenosine-5‘-triphosphate; Ca2þ, calcium.

phosphatases. This receptor isoform has also been docu-mented to trigger the kinin-NO-cGMP pathway.8 However,it is through the AT1 receptor with which the majority ofphysiologic and pathologic actions of angiotensin II is me-diated, and will be the focus of the signaling in this review.

The angiotensin II signaling cascade via the AT1 receptoris a consequence of the particular G-protein or signalingcascade that is activated: Gq/11, Gi, G12, and G13.5,6 TheAT1 receptor signals through three main pathways: classicalphospholipase C (PLC) signaling leading to inositol tri-sphosphate (IP3) and diacylglycerol (DAG) cleavage, phos-pholipase D, also culminating in DAG generation; andphosphatidylcholine, phosphatidic acid and phospholipaseA2, which is responsible for arachidonic acid and prosta-glandin production. Tyrosine phosphorylation, ie, mitogenactivated protein kinase (MAPK), which is distinctive forits role in growth factor and cytokine activation, has beenassociated with increased levels of AT1 receptor activation.Nonreceptor tyrosine kinase activity has also been associ-ated with angiotensin II, including but not limited to: theSrc family of kinases, proline-rich tyrosine kinases(Pyks), extracellular signal-regulated kinases (ERKs),paxillin, focal adhesion kinase (FAK) phosphoinositide 3kinase (PI3K) and an inflammatory cytokine pathway, januskinases/signal transducers and activators of transcription(JAK/STAT).7 Angiotensin II is a pleiotropic signaling mol-ecule, whose varied pathways is complex and specific, butyet also converges to bring forth multiple responses. Thisreview will focus on the PLC pathway, culminating in vas-cular smooth muscle contraction.

Recent literature suggests that the AT1 receptor couplesto Gq to mediate vasoconstriction, which is the pathwayleading to smooth muscle contraction via PLC.9 In addition,angiotensin II was found to elicit a contractile response viaactivation of G12/G13 G proteins and the regulator of G-pro-tein signaling-Rho-guanine nucleotide exchange factor(RGS-RhoGEF) signaling pathway. Recently, insights into

ontraction. In smooth muscle, contraction is initiated by a Ca2þ-hain phosphorylation, the actin and myosin filaments are capablemooth muscle; with contraction, inorganic phosphate leaves theosphatase, which dephosphorylates myosin light chain, inactivat-

Figure 2. Molecular mechanisms of smooth muscle contraction. Vascular smooth muscle contraction is the summation of MLCK andMLCP activity. With receptor binding, an increase in intracellular Ca2þ occurs, both via channels located in the membrane and intracel-lular stores in the sarcoplasmic reticulum. The Ca2þ interacts with calmodulin, forming a Ca2þ-calmodulin complex, which activatesMLCK. MLCK can then p-MLC, allowing for the close interaction of the actin and myosin filaments for force generation. Relaxationoccurs with MLCP dephosphorylating MLC. In some instances, force generation can be tonic; this is mediated in a Ca2þ-independentmanner. Rho-kinase becomes activated via the small, activated RhoA protein, which subsequently phosphorylates MLCP, renderingthe enzyme inactive and incapable of de-phosphorylating MLC. In addition, PKC and Rho kinase work in concert to activate CPI-17,which inhibits MLCP. Thus, the vascular smooth muscle cannot relax and a tonix contraction occurs. Ca2þ, calcium; MLCK, myosin lightchain kinase; MLCP, myosin light chain phosphatase; p-MLC, phosphorylate myosin light chain.

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this mechanism were discovered and will be discussed ina later section.10,11 After Gq/G11 GPCR activation, secondmessengers are generated, beginning with PLC activation.PLC is a membrane-bound enzyme responsible for cleavingmembrane lipid phosphoinositide 4, 5- bisphosphate, result-ing in the generation of IP3 and DAG.2,12 IP3 and DAG reg-ulate two distinct but parallel pathways leading to increasedintracellular Ca2þ and culminating in MLCphosphorylation.2,13,14

IP3 regulates calcium release into the cytosol from thesarcoplasmic reticulum (SR), mediated via the ryanodinereceptor (RyR). The RyR, which has been shown to be lo-calized centrally as well as peripherally on the SR, isthought to facilitate optimal binding of IP3.15,16 Activationof the RyR opens Ca2þ channels, causing a rapid increasein Ca2þ concentration. After cytosolic Ca2þ concentrationspeak and then decline, there is a sustained elevation ofCa2þ above that of basal levels.2 This sustained increasein Ca2þ is due to receptor-operated Ca2þ channels on

plasma membrane facilitating increased cytosolic Ca2þ

levels from the extracellular space.2,14 The EF handCa2þ-binding protein, calmodulin, is the primary targetfor elevated intracellular Ca2þ. The Ca2þ-calmodulin com-plex is then able to activate MLCK.2 It is also of importanceto note that the Ca2þ-tension relationship changes for stim-ulation type and during the time of the contraction. In gen-eral, studies show that the receptor-mediated stimulationsproduce a greater tension for a given Ca2þ concentration.Moreover, during the sustained phase of contraction, lowerlevels of Ca2þ are needed, which is referred to as Ca2þ sen-sitization. These are two differential points of regulation forvascular smooth muscle contraction17 (Figure 2).

DAG mediates vascular smooth muscle contraction viaactivation of protein kinase C (PKC). PKC is a serine/thre-onine kinase known to exist in several isoforms, all whichcontribute to various physiological activities and are acti-vated differentially. Of the three known vascular smoothmuscle isoforms, PKCa and PKCb are dependent on

Figure 3. The concept of ROC channel, VOC channel, and SOC channel. ROC channels are activated via receptor stimulation of GPCRsdirectly or through the production of second messengers such as IP3 or DAG. IP3 can then bind to and activate the IP3 receptor on thesarcoplasmic reticulum membrane, causing discharge and release of the stored Ca2þ. The release of Ca2þ from the sarcoplasmic reticuluminduces Cl� efflux or the influx of Naþ and Caþ from the ROC channels, which can then activate VOC channels. The second messenger, IP3,activates the IP3 receptor on the sarcoplasmic reticulum membrane, causing discharge and release of the stored Ca2þ. After Ca2þ depletionfrom the sarcoplasmic reticulum, the SOC channels are activated. A, agonist; Ca2þ, calcium; DAG, diacylglycerol; GPCRs, G-protein–coupled receptors; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipaseC; R, receptor; ROC, receptor-operated channels; TK, tyrosine kinase. SOC, store-operated channels; VOC, voltage-operated channels.

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DAG activation and intracellular Ca2þ concentrations,whereas PKCe is DAG-dependent only. PKC modulatesvascular smooth muscle contraction by directly phosphory-lating MLCK, which subsequently phosphorylates MLC.Furthermore, PKC activation leads to the activation ofseveral other target proteins promoting smooth musclecontraction. PKC phosphorylates and activates ERK1/2,Rho-kinase (p160ROCK), and calmodulin-dependentprotein kinase II. In addition, intracellular Ca2þ elevationwas suggested to regulate angiotensin II-induced epidermalgrowth factor receptor and ERK activation.11 Various ionchannels and ion transporters have been demonstrated asdownstream targets of PKC. This phenomenon has beenillustrated with the use of the specific PKC agonists (ie,phorbol esters) which induce smooth muscle contraction.2

All the earlier events described culminating in MLCKactivation, resulting in contraction via the phosphorylationof Ser19 of the 20 kDa regulatory protein of MLC. Thephosphorylation of MLC allows for the interaction betweenactin and myosin filaments. Intrinsic adenosine-5‘-triphos-phatase (ATPase) activity of myosin is crucial for this pro-cess, because the hydrolysis and subsequent release of ATPresults in the cycling of the myosin cross-bridges withactin, causing force generation.3,18–21

Smooth muscle cells also contain Ca2þ-independentmechanisms to regulate contractility. The process of force

generation is mediated by MLCK activation, and subse-quent actin-myosin cross-bridging, but the process of forcemaintenance is mediated in a Ca2þ-independent man-ner.2,18,22 MLCP is the contender, regulating the inactiva-tion of MLC by removing the high-energy phosphategroup, promoting smooth muscle relaxation. MLCP is com-posed of two subunits; one is the catalytic 38 kDa subunitof type 1 protein phosphatase (PP1c, d isoform) and twoother noncatalytic subunits.12,17 This mechanism generatesforce maintenance by means of Ca2þ-sensitization of thecontractile proteins. This is signaled by the RhoA/Rho ki-nase pathway, which inhibits the dephosphorylation ofMLC by MLCP. RhoA is a small, monomeric G protein,which is regulated by the binding of GTP. This process isfacilitated by nucleotide exchange factors, RhoGEFs,which enable the exchange of guanosine diphosphate(GDP) for guanosine triphosphate (GTP), activatingRhoA. Upon RhoA activation, the RhoA-GTP complex isable to activate Rho kinase, a serine/threonine kinase.Rho kinase can then phosphorylate the myosin-bindingsubunit of MLCP, inhibiting it, and thus preventing the de-phosphorylation of MLC, causing maintenance of contrac-tion.2,23,24 In addition to the regulatory effects of Rhokinase, CPI-17 is another protein that regulates myosinphosphatase (MYPT) phosphorylation status by inhibitingPP1c, thus inactivating MLCP. CPI-17 phosphorylation

Figure 4. New pathways in vascular smooth muscle signaling. Orai1 and STIM1 work together to function as Ca2þ sensors within thecell. Orai1 makes up the pore of the CRAC channel, allowing for Ca2þ entry, whereas STIM1 senses Ca2þ levels within the SR. Theirinteraction facilitates this process. The IKCa channels are intermediate conductance Ca2þ-activated Kþ channels which are of impor-tance in small resistance vessels. G12/13 signaling through LARG was recently found to be a mechanism for RhoA/Rho kinase activation,leading to MLCP inactivation. Ca2þ, calcium; CRAC, Ca2þ-release activated Ca2þ; IKCa, intermediate conductance Ca2þ-activated Kþ

channel; p-MLC, myosin light chain phosphatase; STIM, stromal-interacting molecule.

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occurs in a Ca2þ-dependent and Ca2þ-independent mecha-nism. Ca2þ-dependent PKC was suggested to regulate therapid phosphorylation, whereas phosphorylation by PKCand Rho kinase occurs independently of Ca2þ in the laterphase. This has been demonstrated with the use of Rho ki-nase and PKC inhibitors; however, the effects were depen-dent on the type of agonist and tissue used. Its activity andexpression is a factor in the contractile state of vascularsmooth muscle and has also been shown to be of impor-tance in Ca2þ sensitization.12,17 Much needs to be exploredregarding MLCP regulation, but regardless, Rho kinase andCPI-17 are acknowledged to be of importance in vascularsmooth muscle contraction and force maintenance(Figure 2).

Endothelin-1 Signaling

Endothelin-1, which is a 21-amino acid peptide, isknown as one of the most potent endogenous vasoconstric-tors as described by Yanagisawa et al in 1988.25–27 Since itsdiscovery, the endothelin-1 system has become increasinglycomplex; several endothelin-1 isoforms and receptors havebeen identified in a variety of tissues, including neuronal,renal, and vascular tissues. Interestingly, although somedata show a correlation between endothelin-1 levels and hy-pertension, these data are not conclusive.4 Overall, endothe-lin-1 signaling in the vasculature is essentially the same asfor angiotensin II signaling. A brief overview will be dis-cussed, but the reader is directed to angiotensin II-GPCR

coupled signaling pathways for a more in depth explana-tion. Differing pathways regarding to endothelin-1 signal-ing itself will be discussed further.

Endothelin-1 is the main isoform secreted by the endo-thelium, and has been shown to act in a paracrine or auto-crine manner on its receptors in vascular smooth muscle.There are three known endothelin-1 receptors that havean assorted tissue distribution and functional role: ETA

and ETB are present in mammals and a putative ETC innon-mammals. The typical receptor found on vascularsmooth muscle cells is the ETA receptor, which mediatesthe vasoconstrictor effects of endothelin-1.4,25,28 Endothe-lin-1 receptor activation can lead to diverse responses inthe cell through interaction with pathways that are both per-tussis toxin-sensitive and toxin-insensitive, leading to theconclusion that endothelin-1 acts through several GPCRs.Generally, ETA receptors have been associated with vaso-constriction and cell growth, whereas ETB receptors are in-volved in the clearance of endothelin-1, inhibition ofendothelial cell apoptosis, the release of NO and prosta-glandins leading to vasorelaxation, and inhibition of the ex-pression of endothelin-1 converting enzyme; however, bothreceptors have been found to elicit vasoconstriction.29–31

ETA receptors have been shown to be functionallycoupled to the Gq/11 protein leading to PLC-b activation;Gs linked leading to increased cyclic adenosine monophos-phate (cAMP) and also to Gi, thus inhibiting adenylate cy-clase.30,32–34 Activation of Gq/11, ending in IP3 and DAGcleavage, can stimulate Ca2þ release intra- and

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extracellularly, as described for angiotensin II signaling. Inaddition to second messenger generation, endothelin-1 hasalso been demonstrated to activate Ca2þ channels on theplasma membrane and stimulate Ca2þ flux from the extra-cellular space.4,35 There is speculation that ETA receptoractivation can activate the Naþ/Hþ exchanger and activatethe enzymes phospholipase D generating DAG and phos-pholipase A2 releasing arachidonic acid.4 ETB receptorsin rat carotid were shown to mediate vasorelaxation viaa cyclic guanosine monophosphate-nitric oxide (cGMP-NO) pathway, the production of vasodilator cyclooxyge-nase products and the activation of voltage-activated Kþ

channels. Recently, endothelin-1 was shown to inhibit nic-otinamide adenine dinucleotide phosphate (NADPH) oxi-dase activity and superoxide generation via ETB1

receptors.36 The details of these mechanisms are still poorlyunderstood.31

Both agonists, angiotensin II and endothelin-1, mediatevasoconstriction via an increase in cytosolic Ca2þ althoughmechanisms of this process are different between the twoagonists. Endothelin-1 increases intracellular Ca2þ by stim-ulating influx through Ca2þ channels and is known to pro-duce a much more sustained contraction by the vascularsmooth muscle cells, whereas angiotensin II elicits a potentbiphasic response that is generated primarily by mobiliza-tion of Ca2þ from an intracellular store. Consequently,the actions of angiotensin II actions are relatively rapid incomparison.37–41

Given that both of these agonists mediate downstream sig-naling pathways via the same second messengers, it is easy toappreciate how cross-talk occurs between them. Both endo-thelin-1 and angiotensin II signal through distinct Gq-coupledGPCRs leading to phosphoinositide production and MAPKactivation. However, the mechanism by which this occursvaries at some points. It has been demonstrated that endothe-lin-1 and angiotensin II both stimulate this pathway via Ras-Raf; yet, angiotensin II produces phosphorylation of ERK1/2, SAPK/JNK, and p38MAPK via c-Src–dependent path-ways. In contrast, endothelin-1 was shown to induceMAPK phosphorylation through c-Src–independentpathways42 (Figure 3).

Calcium Mobilization

Given that Ca2þ is the trigger for smooth muscle contrac-tion, it is necessary to understand the mechanisms by whichintracellular Ca2þ levels increase. Hence, this is the focusof much research, and the subject of the remainder of thisreview. In vivo, arterial smooth muscle cells exist with anaverage intracellular Ca2þ concentration that is several or-ders of magnitude lower than that in the extracellular fluid.Intracellular Ca2þ concentration levels do not exist homo-genously throughout the cell, but exhibit dynamic changesin the cell, temporally and spatially, because of Ca2þ flux.The changes seen in Ca2þ flux are a component of several

different events, which are all dependent on intracellular ul-tra-structure as well as the spatial relationship of ion pumpsand channels in the plasma membrane.4

Ca2þ entry through channels in the plasma membraneand release from the SR are the major sources of intracel-lular Ca2þ.1,4,15 Of the different Ca2þ entry channels, thevoltage-operated Ca2þ (VOC), receptor-operated Ca2þ

(ROC), store-operated Ca2þ (SOC) channels, and a Ca2þ-permeable nonselective cation channels (NSCC) modulatemost Ca2þ mobilization within the cell. We will discussthese particular vascular smooth muscle Ca2þ entry chan-nels further in the following section (Figure 3).

Voltage-Operated Ca2D Channels

VOC channels represent a major route by which Ca2þ

enters vascular smooth cells.43 VOC channel function isregulated by membrane potential such that hyperpolariza-tion closes them and depolarization opens them; the latterleading to vasoconstriction.1

The majority of agonist-induced Ca2þ influx probablyoccurs through L-type VOC channels. Dihydropyridine-sensitive, L-type VOC channels are regulated by vasocon-strictors that are known to activate the PKC pathway. Vaso-dilators have also been shown to inactivate these channels,but through cAMP production.1,44 These gated channelsalso depolarize in response to membrane stretch, lendingsupport to the hypothesis that these channels are importantfor the myogenic response and vascular tone. Inhibition ofVOC channels can occur primarily because of increasingintracellular Ca2þ concentration and activation of cGMP-dependent protein kinase.1,2,44

Endothelin-1 has been shown to activate the VOC chan-nel in smooth muscle cells from porcine coronary arteries,and has also been shown to augment Ca2þ channel currentsin the smooth muscle cell membrane of guinea pig portalvein. In a model of cultured thoracic aorta vascular smoothmuscle cells, Kawanabe et al demonstrated that endothelin-1 produces a sustained increase in intracellular Ca2þ levelsfrom VOC channels, as well as others.41,45

Although endothelin-1 increases intracellular Ca2þ bystimulating influx through Ca2þ channels, the role ofVOC channels is limited, and current data suggest thatother channels are more important; namely the NSCC, asevidenced by experiments using nifedipine.41,46 Eventhough described more than 60 years ago, the exact mech-anisms of action of this peptide are not completelyunderstood.

Angiotensin II-GPCR stimulation leading to PLC is anaccepted mechanism acknowledged to enhance the L-typeCa2þ channel. Interestingly, intracellular angiotensin IIhas been demonstrated to stimulate VOC channels simi-larly, but yet independently of extracellular ligand bindingof angiotensin II to the AT1 receptor, possibly through theactions of PLC, PKC, and/or tyrosine kinase.47,48

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As shown in the smooth muscle cells of the anococcy-geus, there are two very distinct mechanisms for inwardCa2þ currents and Ca2þ store depletion.49 The increase inCa2þ concentration after the activation of VOC channelscan also lead to a phenomenon known as Ca2þ inducedCa2þ release, where the increase in Ca2þ concentrationleads to Ca2þ release from intracellular stores. The RyRon the SR mediates this process, as they are activated byCa2þ.50 This produces a membrane depolarization that pro-motes further VOC channel opening and Ca2þ entry.

Receptor-Operated Ca2D Channels

Only 20 years ago, the exact mechanism of Ca2þ channelactivation by extracellular ligands, such as hormones andneurotransmitters, was largely unknown. In addition, therewas still uncertainty concerning the mechanism by whichCa2þ entry was mediated until a model, proposed by Put-ney et al, detailing the mechanism by which activation ofsurface membrane receptors causes a sustained Ca2þ entryinto cells from the extracellular space.51 Furthermore, Ben-ham and Tsien suggested that agonists might activate Ca2þ

influx directly, by receptor interaction with Ca2þ-perme-able NSCC.52 Currently, ROC channels are defined by thefollowing: channels where molecules are separate fromthe ligand-binding protein are capable of activating a rangeof GPCRs via circulating agonists, and are neither VOC norSOC channels.28 After ligand binding, ROC channels aregenerally thought to be activated by GPCRs, which are cou-pled to PLC, leading to IP3 and DAG generation.53,54

Several members of the transient receptor potential chan-nel (TRPC) family are accepted to form ROC channels,including TRPC3, TRPC6, and TRPC7. Angiotensin IIhas been proposed to activate TRPC6 in rabbit mesentericarteries, whereas endothelin-1 activates TRPC3 andTRPC7.55–57

Store-Operated Ca2D Channels

SOC channels represent yet another mechanism of excita-tion-contraction coupling in smooth muscle. SOC channelsare activated by means of intracellular Ca2þ level depletion,and, as a result, are inhibited when Ca2þ stores are filled.This process was called capacitative Ca2þ entry, beforestore-operated Ca2þ entry (SOCE) was coined. It shouldbe noted that SOCE does not denote any one mechanismof Ca2þ entry, nor does it refer to any particular chan-nel.1,58–62 These channels are highly selective for Ca2þ,and have been found to be bradykinin and ATP sensitive.60

RyR- or IP3-sensitive stores can induce Ca2þ release se-quentially, possibly amplifying the rise in intracellularCa2þ and further stimulating Ca2þ channel-dependent Cl�

channels. SOC channels were described in 1986, when Put-ney et al demonstrated that the same Ca2þ entry mechanismnormally activated as a result of Ca2þ-mobilizing agonists

can be triggered equally as well by depleting the intracellu-lar Ca2þ pool, even in the absence of receptor activation orelevated cellular levels of inositol polyphosphates.63–65 SOCchannel entry has been proposed to increase cytoplasmicCa2þ levels not only for contraction, but also cell prolifera-tion and apoptosis.4 SERCA pumps, which are found on themembrane of the sarcoplasmic reticulum, function by pump-ing the Ca2þ ions back into the SR after a contractile eventreplenishing Ca2þ concentrations.14,15 SERCA pumps canbe inhibited by cyclopiazonic acid or thapsigargin, whichdeplete intracellular Ca2þ stores and thereby activate SOCchannels.66,67 Ca2þ entering through the SOC channelscan then be pumped into the stores, replenishing them. Ac-cording to functional and pharmacologic experiments, SOCchannel entry elicited by specific inhibitors of SERCApumps induced a sustained elevation of intracellular Ca2þ,which is also dependent on extracellular Ca2þ entry and isattributed to SOCE.68 Thus, Ca2þ signals generated in re-sponse to receptors involve two coupled components: a tran-sient release of Ca2þ stored in the SR, followed by a slowlydeveloping extracellular Ca2þ entry.14,15,69,70 With depletedCa2þ stores, Ca2þ influx factor is generated and diffuses tothe plasma membrane where, through a series of reactions,Ca2þ influx factor activates Ca2þ-independent phospholi-pase A2. This generation of lysophospholipids, in turn, acti-vates the SOC channels.62

The family of TRPC is well known for their role inSOCE, although the mechanism allowing for TRPC activa-tion from store depletion is largely unknown. There isevidence for and against TRPCs actually being a SOCchannel, including TRPC1.56,58,62 Also, interesting datafrom recent high throughput RNAi screens of thapsigar-gin-activated Ca2þ entry revealed a stromal-interactingmolecule (STIM) in Drosophila S2 cells and in mammalianHeLa cells, which are now thought to play an essential rolein SOCE and conductance through Ca2þ-release activatedCa2þ (CRAC) channels.71–73 This single membrane span-ning protein is now thought to activate the SOC channelsby actually sensing Ca2þ within the stores. Pharmacologicexperiments showed that the actual contribution of SOCEto excitation/contraction coupling seems to depend on thesmooth muscle type, and may be more important in tonicsmooth muscle.62 A canonical TRP channel, TRPC7, whichis activated by AT1-coupled GPCR activation andDAG, was recently hypothesized to mediate angiotensinII–induced myocardial apoptosis. Moreover, angiotensinII has been postulated to activate TRPC1, as evidencedby data showing that angiotensin II stimulation increasedSOCE together with TRPC1 expression. These data wereobtained using a cell culture model, but given the heteroge-neity of TRPC channels in vascular smooth muscle, there isno reason to believe that this occurrence does not exist invivo.74 In a recent study in vascular smooth muscle cells,endothelin-1 activation of TRPC1 was found to not onlybe involved in a SOCE, but also in a ROC channel entry,

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which requires IP3 receptor activation. A possible explana-tion for this exciting phenomenon is the report that TRPCsmay exist in heterotrimeric complexes, possibly accountingfor the varying pharmacologic properties seen.56

Nonselective Cation Channels

The NSCC, so called because it is ‘‘nonselective,’’ beingequally permeable to monovalent cations, such as Naþ andKþ in the extra- and intracellular compartments.25,28 Thischannel has been revealed to be activated by stimulationof native ETA receptors in vascular smooth muscle, andthe majority of endothelin-1 response being mediated byNSCCs; namely, NSCC-1 and NSCC-2.28,41,46,75 It waspreviously shown, pharmacologically and with electrophys-iology, that the response to endothelin-1 is dose-dependent.More specifically, with lower concentrations of endothelin-1, Ca2þ entry via IP3 formation from intracellular stores oc-curs; however, with higher endothelin-1 concentrationsboth the former in addition to extracellular Ca2þ entry oc-curs as well.25,28,38,76,77

The molecular mechanisms of NSCC activation are stillnot entirely clear. Recent evidence in rabbit internal carotidartery shows that endothelin-1 may induce an intracellularresponse after GPCR activation via Pyk2 and the ETA

receptor. Data from the same laboratory indicate thatPI3K also regulates the activation of Ca2þ entry after endo-thelin-1 GPCR activation and stimulation of NSCC-2.46,78

Hot Topics in Vascular Smooth Muscle Signaling

Within just the past year, exciting new insights into vas-cular smooth muscle signaling have been discovered. Wewill discuss several recent advancements in the field thathave been especially stimulating (Figure 4).

As described previously, GPCR signaling can elicit stim-ulatory or inhibitory signals in the cell. With regards to vas-cular smooth muscle, the Gq/G11 G proteins have beenaccepted as producing contraction via the Ca2þ-dependentPLC pathway; however, Ca2þ-independent mechanisms,not only for force maintenance, but for contraction haveemerged. By means of a novel murine knockout modelfor Gq/G11 and G12/G13 G proteins in smooth muscle cells,Wirth et al elaborated upon the knowledge of how angio-tensin II and endothelin-1 mediate contraction via G pro-teins. Using aortic segments, they demonstrated that inG12/G13 knockout mice, angiotensin II and endothelin-1were able to elicit a contraction; however, both potencyand efficacy were affected. In the Gq/G11 knockout mice,although endothelin-1 still produced a severely reducedcontraction, angiotensin II was not capable of elicitingany contraction. It was also demonstrated that G12/G13 cou-pled G proteins activate the RhoA/Rho kinase signalingpathway via interaction of RhoGEFs; in particular,

leukemia-associated RhoGEF (LARG). Using a LARG mu-rine knockout model, they showed that LARG is necessaryfor full contraction with angiotensin II and endothelin-1, asdemonstrated by the severely restricted constriction elicitedin aortic segments from LARG knockout mice.10,79

As described previously, Pyk2 is a Ca2þ-sensitive nonre-ceptor protein tyrosine kinase that is known to associatewith focal adhesion sites and is a downstream effector ofangiotensin II and endothelin-1 GPCR stimulation.7,15,80,81

Pyk2 phosphorylation and activation has been postulated inseveral different pathways, including ERK1/2 signaling incardiomyocytes, p38MAPK in mesangial cells and recentlyin RhoA/Rho kinase activation via PDZ-RhoGEF vascularsmooth muscle cell migration.15,80,82,83 It has even beensuggested that tyrosine kinase pathways such as thesemay even be important in angiotensin II signaling leadingto vasoconstriction.84 These alternative signaling pathwaysin angiotensin II signaling should be a new directive for in-vestigation into increased vascular smooth muscle reactiv-ity in conditions such as hypertension. This was a focusof a recent article from Giachini et al.80 It is known that in-creased vascular reactivity to contractile stimuli is presentin vessels from deoxycorticosterone acetate (DOCA)-salthypertensive mice. Using this model with a pharmacologi-cal approach, mechanistic studies were performed to deter-mine the role that Pyk2 plays in the hyperreactivityexhibited in DOCA-salt hypertension. In mesenteric ar-teries and aorta, Pyk2 inhibition attenuated the increasedvascular constriction to phenylephrine and Western blotdata showed increased Pyk2 and phospho-Pyk2 in vesselsfrom DOCA-salt treated mice vs. sham, thus confirminga role for Pyk2 in hypertension.80

A recent discovery of a transcription factor named therepressor element 1-silencing transcriptional (REST)factor, was shown to have a consensus sequence, KCNN4,which is known to encode for the intermediate conductanceCa2þ-activated Kþ channels (IKCa). These channels are ofthe utmost importance in small resistance vessels wherea primary vasodilating agent is endothelium-derived hyper-polarizing factor. Endothelium-derived hyperpolarizing fac-tor not only remains elusive as a factor, but the mechanism bywhich it produces vasodilation is only speculation. One suchhypothesis is that Kþ efflux from endothelial cells via theseIKCa channels in coordination with small-conductanceCa2þ-activated Kþ channels, activates inward rectifier Kþ

channels. This would lead to vascular smooth muscle relax-ation. Unpublished results from the laboratory of Webb andTostes show for the first time that REST expression is closelyassociated with IKCa expression in arteries from hypertensiveanimals. Giachini et al demonstrated that REST expressionwas downregulated, whereas IKCa channels were overex-pressed in arteries obtained from spontaneously hypertensivestroke-prone rats, when compared to their Wistar-Kyotocontrols. Protein levels of small-conductance Ca2þ-activated

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Kþ channels and IKCa were also assessed, and alterationswere observed in mesenteric arteries from hypertensiveanimals. It seems as though REST may be a negative modu-latory mechanism, which controls the levels of IKCa in thespontaneously hypertensive stroke-prone rat vasculature.

As mentioned previously, the STIM molecule was shownto play an essential role for SOCE and conductance throughCRAC channels.71 The recently discovered subunit of theCRAC channel pore, termed Orai1, is essential for CRACchannel activation.62,72,85,86 However, the exact mechanismfor STIM/Orai1 signaling is not yet known. STIM can un-dergo phosphorylation at specific serine/threonine sites, aswell as undergo N-linked glycosylation.87 Sobolff et al de-termined that although STIM1 is expressed at the cell sur-face and within the endoplasmic reticulum (ER), theSTIM2 protein is expressed only intracellularly, whichlikely reflects an ER-retention signal that is present inSTIM2 but not STIM1.85–89 It was apparent that suppressedSTIM1 expression, but not STIM2, was able to preventSOCE and eliminate the store-dependent activation ofCRAC channels.71,73 Thus, the function of STIM1 is postu-lated to act as a Ca2þ sensor in the ER.72

On a final note, the question of ‘‘blame’’ for hyperten-sion is argued between two schools of thought: cardiovas-cular and renal physiologists; the former believing thathypertension is due to increased vascular resistance andan overall hyperreactivity of vascular tissue, the latter be-lieving that the kidney has the final say in BP control. Untilrecently, renal physiologists everywhere cited articles byGuyton, who was the first to clearly demonstrate the phe-nomenon of ‘‘pressure natriuresis’’ and argue for the centralrole for the kidney in BP control, and Coffman, who usesa model of AT1A receptor knockout to illustrate the neces-sity of the RAS in hypertension.90,91 These elegant studiesused the AT1 receptor knockout mice and kidney cross-transplantation to show that the AT1A receptor is crucialto basal BP regulation, as well as hypertension, and thatAT1A receptors in the kidney are paramount in hypertensionand the renal response to hypertension. However, thesestudies also demonstrated that BP regulation by AT1A re-ceptors in nonrenal tissues (ie, vasculature) were also a ma-jor contributor to systemic BP.92,93 An article recentlypublished in PNAS by Michael et al complements thesestudies, using a knocking mutant of the cGMP-dependentprotein kinase, PKGIa. PKGI is expressed in vascular andsmooth muscle cells and has been shown to regulate vascu-lar relaxation via endothelial-derived NO and other nitrova-sodilators. The most remarkable data from this study showthat the LZM-PKGIa mutant mice exhibit elevated BPs,even in the presence of normal renal function and normalrenal salt handling, suggesting an important mechanismfor vascular smooth muscle in the normal and pathophysi-ologic control of BP.94 Overall, these studies reiterate ourawareness of the complexity of hypertension, and thenecessity for systemic integration.

Conclusion

Arterial vascular smooth muscle cells constitute the ma-jority of the arterial wall, playing a foremost role in vascu-lar resistance and blood flow. Angiotensin II andendothelin-1 are potent agonists inducing contraction ofvascular smooth muscle. The contractile apparatus of vas-cular smooth muscle, actin, and myosin, can be activatedin a Ca2þ-dependent and Ca2þ-independent manner. Via li-gand binding to plasma membrane GPCRs, second messen-gers are generated and induce the release of Ca2þ throughchannels located on the plasma membrane or on the SRproducing a rapid and transient increase in intracellularCa2þ. Channels discussed in this review are activatedthrough ligand binding, store depletion, and membrane de-polarization. In the form of a Ca2þ-calmodulin complex,subsequent activation of MLCK occurs, inducing contrac-tion via actin-myosin cross-bridges. Contraction also oc-curs, Ca2þ independently, through the activation of theRhoA/Rho kinase pathway leading to MLCP inactivation,and the maintenance of contraction.

Given the varied mechanisms for smooth muscle contrac-tion, one can imagine the wide range of areas where dereg-ulation can occur, leading to increased BP or hypertension.On a molecular level, alterations in various cellular signal-ing components, can lead to over stimulation, causing anincreased and maintained vasoconstriction, decreased re-laxation, and consequently, elevation of systemic BP.

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