R. Clinton Webb
Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912
This brief review serves as a refresher on smooth muscle physiologyfor those educators who teach in medical and graduate coursesof physiology. Additionally, those professionals who are inneed of an update on smooth muscle physiology may find thisreview to be useful. Smooth muscle lacks the striations characteristicof cardiac and skeletal muscle. Layers of smooth muscle cellsline the walls of various organs and tubes in the body, andthe contractile function of smooth muscle is not under voluntarycontrol. Contractile activity in smooth muscle is initiatedby a Ca2+-calmodulin interaction to stimulate phosphorylationof the light chain of myosin. Ca2+ sensitization of the contractileproteins is signaled by the RhoA/Rho kinase pathway to inhibitthe dephosphorylation of the light chain by myosin phosphatase,thereby maintaining force generation. Removal of Ca2+ from thecytosol and stimulation of myosin phosphatase initiate the processof smooth muscle relaxation.
Key words: review ; cell signaling
ADV PHYSIOL EDUC 27:201-206, 2003.
Sheets or layers of smooth muscle cells are contained in thewalls of various organs and tubes in the body, including theblood vessels, stomach, intestines, bladder, airways, uterus,and the penile and clitoral cavernosal sinuses. When made tocontract, the smooth muscle cells shorten, thereby propellingthe luminal contents of the organ, or the cell shortening variesthe diameter of a tube to regulate the flow of its contents.There are also bundles of smooth muscle cells attached to thehairs of the skin and to the iris and lens of the eye. Whenthese bundles contract, the hairs become erect and the lensof the eye changes shape to focus light on the retina.
Smooth muscle cells lack the striated banding pattern foundin cardiac and skeletal muscle, and they receive neural innervationfrom the autonomic nervous system. In addition, the contractilestate of smooth muscle is controlled by hormones, autocrine/paracrineagents, and other local chemical signals. Smooth muscle cellsalso develop tonic and phasic contractions in response to changesin load or length. Regardless of the stimulus, smooth musclecells use cross-bridge cycling between actin and myosin to developforce, and calcium ions (Ca2+) serve to initiate contraction.
This brief review will serve as a refresher for those educatorswho teach in medical and graduate courses of physiology. Additionally,those professionals who are in need of an update on smooth musclephysiology may find this review to be useful. New concepts aboutregulatory mechanisms are presented to add depth to the understandingof the integrated responses of contraction and relaxation insmooth muscle. For those individuals desiring a more in-depthtreatment of the subject, several recent reviews are recommended(1, 3, 5, 7, 9, 10, 18).
The contractile mechanism
In the intact body, the process of smooth muscle cell contractionis regulated principally by receptor and mechanical (stretch)activation of the contractile proteins myosin and actin. A changein membrane potential, brought on by the firing of action potentialsor by activation of stretch-dependent ion channels in the plasmamembrane, can also trigger contraction. For contraction to occur,myosin light chain kinase (MLC kinase) must phosphorylate the20-kDa light chain of myosin, enabling the molecular interactionof myosin with actin. Energy released from ATP by myosin ATPaseactivity results in the cycling of the myosin cross-bridgeswith actin for contraction. Thus contractile activity in smoothmuscle is determined primarily by the phosphorylation stateof the light chain of myosin—a highly regulated process.In some smooth muscle cells, the phosphorylation of the lightchain of myosin is maintained at a low level in the absenceof external stimuli (i.e., no receptor or mechanical activation).This activity results in what is known as smooth muscle toneand its intensity can be varied.
Ca2+-dependent contraction of smooth muscle
Contraction of smooth muscle is initiated by a Ca2+-mediatedchange in the thick filaments, whereas in striated muscle Ca2+mediates contraction by changes in the thin filaments. In responseto specific stimuli in smooth muscle, the intracellular concentrationof Ca2+ increases, and this activator Ca2+ combines with theacidic protein calmodulin. This complex activates MLC kinaseto phosphorylate the light chain of myosin (Fig. 1). CytosolicCa2+ is increased through Ca2+ release from intracellular stores(sarcoplasmic reticulum) as well as entry from the extracellularspace through Ca2+ channels (receptor-operated Ca2+ channels).Agonists (norepinephrine, angiotensin II, endothelin, etc.)binding to serpentine receptors, coupled to a heterotrimericG protein, stimulate phospholipase C activity. This enzyme isspecific for the membrane lipid phosphatidylinositol 4,5-bisphosphateto catalyze the formation of two potent second messengers: inositoltrisphosphate (IP3) and diacylglycerol (DG). The binding ofIP3 to receptors on the sarcoplasmic reticulum results in therelease of Ca2+ into the cytosol. DG, along with Ca2+, activatesprotein kinase C (PKC), which phosphorylates specific targetproteins. There are several isozymes of PKC in smooth muscle,and each has a tissue-specific role (e.g., vascular, uterine,intestinal, etc.). In many cases, PKC has contraction-promotingeffects such as phosphorylation of L-type Ca2+ channels or otherproteins that regulate cross-bridge cycling. Phorbol esters,a group of synthetic compounds known to activate PKC, mimicthe action of DG and cause contraction of smooth muscle. Finally,L-type Ca2+ channels (voltage-operated Ca2+ channels) in themembrane also open in response to membrane depolarization broughton by stretch of the smooth muscle cell.
Ca2+ Sensitization mechanism and contraction of smooth muscle
In addition to the Ca2+-dependent activation of MLC kinase,the state of myosin light chain phosphorylation is further regulatedby MLC phosphatase [aka myosin phosphatase (1, 4, 9, 11–16)],which removes the high-energy phosphate from the light chainof myosin to promote smooth muscle relaxation (Fig. 1). Thereare three subunits of MLC phosphatase: a 37-kDa catalytic subunit,a 20-kDa variable subunit, and a 110- to 130-kDa myosin-bindingsubunit. The myosin-binding subunit, when phosphorylated, inhibitsthe enzymatic activity of MLC phosphatase, allowing the lightchain of myosin to remain phosphorylated, thereby promotingcontraction. The small G protein RhoA and its downstream targetRho kinase play an important role in the regulation of MLC phosphataseactivity. Rho kinase, a serine/threonine kinase, phosphorylatesthe myosin-binding subunit of MLC phosphatase, inhibiting itsactivity and thus promoting the phosphorylated state of themyosin light chain (Fig. 1). Pharmacological inhibitors of Rhokinase, such as fasudil and Y-27632, block its activity by competingwith the ATP-binding site on the enzyme. Rho kinase inhibitioninduces relaxation of isolated segments of smooth muscle contractedto many different agonists. In the intact animal, the pharmacologicalinhibitors of Rho kinase have been shown to cause relaxationof smooth muscle in arteries, resulting in a blood pressure-loweringeffect (2, 17).
An important question facing the smooth-muscle physiologistis: what is the link between receptor occupation and activationof the Ca2+-sensitizing activity of the RhoA/Rho kinase-signalingcascade? Currently, it is thought that receptors activate aheterotrimeric G protein that is coupled to RhoA/Rho kinasesignaling via guanine nucleotide exchange factors (RhoGEFs;Fig. 1). Because RhoGEFs facilitate activation of RhoA, theyregulate the duration and intensity of signaling via heterotrimericG protein receptor coupling. There are 70 RhoGEFs in the humangenome, and three RhoGEFs have been identified in smooth muscle:PDZ-RhoGEF, LARG (leukemia-associated RhoGEF), and p115-RhoGEF.Increased expression and/or activity of RhoGEF proteins couldaugment contractile activation of smooth muscle and thereforeplay a role in diseases where an augmented response contributesto the pathophysiology (hypertension, asthma, etc.).
Several recent studies suggest a role for additional regulatorsof MLC kinase and MLC phosphatase (13–16). Calmodulin-dependentprotein kinase II promotes smooth muscle relaxation by decreasingthe sensitivity of MLC kinase for Ca2+. Additionally, MLC phosphataseactivity is stimulated by the 16-kDa protein telokin in phasicsmooth muscle and is inhibited by a downstream mediator of DG/proteinkinase C, CPI-17.
Smooth muscle relaxation
Smooth muscle relaxation occurs either as a result of removalof the contractile stimulus or by the direct action of a substancethat stimulates inhibition of the contractile mechanism (e.g.,atrial natriuretic factor is a vasodilator). Regardless, theprocess of relaxation requires a decreased intracellular Ca2+concentration and increased MLC phosphatase activity (Fig. 2)(10, 16). The mechanisms that sequester or remove intracellularCa2+ and/or increase MLC phosphatase activity may become altered,contributing to abnormal smooth muscle responsiveness.
A decrease in the intracellular concentration of activator Ca2+elicits smooth muscle cell relaxation. Several mechanisms areimplicated in the removal of cytosolic Ca2+ and involve thesarcoplasmic reticulum and the plasma membrane. Ca2+ uptakeinto the sarcoplasmic reticulum is dependent on ATP hydrolysis.This sarcoplasmic reticular Ca,Mg-ATPase, when phosphorylated,binds two Ca2+ ions, which are then translocated to the luminalside of the sarcoplasmic reticulum and released. Mg2+ is necessaryfor the activity of the enzyme; it binds to the catalytic siteof the ATPase to mediate the reaction. The sarcoplasmic reticularCa,Mg-ATPase is inhibited by several different pharmacologicalagents: vanadate, thapsigargin, and cyclopiazonic acid. Sarcoplasmicreticular Ca2+-binding proteins also contribute to decreasedintracellular Ca2+ levels. Recent studies have identified calsequestrinand calreticulin as sarcoplasmic reticular Ca2+-binding proteinsin smooth muscle.
The plasma membrane also contains Ca,Mg-ATPases, providing anadditional mechanism for reducing the concentration of activatorCa2+ in the cell. This enzyme differs from the sarcoplasmicreticular protein in that it has an autoinhibitory domain thatcan be bound by calmodulin, causing stimulation of the plasmamembrane Ca2+ pump.
Na+/Ca2+ exchangers are also located on the plasma membraneand aid in decreasing intracellular Ca2+. This low-affinityantiporter is closely coupled to intracellular Ca2+ levels andcan be inhibited by amiloride and quinidine.
Receptor-operated and voltage-operated Ca2+ channels locatedin the plasma membrane are important in Ca2+ influx and smoothmuscle contraction, as previously mentioned. Inhibition of thesechannels can elicit relaxation. Channel antagonists such asdihydropyridine, phenylalkylamines, and benzothiazepines bindto distinct receptors on the channel protein and inhibit Ca2+entry in smooth muscle.
Abnormal contractile regulation of smooth muscle
Alterations in the regulatory processes maintaining intracellularCa2+ and MLC phosphorylation have been proposed as possiblesites contributing to the abnormal contractile events in smoothmuscle cells of various organs and tissues (2, 5, 8, 9). Inaddition, alterations in upstream targets that impact Ca2+ andMLC phosphorylation have also been implicated. For example,changes in the affinity, number, or subtype of -adrenergic receptorsleading to enhanced vasoconstriction have been characterizedin arterial smooth muscle cells in some types of hypertension.Increases in the activity of RhoA/Rho kinase signaling leadto increased contractile responses that may contribute to erectiledysfunction in the penis and clitoris. Increased activity ofthe RhoA/Rho kinase-signaling pathway may also contribute toaugmented contraction or spastic behavior of smooth muscle indisease states such as asthma or atherosclerosis.
Impaired function may occur as the result of a change in thedirect action of a substance that stimulates inhibition of thecontractile mechanism. For example, decreased relaxation responsescan be due to a reduction in cyclic nucleotide-dependent signalingpathways coupled with reductions in receptor activation (ß-adrenergicreceptors and cyclic AMP) or agonist bioavailability (endotheliumdysfunction, reduced nitric oxide and cyclic GMP). Importantly,it is the complexity and redundancy of these cell signalingpathways regulating intracellular Ca2+ and MLC phosphorylationin smooth muscle that provide therapeutic potential for dysfunction.
Smooth muscle derives its name from the fact that it lacks thestriations characteristic of cardiac and skeletal muscle. Layersof smooth muscle cells line the walls of various organs andtubes, and the contractile function of smooth muscle is notunder voluntary control. Contractile activity in smooth muscleis initiated by a Ca2+-calmodulin interaction to stimulate phosphorylationof the light chain of myosin. A Ca2+ sensitization of the contractileproteins is signaled by the RhoA/Rho kinase pathway to inhibitthe dephosphorylation of the light chain by myosin phosphatase,maintaining force generation. Removal of Ca2+ from the cytosoland stimulation of myosin phosphatase initiate the process ofsmooth muscle relaxation.
This work was supported by grants from the National Heart, Lung,and Blood Institute (HL-18575 and HL-71138).
Address for reprint requests and other correspondence: R. C.Webb, Dept. of Physiology, Medical College of Georgia, 1120Fifteenth St., Augusta, GA 30912–3000 (E-mail: firstname.lastname@example.org ).
Received for publication August 4, 2003. Accepted for publication August 19, 2003.
ReferencesBarany M. Biochemistry of Smooth Muscle Contraction. San Diego, CA: Academic, 1996. Chitaley K, Weber DS, and Webb RC. RhoA/Rho-kinase, vascular changes and hypertension. Curr Hypertension Rep 3: 139–144, 2001. Feletou M and Vanhoutte PM. Endothelium-dependent hyperpolarization of vascular smooth muscle cells. Acta Pharmacol Sin 21: 1–18, 2000. Fukata Y, Mutsuki A and Kaibuchi. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non muscle cells. Trends Physiol Sci 22: 32–39, 2001. Jin L, Linder AE, Mills TM, and Webb RC. Inhibition of the tonic contraction in the treatment of erectile dysfunction. Exp Opin Therapeutic Targets 7: 265–276, 2003. Mehta S, Webb RC, and Dorrance AM. The pathophysiology of ischemic stroke: a neuronal and vascular perspective. J Med Sci 22: 53–62, 2002. Meiss RA. Mechanics of smooth muscle contraction. In: Cellular Aspects of Smooth Muscle Function, edited by Kao CY and Carsten ME. New York: Cambridge Univ. Press, 1997, p. 169–201. Mills TM, Lewis RW, Wingard CJ, Chitaley K, and Webb RC. Inhibition of tonic contraction—a novel way to approach erectile dysfunction. J Androl 23: S5–S9, 2002. Mitchell BM, Chitaley KC, and Webb RC. Vascular smooth muscle contraction and relaxation. In: Hypertension Primer: The Essentials of High Blood Pressure, edited by Izzo JL and Black HR. Dallas, TX: Am. Heart Assoc., 2003, p. 97–99. Morgan K. The role of calcium in the control of vascular tone as assessed by the Ca++ indicator Aequorin. Cardiovasc Drugs Ther 4: 1355–1362, 1990. Ridley A. Rho: theme and variations. A review. Curr Biol 6: 1256–1264, 1996. Sah VP, Seasholtz TM, Sagi SA, and Brown JH. The role of rho in g protein-coupled receptor signal transduction. Annu Rev Pharmacol Toxicol 40: 459–489, 2000. Solaro RJ. Myosin light chain phosphatase: a Cinderella of cellular signaling. Circ Res 87: 173–175, 2000. Somlyo AP and Somlyo AV. From pharmacomechanical coupling to G-proteins and myosin phosphatase. Acta Physiol Scand 164: 437–448, 1998. Somlyo AP and Somlyo AV. Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177–185, 2000. Somlyo AP, Wu X, Lalker LA, and Somlyo AV. Pharmacomechanical coupling: the role of calcium, G-proteins, kinases and phosphatases. Rev Physiol Biochem Pharmacol 134: 201–234, 1999. Uehata M, Ishizuki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990–994, 1997. Woodrum DA and Brophy CM. The paradox of smooth muscle physiology. Mol Cell Endocrinol 177: 135–143, 2001.
FIG. 1 Regulation of smooth muscle contraction. Various agonists (neurotransmitters, hormones, etc.) bind to specific receptors to activate contraction in smooth muscle. Subsequent to this binding, the prototypical response of the cell is to increase phospholipase C activity via coupling through a G protein. Phospholipase C produces two potent second messengers from the membrane lipid phosphatidylinositol 4,5-bisphosphate: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). IP3 binds to specific receptors on the sarcoplasmic reticulum, causing release of activator calcium (Ca2+). DG along with Ca2+ activates PKC, which phosphorylates specific target proteins. In most smooth muscles, PKC has contraction-promoting effects such as phosphorylation of Ca2+ channels or other proteins that regulate cross-bridge cycling. Activator Ca2+ binds to calmodulin, leading to activation of myosin light chain kinase (MLC kinase). This kinase phosphorylates the light chain of myosin, and, in conjunction with actin, cross-bridge cycling occurs, initiating shortening of the smooth muscle cell. However, the elevation in Ca2+ concentration within the cell is transient, and the contractile response is maintained by a Ca2+-sensitizing mechanism brought about by the inhibition of myosin phosphatase activity by Rho kinase. This Ca2+-sensitizing mechanism is initiated at the same time that phospholipase C is activated, and it involves the activation of the small GTP-binding protein RhoA. The precise nature of the activation of RhoA by the G protein-coupled receptor is not entirely clear but involves a guanine nucleotide exchange factor (RhoGEF) and migration of RhoA to the plasma membrane. Upon activation, RhoA increases Rho kinase activity, leading to inhibition of myosin phosphatase. This promotes the contractile state, since the light chain of myosin cannot be dephosphorylated.
FIG. 2 Relaxation of smooth muscle. Smooth muscle relaxation occurs either as a result of removal of the contractile stimulus or by the direct action of a substance that stimulates inhibition of the contractile mechanism. Regardless, the process of relaxation requires a decreased intracellular Ca2+ concentration and increased MLC phosphatase activity. The sarcoplasmic reticulum and the plasma membrane contain Ca,Mg-ATPases that remove Ca2+ from the cytosol. Na+/Ca2+ exchangers are also located on the plasma membrane and aid in decreasing intracellular Ca2+. During relaxation, receptor- and voltage-operated Ca2+ channels in the plasma membrane close resulting in a reduced Ca2+ entry into the cell.