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Pathways controlling healthy and diseased arterial smooth muscle
Pathways Controlling Healthy and Diseased Arterial Smooth Muscle Richard A. Cohen,
The cells within the vascular wall act as a unit of smooth muscle regulating the co&action cells. In arteries the endothellum and autonomk nerves provide the major factors that regulate intmcelkdar caklum In smooth muscle cells, whkh determines wntractiletone.Thellum provldes a major InhlMtory influence, whkh Itself is modulated by shear forces wtthln the vascular lumen reguktlng endothetlal cell caklum and the release ofendothellum-derlved relaxing factors. Ofthe major mechanisms controlling smooth muscle caklum, K has been suggested that voltagedpemht calcium channels are among the most Important In medlatlng the lnhlbltory Influence of the endothellum. Smooth musde potassium channels and sodlumpotasslum adenosine trtphosphatase (Na+,K+-AlPase) are Important regulators of membrane potential, and each Is affeded by the endothellum. Because the activtty of each hyperpolarkes the membrane potential, they counter the Influence on vottagedependent caklum channels and lnhlblt contractkm. Both of these counterregulatory mechanisms have recently been shown to be impalred In diseased arteries. This may help to explain the dlminlshed etfectlveness of the endothellum on the smooth muscle. Thus, vascular disease may cause dlmlnlshed release, increased destruction, or llmtted effectiveness of emkthellum&rlved relaxing factors. The failure of the lnhlbttory lnfluence of the endothellum may be the prlnclpal mechanism by whkh vascular risk factors and disease increase vasoconstrktor tone, possiMy contrlbutlng to hypertenskn and the pm of atherosclerosis. (Am J Cardlol139~72:33C47C)
From the Vascular Biology Unit, Robert Dawson Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts. This review discusses work supported by National Institutes of Health grants HL31607, HL38731, and HL47124, as well as by an American Heart Association Established Investigator Award to Dr. Cohen. Address for reprints: Richard A. Cohen, MD, Vascular Biology Unit, E401 University Hospital, 88 E. Newton Street, Boston, Massachusetts 02118.
MD
T
he arterial wall consists of an integrated unit composed not only of vascular smooth muscle, but also of other cellular components that regulate vascular function. The endothelium bordering the luminal aspect of the blood vessel wall provides the major local control mechanism for the vascular smooth muscle, and the autonomic nerves on the adventitial aspect of the blood vessel provide the major extrinsic input. Also present are other cell types, including fibroblasts, leukocytes, platelets, and macrophages, that contribute to the structure and function of the whole. This article reviews the principal factors controlling arterial smooth muscle under physiologic and pathophysiologic conditions. COMPONEN7S
OF THE ARlERlAL
WAIL
Before considering physiologic studies of blood vessels, the major mechanisms controlling vascular smooth muscle tone will be discussed in terms of how the endothelium and autonomic nerves regulate vascular smooth muscle function. Vascular dysfunction in disease cannot be fully understood without understanding the interaction of these cells, which compose the basic vascular wall unit. Vascular smooth muscle: Figure 1 shows the mechanisms that regulate the level of intracellular free calcium, the predominant determinant of smooth muscle contraction. Free intracellular calcium initiates a cascade of events by binding to calmodulin, which is followed by myosin lightchain phosphorylation and actin-myosin crossbridge cycling, resulting in contraction.l,* Figure 1 also depicts 3 major mechanisms that increase cell calcium. Perhaps the most important physiologic regulator of calcium entry into the muscle cell is the voltage-dependent calcium channel, shown at the upper right of the smooth muscle cell. Depolarization of the cell membrane caused by endothelial or autonomic nerve-released contractile factors increases the probability of “open” calcium channels, allowing influx of the ion. Within the box to the left of the calcium channel in Figure 1 are two important physiologic voltage regulatory A SYMPOSIUM: MANAGING MYOCARDIAL ISCHEMIA
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FlalNEl.Dlagramsbouilngcon. trol ofvascular smooth muscle VW by endotbdlum (top) and autonomknarveendngs(lJottOflt).-d~murde cdl Is regulated by bltraceKular caklum,whkbblndstocalnoduIln, leadng to myosln ll@t chain Wylatkn(MW)=f=tln4nyosln cross-llnldng. Calcium entersthecellbytlK+voltagedependantcaklumchannelat the upper left ofthe smooth mus--b-~bWtvelyre(pllatedbydepolaiizatlonastilas Iry dv (DAD)dent protein klnase C actMy. lhe4caklumchamdrarenega~a~u--w(ATP)+kpembt sz ~(Kbda~by*~ geak Na+,K+-AlPase, whkh tend tohyperpdarlzethesnlootbmuscls membrane. Cal&m also eb izlb~~~~ caklum channels. Caklum ls also -vlareceptoroperated,G-proteiP~phollpscreCO~, In addtlom to DAD, fonnr lnosltol trlphosphate (IPJ, wldch r&as6scaklumfromtlb8sarcoplas4n@retkulum(SR).The rdeasesendotbellumderlvedhyperlntracdhdar caldum cham -b-mmby-erWthellumdewlved mlaxlng factors (EDRFs, lncludlng nltrk oxide [NOD, and embthellum-de m-(EDHF), pw&agbdln endoperoxlde [PDl&l, sqwoxlde anion [4-l, and endotheln rhfedcontractlngfactors(EDCFs,I~ [ETI). EDRFNO acth&es smooth muscle guanylate cyclase (DC) to form cyclk guanoslne monophosphate (cGMP), nervescantrolsmoothmusclebyreleaslng caklwnentryandcaklumrekase.Autonomk wbkbnegatlvdylnnuences -tine (NE), AlP, peptldes such as cakttonln g-related peptlde (CDKP), and NO.
mechanisms-potassium channels and the sodiumpotassium adenosine triphosphatase (Na+,K+ATPase) pump. The open state of different potassium channels is regulated by voltage, intracellular calcium, or intracellular ATP levels. The open potassium channel allows potassium to leave the
NTG E-
PlGlJRE2.Dlagramshowlngshear~,e1+ dothehm-derhfed nlhlc oxide (NO) production from arglnine by NO syntbase (NOS), NO releew, and action on vascularsmoothmusck.Shearstresslsshownregulating endothebl cdl caklum by ragulathg a caklum charnel. The~edmechanlsmbywldchhormones suds as ac&ylhMne regulate endothellal cell caklum Is alsoshown.NOInsmoothmusclelsshownrMhtbu?guapilate4(chMP)hnlgua~trlpl;orphate(DlP).For otberabkevbtlons,seePlgurel. mc
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
cell on a steep chemical gradient and results in hyperpolarization of the cell membrane, which thus tends to close the calcium channel.3 Activity of the sodium pump is “electrogenic,” also resulting in membrane hyperpolarization. Thus, the activity of these 2 mechanisms counterregulates the voltagedependent calcium channel. Of the 3 mechanisms responsible for increasing intracellular calcium, 2 are activated by cell membrane receptors stimulated by endothelial or neural vasoactive factors. One type of receptor causes G-protein-linked activation of phospholipase C, resulting in the formation of inositol triphosphates, whose principal action is to release calcium stores from the sarcoplasmic reticulum. A byproduct, diacylglycerol, has additional important cellular functions by activating protein kinase C, including stimulation of voltage-dependent calcium channels. Another type of receptor is directly linked by G-proteins to calcium channels. Not shown in Figure 1 are the mechanisms that lower intracellular calcium, such as pumps that extrude calcium from the cell and the reuptake of calcium into the sarcoplasmic reticulum. Endothelium: The endothelium regulates smooth muscle function in several ways, including the release of vasoactive factors. Endothelial cell calcium regulates the activity of nitric oxide (NO) SEPTEMBER 9, 1993
synthase, which metabolizes L-arginine to NO (Figures 1 and 2), the most important endotheliumderived relaxing factor (EDRF).4 After diffusing to the smooth muscle, NO exerts its effects by stimulating guanylate cyclase-catalyzed production of cyclic guanosine monophosphate (GMP). Cyclic GMP down-regulates the contractile process by negatively influencing calcium release from the sarcoplasmic reticulum and its entry through calcium channels, as well as by inhibiting the calciumactivated contractile process itself.5-x It is important to understand the mechanisms by which NO relaxes vascular smooth muscle if one is to understand the mechanisms of all nitrovasodilators, because they all break down to release NO.” The endothelium releases additional important factors that are less well described. Endotheliumderived hyperpolarizing factor is thought to activate smooth muscle potassium channels, thereby inhibiting voltage-dependent calcium channels and contraction.1° Endothelium-derived hyperpolarizing factor is thus another EDRF. Endotheliumderived contracting factors include prostaglandin endoperoxides, endothelin, and superoxide radicals.ll Prostaglandin endoperoxides and endothelin activate smooth muscle receptors. The action of superoxide radical is poorly understood, but it may cause contraction by interfering with the action of NO. Although most of the experimental evidence for the actions of these factors has resulted from stimulating endothelial cells with hormones and neurotransmitters, such as acetylcholine and bradykinin, the likely principal physiologic regulator is the variation of shear stress at the endothelial cell surface, which has been shown to regulate endothelial cell calcium levels. l2 Autonomic nerves: Figure 1 also shows several neurotransmitters, released by vascular nerves, that regulate vascular smooth muscle.” The classic
sympathetic transmitter, norepinephrine, contracts most vascular smooth muscle by activating (Yadrenoceptors. An important exception is coronary artery smooth muscle, which norepinephrine relaxes by activating B adrenoceptors. ATP is a cotransmitter with norepinephrine and contracts smooth muscle in some blood vessels. Peptidergic nerves innervate certain blood vessels; calcitonin gene-related peptide is one of several neuropeptides that regulate vascular smooth muscle.14 NO, which is also produced within vascular nerve endings, has been suggested to be a nonadrenergic, noncholinergic, vasodilatory neurotransmitter.15 FUNCTION OF THE VASCULAR WALL UNIT
To discuss factors that are important in controlling smooth muscle as the muscle functions within the vascular wall unit that it shares with endothelium and nerve, this review will focus on the rabbit carotid artery.16J7This artery is a typical peripheral artery with cy-adrenergic sympathetic innervation. Figure 3 shows that when the nerves of the isolated artery are stimulated, the stimulation of 011adrenoceptors by neurally released norepinephrine causes contractions.17 The endothelium inhibits the contractions primarily as a result of the continuous release of endothelium-derived NO. The endothelium thereby provides a tonic counterregulatory influence to neurogenic vasoconstriction, which is a principal physiologic means whereby constrictor tone of the peripheral vasculature is regulated. Myogenic tone, important in resistance vessels, is also tonically inhibited by the endothelium. Because the failure of the endothelium to inhibit the contraction of smooth muscle is a primary characteristic of diseased blood vessels, the remainder of this review will discuss the mechanisms that may be important in the inhibitory influence of the endothelium on vasoconstriction.
__ ------FlGURE 3. Recordlr@s of lsometrktenslonof2MgsofrabbRcarotld arteq wRh endothellum ENDl;;Iyfi / ((top) and denuded emlothellum (bottom) demonstrating the InhlbRory effect ofthe endothellum on w neurotransmlsslon. lhe rbqgs are first co&acted with noreplnephrlne (NE), and aaetylDENUDED ;It..,r’l-ir 5gLn cholhw (ACH) Is addsd to show that ths rlqg wtth endothellum 0.5 t relaxes,whereastherhlgwlthout E 8 QNP K+lZOmM endothedlumdoesnotrelax.The AWlog M FREQUENCY.Hz I--.--__ sympathetk adrenergk nerves In the artedal Mgs are then sthnulatedekctronkallyat lncread~wqu8dss, revealing that the artery wRh endothellum contracts less than the one wRhout edothellum. The artedes are then contracted euuall~ by deuolarlzlng wtth elevated potassium (K++).Concentratlons are @ven as the negattve IogarRhm of the molar coke&k (-log M)or mlllhnolar (d). SNP =‘saklum nRroprusside. (ReprInted with permlsslon from Cohen.9 A SYMPOSIUM:
MANAGING
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41c
abrogated.‘* The channel activator was used under conditions in which it had little influence on L CONTROL . o -A BAY K 6644 . 60. arteries denuded of endothelium, suggesting that its inhibition of endothelial cell action was specific. Further, voltage-dependent calcium channels are thought to be absent from endothelial cells, indicating that the drug’s site of action is smooth muscle. Although the results of these studies await electrophysiologic confirmation, they suggestthat smooth muscle voltage-dependent calcium channels are tonically inhibited by the endothelium. 1 2 4 a FREQUENCY. Hz Shear stress: Under physiologic conditions, the release of NO is regulated by shear forces on PlOURE4.3ummaydataobtahdbythemethodds endothelial cells. This is thought to occur by way of scrbedlnFigure3showlngthelnhbltofyinRuenceofthe &dothe3umofrabbRcarotldartwksandthedfectof the shear-dependent influx of extracellular calcium voe baliSum channel acthmtlon by RAY K contributing to calcium levels in the cell, which in turn regulates NO synthase activity (Figure 2).19In perfused rabbit carotid arteries, increases in shear stress caused by elevated viscosity of the bathing ( - Tat all Weqwdwklnthepresenceofthev~ solution releases EDRF from the endothelium c&lumchannelacthkt6rRAYK3644,thecoWadhsd (Figure 5).20Thus, this shear-dependent release of artwleswRhorwRhout edoth@umareslmllartothose EDRF tonically inhibits neurogenic vasoconstricofartudeswRho4R edothdlumIntheabsenceof~heactlvator.(ReprlnteawRh --~Jphydol.9 tion (Figure 6). The shear-dependent release of EDRF is likely an important regulator of vascular Vdtage-clependent caklum dfannek It is tone in vivo, as demonstrated by inhibitors of NO widely recognized that the basal release of NO infused in animals and humans.21Thus, endotheinhibits smooth muscle by increasing cyclic GMP lium and nerves normally counterregulate vascular levels, and pharmacologic studies have indicated smooth muscle continuously in vivo. that voltage-dependent calcium channels are probably modulated by cyclic GMP.6 Figure 4 shows VASCULAR RISK FACTORS AND DISEASE that when the activation of voltage-dependent A common feature of vascular diseaseis dysfunccalcium channels is enhanced in the rabbit carotid tion of the endothelium early in the course of the artery by the specific channel activator BAY K disease. Endothelial cell dysfunction has been 8644, the inhibitory influence of the endothelium is demonstrated in animals and in patients with the 7.
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THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
SEPTEMBER 9, 1993
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may be important in initiating atherosclerosis. The inhibitory influence of the endothelium on neurogenie vasoconstriction disappears rapidly in rabbits with hypercholesterolemia, diabetes, or hyperten-
major risk factors for atherosclerosis (i.e., hypercholesterolemia, diabetes mellitus, and hypertension). Because such dysfunction may occur even before the presence of any discernible atherosclerosis, it
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A SYMPOSIUM: MANAGING MYOCARDIAL ISCHEMIA
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sion. In the carotid artery, this may occur even before endothelium-dependent relaxations stimulated by agents such as acetylcholine have become abnormal, implying the importance of the failure of the tonic inhibitory mechanism as an early event in the pathogenesis of vascular disease. mlemla: In rabbits fed a 2% cholesterol diet for 13-15 weeks, the plasma cholesterol was elevated to >2,000 mg/dL, but in the carotid arteries, the endothelium-dependent relaxation to acetylcholine did not differ from that in normal rabbits.22However, the inhibitory influence of the endothelium on neurogenic contractions was nearly absent (Figure 7). d4C
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
Diabetes mellttus: The diabetogenic agent alloxan was used to increase blood glucose levels in rabbits to approximately 350 mg/dL for 6 weeks.23 In the carotid artery of these rabbits, the acetylcholine relaxation was normal, but the inhibition of neurogenic contraction by the endothelium was also nearly absent (Figure 8). Hypertension: A few observations in Watanabe heritable hyperlipidemic rabbits suggestthat hypertension may also cause a deterioration in endothelial cell inhibition of neurogenic contractions. Compared with those of normotensive New Zealand white rabbits, carotid arteries of 12-month-old Watanabe rabbits demonstrated normal endothelial cell function. Compared with rabbits fed cholestero1,22 the Watanabe rabbits had much lower plasma cholesterol levels (400-700 mg/dL), possibly explaining why endothelial function was normal. However, in Watanabe rabbits made hypertensive for 1 month, endothelial cell inhibition was decreased (Figure 9). Potential mechanisms: Endothelial cell dysfunction in response to risk factors can occur in severalways. For instance, the production or effectiveness of EDRFs can decrease. Recent studies suggest that decreased effectiveness may be more important than decreased release of EDRFs in both hypercholesterolemia24 and diabetes.25 Decreased effectiveness may result from increased destruction of EDRFs, increased production of counteracting endothelium-derived contracting factors, or altered responsivenessof the smooth muscle to EDRF. Vascular disease may alter the responsiveness of the smooth muscle to the endothelium through 2 recently discoveredand potentially important mechanisms. Both mechanisms suggest an impaired counterregulation of smooth muscle voltage-activated calcium channels by EDRFs. It was recently demonstrated that the addition of cholesterol to smooth muscle membranes can inhibit the function of calcium-activated potassium channels in just a few hours.26Since these studies were performed on single channels in isolated membrane patches, the effect may have been due to a fundamental molecular interaction between cholesterol and the channels in the cell membrane. The function of calcium-activated potassium channels in cells from atherosclerotic human aorta (which also have increased membrane cholesterol) are also inhibited compared with those from nonatherosclerotic aorta (Figure 1O).27Insofar as the impaired activity of potassium channels depolarizes the smooth muscle cells, voltage-dependent
SEPTEMBER 9, 1993
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calcium channels may tend to be more active in cells with increased membrane cholesterol, and, as a result, the endothelial cell inhibitory influence would be lessened. In arteries of diabetic patients, the activity of Na+,K+-ATPase is impaired,28 making it more likely that the artery is relatively depolarized, which would similarly limit endothelial cell inhibition by increasing the activity of voltage-dependent calcium channels. Studies have also shown that elevated glucose exposure inhibits the vascular sodium pump of isolated rabbit arteries in as little as 3 hours (Figure 11).28 A similar degree of inhibition of the sodium pump was also observed after NO production was pharmacologically blocked (Figure 11). These data suggest that elevated glucose prevents endothelial cell-derived NO from stimulating the sodium pump. Although the means by which NO influences the sodium pump is not yet known, the inhibitory action of glucose may explain the augmented vascular contractions of diabetic arteries shown in Figure 8.
stand the mechanisms by which vascular risk factors interfere with the physiologic function of the endothelium may lead to the development of adjunctive treatments to ameliorate the deleterious effects of these factors on the vasculature. These studies will require not only further cellular investigations to understand the regulation of smooth muscle cell contraction but, perhaps more importantly, will have to relate these findings to the function of the vasculature as an integrated whole. Only then will therapeutically useful advances be made to control vascular disease. One intriguing 0.25
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Although some effort must be made to relate the in vitro data on isolated arteries discussed in this brief review to in vivo vascular function, the mechanisms involved are likely to be similar. We know that sympathetic vasoconstrictor nervesmaintain the tone of the peripheral vasculature, and we can infer, from the vasoconstrictor effects of inhibitors of N0,21 that the endothelium exerts a tonic inhibitory influence on the vasculature. Because risk factors for vascular disease in humans are associated with hypertension and with diminished inhibitory influence of the endothelium, the effect of risk factors on the endothelium may be key to both the hypertension that results and the progression of vascular disease. Further studies to under-
0.00 5.5 mM Glucose
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FlGUREllDatashowl~thel~uenceof-glucase exposure and/or InhlbRlng nRrk ox&k (NO) on ouabalnensRlw dldlum-38 f%b) uptake, rdbtlngsodluwpotasdm adenodne trlphosphatare (Na+,K+ATPase) actlvRy In rings of the rabbR aorta. Lefk InhlbRlng ~-a@nlne (L-NMMA) NoprodwtlonwRhL-n-monomethyl for3OmlnutesInrlngs IncuMtd In 5.5 mlW (100 mg%) glucosedweasesthesodlumpumpacthfRybyapproxC matdySO%.lhe4dataonthettghtarefromrlngslncubated for3hoursIn44nH@OOmg?4)gbcose.~ebvated@ucase exposure IdbRed the sadum pulnp acthMy to an extent slmllar to that obtalned wRh L-NMMA In b.b mM glucow. Furthermore, L-NMMA had no slgnt?lcant effect In therhgsexposedtoelevated~,~esthgthat devatedglucoseand~-NMMAactbyacommonmachanlsm, presumablyvla InhbRlon oftheacUvRyofN0. *=p
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A SYMPOSIUM:
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therapeutic insight resulting from research into endothelium-dependent control of vascular smooth muscle is that nitrate therapy, whose use preceded research by decades, may amount to “replacement therapy” for diminished EDRF in diseased arteries. Bnt: The author would like to acknowledge the dedicated work of his coinvestigators and of Karen Hennessey in helping to assemble this review. REFERENCES l. Morgan KG. The role of calcium in the control of vascular tone as assessed by the Ca*+ indicator aequorin. CanliowcscDmgr l&r 1990;4:1355-1362 2. Karaki H, WeissGB. Mireview calcium release in smooth muscle.L@ Sci 1988,42111-122. 2. Nelson MT, Patlak JB, Worley JF, Standen NB. Calcium channels, potassium channels, and voltage dependenceof arterial smooth muscle tone. Am Physiol Sm 1990,9OC3-C18. 4. Furchgott RF, Vanhoutte PM. Endotheliumderived relasing and contracting factors. F&ES I 1989;3:2097-2018. 5.Twort CHC, van Breemen C. Cyclic guanosine monopbosphate-enhanced sequestration of Caz+ by sarcoplasmic reticulum in vascuku smooth muscle. Clrr Res 1988;62%1-964. Collins P, Griffith TM, Henderson AH, Lewis MJ. Endotheiiumderived relaxing factors alters calcium fluxea in rabbit aorta: a cyclic guanosinemonophosphate-mediatedeffect Jphydd (bnd) 1986;381:427437. 7. Godfmind T. EDRF and cGMP control gatmgof receptor-oIwated calcium channelsin vascular smooth muscle.Eur I Pharmccol1986;126:341-343. 8. LincoIn TM, Cornwell TL, Taylor AE. cGMPdependent protein kinase mediatesthe reduction of Car+ by CAMP in vascular smooth musclecells.Am J Physiol199@258:c399-c4tn. 9. Wakhnan SA, Murad F. Cyclic GMP synthesisand function. pharmecol Rev 1981;39:163-196. lO.Komori K, Vanhoutte PM. Endotheliumderived hyperp&rizing factor. Blbod Vmh 199@27238-as. iL Vanhoutte PM, Luscher TF, Graser T. Endothelium-dependent contractions. Bhd Vessels 1991;28:7483. 12. Bevan JA, Iaher I. Pressureand flowdependent vascular tone. FASEB I 1991;5:2267-2273. l& Burnstock G. The changing face of autonomic neurotransmissii. Acra t’h@l Stand 19%;126:67-91. l4. Bevan JA, Brayden JE. Nonadrenergic neural vasodilator mechanisms. CiJcRe.91987;60:30!&326. 16 Bult I-H, Boeclastaens GE, PelckmansPA, Jordaens FH, Van Maercke YM, Herman AG. Nitric aside as an inhibitory non-adrenergic non-chdmergic neurotransmitter.Nature 1990,345:~347. l&Cohen RA, Tesfamariam B, Weisbmd RM. The endothelium inhibits adrenergic neurotransmimion.In: Vanhoutte PM, Rubanyi GM, eds. Proceedings of the First International Symposiumon EndotheliumDerived Vasoactive Factors. New York: Karger, 1990:24l6-212. 17. Cohen RA. The role of the endothehumin vascuku adrenergicneurotransmission. In: Ryan U, Rubanyi G, eds. Endothelial Regulation of Vascular Tone. New York: Marcel Dekker, 1992:1.5~169. lK.Tesfamatiam B, Weisbmd RM, Cohen RA. The endothelium inhibits activation by calcium of vascular neurotransmimion.Am J Physic 1989;257: Hlgll-H1877. lS. Dull RO, Davies PF. Flow modulation of agonist (ATP>response (Gas+) coupling in vascular endothelial cells.Am J Php-id 1991;3OzH14~H154. ZO.Tesfamariam B, Cohen RA. Inhibition of adrenergic vasoconstriction by endothetial ceUshear stress.Circ Rer 1988,62:720-72.5. 2L Rem DD, Palmer RMJ, Moncada S. Role of endotheliumderived nitric oxide in the regulation of blood pressure.Aoc NatlAcad Sd US.4 1989;86:33753378. 22. TesfamariamB, Weisbrod RM, Cohen RA. Augmentedadrenergiccontractions of carotid arteries from cholesterol-fed rabbits due to endothelial cell dysfunction.J Canliovasc Phannacol1989;13:82&825. 2S. Cohen RA, Tesfamariam B, Weisbrod RM, Zitnay KM. Arterial denervation in rabbits with diabetesmellitus.Am J physic 1990$.59:I-I55-H61. MMinor RL Jr, Myers PR, Guerra R Jr, Bates JN, Harrison DG. DietMC
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Iwe.rr 1990,86:2109-2116. 25. Cohen RA, Tesfamariam B. Diabetes meUitusand the vascular endotheIium. In: Ruderman N, ed. Hyperglycemia, Diabetes and Vascular Disease. New York: Oxford University Press,19924449. 26. Bolotina V, OmelyanenkoV, Heyes B, Ryan U, BregeatovskiP. Variations of membranecholesterol alter the kinetics of Ca*+dependent K+ channelsand membranefluidity in vascular smooth musclecells. Q?igersArch 1989;415:262268. 27. Bolotina V, Gericke M, BregestovskiP. Kinetic differences between Ca*+dependent KC channels in smooth muscle cells isolated from normal and atherosclerotic human aorta. Pmc R Sot Lmd [Biol] 1992;90:~7-732. 28. Gupta S, SussmanI, McArthur CS, Cohen RA, Ruderman NB. Endotheiiurn-dependentinhlbition of NA+-K+ ATPase activity in rabbit aorta by hypergiy cemhx possible role of endotheliumderived nitric oxide. J C/in Invesr 1992,90: 727-732.
DISCUSSION Dr. Thomas W. Smtth (Do&on,
Massachu-
setts): Dr. Cohen, regarding the experiment in which you measured the sodium pump activity by rubidium uptake: as you know, under most circumstances the rate-limiting step in the process of potassium or rubidium uptake is the availability of sodium on the internal side of the membrane. Were those preparations sodium-loaded so that the availability of intracellular sodium was not a variable? Dr. Cohen: They were not. In fact, the data may imply that the sodium levels in the cells have been altered. That has been described in tissues in diabetic animals. Dr. Sm?th: So, it may be that the sodium pump is behaving normally, but there is a change in the sodium homeostasis? Dr. Cohen: That is true. Dr. Phlllp D. Henry (Houston, Texas): Dr. Cohen, in one of your comments, you indicated that the stimulation of endothelial cells with bradykinin did not result in the release of calcium. How do you explain that? In endothelial cells in culture (bovine, rabbit, and rat aortic), there is an increase in calcium activity (fura- method) on stimulation with bradykinin. Dr. Cohen: I did not discuss that in detail. The endothelium was producing nitric oxide (NO). In fact, we have been able to show that we can eliminate the action of endothelium-derived relaxing factor under special circumstances, and we have shown endothelium-dependent relaxation caused by bradykinin, which is inhibited by the NO synthase blockers. In the pig coronary endothelium, it appears that both vasodilator systems are active. It is evident, however, that if the production of NO is blocked, the vessel can still relax adequately by another mechanism. Dr. Peter Danz (Boston, Massachusetts): Dr. Cohen, I would like you to comment on the potassium channel recordings from human athero-
SEPTEMBER 9, 1993
sclerotic arteries. Were they disassociated cells, or was endothelium present in the areas of the recorded smooth muscle cells? Dr. Cohen: The recording was of a single channel in a patch of membrane, and so there were no endothelial cells in the vicinity. Dr. Ganz: Was there an effect of cholesterol on the smooth muscle membrane? Dr. Cohen: In the recordings there was a difference between the channel obtained from the atherosclerotic smooth muscle and that obtained from normal smooth muscle. The data showed that when cholesterol was donated to the membrane, the channel activity was inhibited in a manner similar to that in the channel derived from atherosclerotic smooth muscle. In fact, when cholesterol is removed from the membrane, one obtains the opposite effect. Dr. Ganz: What is the relative role of prostacyclin (PG12) versus NO? Some investigators have been emphasizing the importance of PGIz in the microcirculation. Also, what is the role of bradykinin in flow-mediated dilation? Endothelium releases bradykinin, which acts on the endothelium to release endothelium-derived relaxing factor and PG12,which has obvious implications for angiotensin-converting enzyme (ACE) inhibition therapy. One of the mechanisms of ACE inhibitors could be their effect on bradykinin release during shear stress-mediated dilation. Do you have any comments on that? Dr. Cohen: The size of the vesseland the species from which the vessel is obtained determine which mediator is important. Thus, under certain circumstances NO participates in the response, whereas under other circumstances it may be PGI2. I agree with you that endogenous bradykinin may activate the endothelium under physiologic conditions. Dr. Marc D. Thames (Cleveland, Ohlo): I would like to comment on the modulatory effects of sympathetic activation mediated through NO. Dr. Cohen, your data indicate that we do not see much of an impact on the vasoconstrictor responses to sympathetic activation until the frequency of the electrical stimulation reaches approximately 4 Hz. I mention that because I think it is important to recognize that in normal, resting humans the level of sympathetic activity is extremely low. Even during dynamic exercise the increases in sympa-
thetic nerve activity are very small. The conditions in which large increases in sympathetic nerve activity occur are usually pathologic states in which the endothelium does not work anyway, such as heart failure, atherosclerosis, chronic liver disease, and chronic renal disease. We need to be more cautious about how we interpret the relative roles of these different mechanismsin regulatingvasomotor tone, not only in the coronary circulation but in all circulations. Dr. Cohen: Your comments are well taken. I do not quite agree with your interpretation of the data, however. The data in Figures 3,4,7,8, and 9 show that, from OS-32 Hz, arteries with endothelium contract less than those without endothelium. It is important to realize that sympathetic activity does not occur at a constant rate in any vascular bed. It often occurs in bursts so that there are periods of silence and periods of activity. During periods of activity, the rates can be high. Therefore, it is difficult to make a firm correlation between studies involving isolated blood vessels and those that are physiologically regulated, where actual nerve activity regulates the smooth muscle. Nevertheless, the endothelium inhibits syrnpathetic vasoconstriction. Dr. l’hames: When one records from sympathetic nerves in humans under basal conditions, there is probably less than 1 Hz of resting activity. When a subject does an isometric hand grip or even dynamic muscle exercises, such as a leg exercise, the increases are very modest. That stands in contrast to pathologic states, of course. It is fine to talk about in vitro preparations and about what happens in patients when acetylcholine is injected down coronary arteries. However, we need to be careful about trying to evaluate the relative roles of the different mechanisms that regulate vascular tone because sometimes the answers we get are based on the way the experiments are designed. Dr. Cohen: That is true. As a final comment: when we inject a NO synthase inhibitor, we are preventing the production of NO. Endothelial inhibition is working under physiologic conditions because when we block its effects, sympathetic vasoconstriction increases. Therefore, regardless of the rate at which the sympathetic nerves are functioning, the endothelium is having an inhibitory effect on their action in vivo.
A SYMPOSIUM: MANAGING MYOCARDIAL ISCHEMIA
47c
The cells within the vascular wall act as a unit of smooth muscle regulating the co&action cells. In arteries the endothellum and autonomk nerves provide the major factors that regulate intmcelkdar caklum In smooth muscle cells, whkh determines wntractiletone.Thellum provldes a major InhlMtory influence, whkh Itself is modulated by shear forces wtthln the vascular lumen reguktlng endothetlal cell caklum and the release ofendothellum-derlved relaxing factors. Ofthe major mechanisms controlling smooth muscle caklum, K has been suggested that voltagedpemht calcium channels are among the most Important In medlatlng the lnhlbltory Influence of the endothellum. Smooth musde potassium channels and sodlumpotasslum adenosine trtphosphatase (Na+,K+-AlPase) are Important regulators of membrane potential, and each Is affeded by the endothellum. Because the activtty of each hyperpolarkes the membrane potential, they counter the Influence on vottagedependent caklum channels and lnhlblt contractkm. Both of these counterregulatory mechanisms have recently been shown to be impalred In diseased arteries. This may help to explain the dlminlshed etfectlveness of the endothellum on the smooth muscle. Thus, vascular disease may cause dlmlnlshed release, increased destruction, or llmtted effectiveness of emkthellum&rlved relaxing factors. The failure of the lnhlbttory lnfluence of the endothellum may be the prlnclpal mechanism by whkh vascular risk factors and disease increase vasoconstrktor tone, possiMy contrlbutlng to hypertenskn and the pm of atherosclerosis. (Am J Cardlol139~72:33C47C)
From the Vascular Biology Unit, Robert Dawson Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts. This review discusses work supported by National Institutes of Health grants HL31607, HL38731, and HL47124, as well as by an American Heart Association Established Investigator Award to Dr. Cohen. Address for reprints: Richard A. Cohen, MD, Vascular Biology Unit, E401 University Hospital, 88 E. Newton Street, Boston, Massachusetts 02118.
MD
T
he arterial wall consists of an integrated unit composed not only of vascular smooth muscle, but also of other cellular components that regulate vascular function. The endothelium bordering the luminal aspect of the blood vessel wall provides the major local control mechanism for the vascular smooth muscle, and the autonomic nerves on the adventitial aspect of the blood vessel provide the major extrinsic input. Also present are other cell types, including fibroblasts, leukocytes, platelets, and macrophages, that contribute to the structure and function of the whole. This article reviews the principal factors controlling arterial smooth muscle under physiologic and pathophysiologic conditions. COMPONEN7S
OF THE ARlERlAL
WAIL
Before considering physiologic studies of blood vessels, the major mechanisms controlling vascular smooth muscle tone will be discussed in terms of how the endothelium and autonomic nerves regulate vascular smooth muscle function. Vascular dysfunction in disease cannot be fully understood without understanding the interaction of these cells, which compose the basic vascular wall unit. Vascular smooth muscle: Figure 1 shows the mechanisms that regulate the level of intracellular free calcium, the predominant determinant of smooth muscle contraction. Free intracellular calcium initiates a cascade of events by binding to calmodulin, which is followed by myosin lightchain phosphorylation and actin-myosin crossbridge cycling, resulting in contraction.l,* Figure 1 also depicts 3 major mechanisms that increase cell calcium. Perhaps the most important physiologic regulator of calcium entry into the muscle cell is the voltage-dependent calcium channel, shown at the upper right of the smooth muscle cell. Depolarization of the cell membrane caused by endothelial or autonomic nerve-released contractile factors increases the probability of “open” calcium channels, allowing influx of the ion. Within the box to the left of the calcium channel in Figure 1 are two important physiologic voltage regulatory A SYMPOSIUM: MANAGING MYOCARDIAL ISCHEMIA
39c
FlalNEl.Dlagramsbouilngcon. trol ofvascular smooth muscle VW by endotbdlum (top) and autonomknarveendngs(lJottOflt).-d~murde cdl Is regulated by bltraceKular caklum,whkbblndstocalnoduIln, leadng to myosln ll@t chain Wylatkn(MW)=f=tln4nyosln cross-llnldng. Calcium entersthecellbytlK+voltagedependantcaklumchannelat the upper left ofthe smooth mus--b-~bWtvelyre(pllatedbydepolaiizatlonastilas Iry dv (DAD)dent protein klnase C actMy. lhe4caklumchamdrarenega~a~u--w(ATP)+kpembt sz ~(Kbda~by*~ geak Na+,K+-AlPase, whkh tend tohyperpdarlzethesnlootbmuscls membrane. Cal&m also eb izlb~~~~ caklum channels. Caklum ls also -vlareceptoroperated,G-proteiP~phollpscreCO~, In addtlom to DAD, fonnr lnosltol trlphosphate (IPJ, wldch r&as6scaklumfromtlb8sarcoplas4n@retkulum(SR).The rdeasesendotbellumderlvedhyperlntracdhdar caldum cham -b-mmby-erWthellumdewlved mlaxlng factors (EDRFs, lncludlng nltrk oxide [NOD, and embthellum-de m-(EDHF), pw&agbdln endoperoxlde [PDl&l, sqwoxlde anion [4-l, and endotheln rhfedcontractlngfactors(EDCFs,I~ [ETI). EDRFNO acth&es smooth muscle guanylate cyclase (DC) to form cyclk guanoslne monophosphate (cGMP), nervescantrolsmoothmusclebyreleaslng caklwnentryandcaklumrekase.Autonomk wbkbnegatlvdylnnuences -tine (NE), AlP, peptldes such as cakttonln g-related peptlde (CDKP), and NO.
mechanisms-potassium channels and the sodiumpotassium adenosine triphosphatase (Na+,K+ATPase) pump. The open state of different potassium channels is regulated by voltage, intracellular calcium, or intracellular ATP levels. The open potassium channel allows potassium to leave the
NTG E-
PlGlJRE2.Dlagramshowlngshear~,e1+ dothehm-derhfed nlhlc oxide (NO) production from arglnine by NO syntbase (NOS), NO releew, and action on vascularsmoothmusck.Shearstresslsshownregulating endothebl cdl caklum by ragulathg a caklum charnel. The~edmechanlsmbywldchhormones suds as ac&ylhMne regulate endothellal cell caklum Is alsoshown.NOInsmoothmusclelsshownrMhtbu?guapilate4(chMP)hnlgua~trlpl;orphate(DlP).For otberabkevbtlons,seePlgurel. mc
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
cell on a steep chemical gradient and results in hyperpolarization of the cell membrane, which thus tends to close the calcium channel.3 Activity of the sodium pump is “electrogenic,” also resulting in membrane hyperpolarization. Thus, the activity of these 2 mechanisms counterregulates the voltagedependent calcium channel. Of the 3 mechanisms responsible for increasing intracellular calcium, 2 are activated by cell membrane receptors stimulated by endothelial or neural vasoactive factors. One type of receptor causes G-protein-linked activation of phospholipase C, resulting in the formation of inositol triphosphates, whose principal action is to release calcium stores from the sarcoplasmic reticulum. A byproduct, diacylglycerol, has additional important cellular functions by activating protein kinase C, including stimulation of voltage-dependent calcium channels. Another type of receptor is directly linked by G-proteins to calcium channels. Not shown in Figure 1 are the mechanisms that lower intracellular calcium, such as pumps that extrude calcium from the cell and the reuptake of calcium into the sarcoplasmic reticulum. Endothelium: The endothelium regulates smooth muscle function in several ways, including the release of vasoactive factors. Endothelial cell calcium regulates the activity of nitric oxide (NO) SEPTEMBER 9, 1993
synthase, which metabolizes L-arginine to NO (Figures 1 and 2), the most important endotheliumderived relaxing factor (EDRF).4 After diffusing to the smooth muscle, NO exerts its effects by stimulating guanylate cyclase-catalyzed production of cyclic guanosine monophosphate (GMP). Cyclic GMP down-regulates the contractile process by negatively influencing calcium release from the sarcoplasmic reticulum and its entry through calcium channels, as well as by inhibiting the calciumactivated contractile process itself.5-x It is important to understand the mechanisms by which NO relaxes vascular smooth muscle if one is to understand the mechanisms of all nitrovasodilators, because they all break down to release NO.” The endothelium releases additional important factors that are less well described. Endotheliumderived hyperpolarizing factor is thought to activate smooth muscle potassium channels, thereby inhibiting voltage-dependent calcium channels and contraction.1° Endothelium-derived hyperpolarizing factor is thus another EDRF. Endotheliumderived contracting factors include prostaglandin endoperoxides, endothelin, and superoxide radicals.ll Prostaglandin endoperoxides and endothelin activate smooth muscle receptors. The action of superoxide radical is poorly understood, but it may cause contraction by interfering with the action of NO. Although most of the experimental evidence for the actions of these factors has resulted from stimulating endothelial cells with hormones and neurotransmitters, such as acetylcholine and bradykinin, the likely principal physiologic regulator is the variation of shear stress at the endothelial cell surface, which has been shown to regulate endothelial cell calcium levels. l2 Autonomic nerves: Figure 1 also shows several neurotransmitters, released by vascular nerves, that regulate vascular smooth muscle.” The classic
sympathetic transmitter, norepinephrine, contracts most vascular smooth muscle by activating (Yadrenoceptors. An important exception is coronary artery smooth muscle, which norepinephrine relaxes by activating B adrenoceptors. ATP is a cotransmitter with norepinephrine and contracts smooth muscle in some blood vessels. Peptidergic nerves innervate certain blood vessels; calcitonin gene-related peptide is one of several neuropeptides that regulate vascular smooth muscle.14 NO, which is also produced within vascular nerve endings, has been suggested to be a nonadrenergic, noncholinergic, vasodilatory neurotransmitter.15 FUNCTION OF THE VASCULAR WALL UNIT
To discuss factors that are important in controlling smooth muscle as the muscle functions within the vascular wall unit that it shares with endothelium and nerve, this review will focus on the rabbit carotid artery.16J7This artery is a typical peripheral artery with cy-adrenergic sympathetic innervation. Figure 3 shows that when the nerves of the isolated artery are stimulated, the stimulation of 011adrenoceptors by neurally released norepinephrine causes contractions.17 The endothelium inhibits the contractions primarily as a result of the continuous release of endothelium-derived NO. The endothelium thereby provides a tonic counterregulatory influence to neurogenic vasoconstriction, which is a principal physiologic means whereby constrictor tone of the peripheral vasculature is regulated. Myogenic tone, important in resistance vessels, is also tonically inhibited by the endothelium. Because the failure of the endothelium to inhibit the contraction of smooth muscle is a primary characteristic of diseased blood vessels, the remainder of this review will discuss the mechanisms that may be important in the inhibitory influence of the endothelium on vasoconstriction.
__ ------FlGURE 3. Recordlr@s of lsometrktenslonof2MgsofrabbRcarotld arteq wRh endothellum ENDl;;Iyfi / ((top) and denuded emlothellum (bottom) demonstrating the InhlbRory effect ofthe endothellum on w neurotransmlsslon. lhe rbqgs are first co&acted with noreplnephrlne (NE), and aaetylDENUDED ;It..,r’l-ir 5gLn cholhw (ACH) Is addsd to show that ths rlqg wtth endothellum 0.5 t relaxes,whereastherhlgwlthout E 8 QNP K+lZOmM endothedlumdoesnotrelax.The AWlog M FREQUENCY.Hz I--.--__ sympathetk adrenergk nerves In the artedal Mgs are then sthnulatedekctronkallyat lncread~wqu8dss, revealing that the artery wRh endothellum contracts less than the one wRhout edothellum. The artedes are then contracted euuall~ by deuolarlzlng wtth elevated potassium (K++).Concentratlons are @ven as the negattve IogarRhm of the molar coke&k (-log M)or mlllhnolar (d). SNP =‘saklum nRroprusside. (ReprInted with permlsslon from Cohen.9 A SYMPOSIUM:
MANAGING
MYOCARDIAL
ISCHEMIA
41c
abrogated.‘* The channel activator was used under conditions in which it had little influence on L CONTROL . o -A BAY K 6644 . 60. arteries denuded of endothelium, suggesting that its inhibition of endothelial cell action was specific. Further, voltage-dependent calcium channels are thought to be absent from endothelial cells, indicating that the drug’s site of action is smooth muscle. Although the results of these studies await electrophysiologic confirmation, they suggestthat smooth muscle voltage-dependent calcium channels are tonically inhibited by the endothelium. 1 2 4 a FREQUENCY. Hz Shear stress: Under physiologic conditions, the release of NO is regulated by shear forces on PlOURE4.3ummaydataobtahdbythemethodds endothelial cells. This is thought to occur by way of scrbedlnFigure3showlngthelnhbltofyinRuenceofthe &dothe3umofrabbRcarotldartwksandthedfectof the shear-dependent influx of extracellular calcium voe baliSum channel acthmtlon by RAY K contributing to calcium levels in the cell, which in turn regulates NO synthase activity (Figure 2).19In perfused rabbit carotid arteries, increases in shear stress caused by elevated viscosity of the bathing ( - Tat all Weqwdwklnthepresenceofthev~ solution releases EDRF from the endothelium c&lumchannelacthkt6rRAYK3644,thecoWadhsd (Figure 5).20Thus, this shear-dependent release of artwleswRhorwRhout edoth@umareslmllartothose EDRF tonically inhibits neurogenic vasoconstricofartudeswRho4R edothdlumIntheabsenceof~heactlvator.(ReprlnteawRh --~Jphydol.9 tion (Figure 6). The shear-dependent release of EDRF is likely an important regulator of vascular Vdtage-clependent caklum dfannek It is tone in vivo, as demonstrated by inhibitors of NO widely recognized that the basal release of NO infused in animals and humans.21Thus, endotheinhibits smooth muscle by increasing cyclic GMP lium and nerves normally counterregulate vascular levels, and pharmacologic studies have indicated smooth muscle continuously in vivo. that voltage-dependent calcium channels are probably modulated by cyclic GMP.6 Figure 4 shows VASCULAR RISK FACTORS AND DISEASE that when the activation of voltage-dependent A common feature of vascular diseaseis dysfunccalcium channels is enhanced in the rabbit carotid tion of the endothelium early in the course of the artery by the specific channel activator BAY K disease. Endothelial cell dysfunction has been 8644, the inhibitory influence of the endothelium is demonstrated in animals and in patients with the 7.
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THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
SEPTEMBER 9, 1993
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may be important in initiating atherosclerosis. The inhibitory influence of the endothelium on neurogenie vasoconstriction disappears rapidly in rabbits with hypercholesterolemia, diabetes, or hyperten-
major risk factors for atherosclerosis (i.e., hypercholesterolemia, diabetes mellitus, and hypertension). Because such dysfunction may occur even before the presence of any discernible atherosclerosis, it
RWRE 8. Data showim! that dlahlMtorylnfluenceoftheendothe llum on WC contractions oftherabhltcarotldartely.m carotldatiefyrlngswltheubthellumfronlrabbltsmmledlabetk for8weekrwlthalloxamc
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A SYMPOSIUM: MANAGING MYOCARDIAL ISCHEMIA
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sion. In the carotid artery, this may occur even before endothelium-dependent relaxations stimulated by agents such as acetylcholine have become abnormal, implying the importance of the failure of the tonic inhibitory mechanism as an early event in the pathogenesis of vascular disease. mlemla: In rabbits fed a 2% cholesterol diet for 13-15 weeks, the plasma cholesterol was elevated to >2,000 mg/dL, but in the carotid arteries, the endothelium-dependent relaxation to acetylcholine did not differ from that in normal rabbits.22However, the inhibitory influence of the endothelium on neurogenic contractions was nearly absent (Figure 7). d4C
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
Diabetes mellttus: The diabetogenic agent alloxan was used to increase blood glucose levels in rabbits to approximately 350 mg/dL for 6 weeks.23 In the carotid artery of these rabbits, the acetylcholine relaxation was normal, but the inhibition of neurogenic contraction by the endothelium was also nearly absent (Figure 8). Hypertension: A few observations in Watanabe heritable hyperlipidemic rabbits suggestthat hypertension may also cause a deterioration in endothelial cell inhibition of neurogenic contractions. Compared with those of normotensive New Zealand white rabbits, carotid arteries of 12-month-old Watanabe rabbits demonstrated normal endothelial cell function. Compared with rabbits fed cholestero1,22 the Watanabe rabbits had much lower plasma cholesterol levels (400-700 mg/dL), possibly explaining why endothelial function was normal. However, in Watanabe rabbits made hypertensive for 1 month, endothelial cell inhibition was decreased (Figure 9). Potential mechanisms: Endothelial cell dysfunction in response to risk factors can occur in severalways. For instance, the production or effectiveness of EDRFs can decrease. Recent studies suggest that decreased effectiveness may be more important than decreased release of EDRFs in both hypercholesterolemia24 and diabetes.25 Decreased effectiveness may result from increased destruction of EDRFs, increased production of counteracting endothelium-derived contracting factors, or altered responsivenessof the smooth muscle to EDRF. Vascular disease may alter the responsiveness of the smooth muscle to the endothelium through 2 recently discoveredand potentially important mechanisms. Both mechanisms suggest an impaired counterregulation of smooth muscle voltage-activated calcium channels by EDRFs. It was recently demonstrated that the addition of cholesterol to smooth muscle membranes can inhibit the function of calcium-activated potassium channels in just a few hours.26Since these studies were performed on single channels in isolated membrane patches, the effect may have been due to a fundamental molecular interaction between cholesterol and the channels in the cell membrane. The function of calcium-activated potassium channels in cells from atherosclerotic human aorta (which also have increased membrane cholesterol) are also inhibited compared with those from nonatherosclerotic aorta (Figure 1O).27Insofar as the impaired activity of potassium channels depolarizes the smooth muscle cells, voltage-dependent
SEPTEMBER 9, 1993
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calcium channels may tend to be more active in cells with increased membrane cholesterol, and, as a result, the endothelial cell inhibitory influence would be lessened. In arteries of diabetic patients, the activity of Na+,K+-ATPase is impaired,28 making it more likely that the artery is relatively depolarized, which would similarly limit endothelial cell inhibition by increasing the activity of voltage-dependent calcium channels. Studies have also shown that elevated glucose exposure inhibits the vascular sodium pump of isolated rabbit arteries in as little as 3 hours (Figure 11).28 A similar degree of inhibition of the sodium pump was also observed after NO production was pharmacologically blocked (Figure 11). These data suggest that elevated glucose prevents endothelial cell-derived NO from stimulating the sodium pump. Although the means by which NO influences the sodium pump is not yet known, the inhibitory action of glucose may explain the augmented vascular contractions of diabetic arteries shown in Figure 8.
stand the mechanisms by which vascular risk factors interfere with the physiologic function of the endothelium may lead to the development of adjunctive treatments to ameliorate the deleterious effects of these factors on the vasculature. These studies will require not only further cellular investigations to understand the regulation of smooth muscle cell contraction but, perhaps more importantly, will have to relate these findings to the function of the vasculature as an integrated whole. Only then will therapeutically useful advances be made to control vascular disease. One intriguing 0.25
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Although some effort must be made to relate the in vitro data on isolated arteries discussed in this brief review to in vivo vascular function, the mechanisms involved are likely to be similar. We know that sympathetic vasoconstrictor nervesmaintain the tone of the peripheral vasculature, and we can infer, from the vasoconstrictor effects of inhibitors of N0,21 that the endothelium exerts a tonic inhibitory influence on the vasculature. Because risk factors for vascular disease in humans are associated with hypertension and with diminished inhibitory influence of the endothelium, the effect of risk factors on the endothelium may be key to both the hypertension that results and the progression of vascular disease. Further studies to under-
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A SYMPOSIUM:
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therapeutic insight resulting from research into endothelium-dependent control of vascular smooth muscle is that nitrate therapy, whose use preceded research by decades, may amount to “replacement therapy” for diminished EDRF in diseased arteries. Bnt: The author would like to acknowledge the dedicated work of his coinvestigators and of Karen Hennessey in helping to assemble this review. REFERENCES l. Morgan KG. The role of calcium in the control of vascular tone as assessed by the Ca*+ indicator aequorin. CanliowcscDmgr l&r 1990;4:1355-1362 2. Karaki H, WeissGB. Mireview calcium release in smooth muscle.L@ Sci 1988,42111-122. 2. Nelson MT, Patlak JB, Worley JF, Standen NB. Calcium channels, potassium channels, and voltage dependenceof arterial smooth muscle tone. Am Physiol Sm 1990,9OC3-C18. 4. Furchgott RF, Vanhoutte PM. Endotheliumderived relasing and contracting factors. F&ES I 1989;3:2097-2018. 5.Twort CHC, van Breemen C. Cyclic guanosine monopbosphate-enhanced sequestration of Caz+ by sarcoplasmic reticulum in vascuku smooth muscle. Clrr Res 1988;62%1-964. Collins P, Griffith TM, Henderson AH, Lewis MJ. Endotheiiumderived relaxing factors alters calcium fluxea in rabbit aorta: a cyclic guanosinemonophosphate-mediatedeffect Jphydd (bnd) 1986;381:427437. 7. Godfmind T. EDRF and cGMP control gatmgof receptor-oIwated calcium channelsin vascular smooth muscle.Eur I Pharmccol1986;126:341-343. 8. LincoIn TM, Cornwell TL, Taylor AE. cGMPdependent protein kinase mediatesthe reduction of Car+ by CAMP in vascular smooth musclecells.Am J Physiol199@258:c399-c4tn. 9. Wakhnan SA, Murad F. Cyclic GMP synthesisand function. pharmecol Rev 1981;39:163-196. lO.Komori K, Vanhoutte PM. Endotheliumderived hyperp&rizing factor. Blbod Vmh 199@27238-as. iL Vanhoutte PM, Luscher TF, Graser T. Endothelium-dependent contractions. Bhd Vessels 1991;28:7483. 12. Bevan JA, Iaher I. Pressureand flowdependent vascular tone. FASEB I 1991;5:2267-2273. l& Burnstock G. The changing face of autonomic neurotransmissii. Acra t’h@l Stand 19%;126:67-91. l4. Bevan JA, Brayden JE. Nonadrenergic neural vasodilator mechanisms. CiJcRe.91987;60:30!&326. 16 Bult I-H, Boeclastaens GE, PelckmansPA, Jordaens FH, Van Maercke YM, Herman AG. Nitric aside as an inhibitory non-adrenergic non-chdmergic neurotransmitter.Nature 1990,345:~347. l&Cohen RA, Tesfamariam B, Weisbmd RM. The endothelium inhibits adrenergic neurotransmimion.In: Vanhoutte PM, Rubanyi GM, eds. Proceedings of the First International Symposiumon EndotheliumDerived Vasoactive Factors. New York: Karger, 1990:24l6-212. 17. Cohen RA. The role of the endothehumin vascuku adrenergicneurotransmission. In: Ryan U, Rubanyi G, eds. Endothelial Regulation of Vascular Tone. New York: Marcel Dekker, 1992:1.5~169. lK.Tesfamatiam B, Weisbmd RM, Cohen RA. The endothelium inhibits activation by calcium of vascular neurotransmimion.Am J Physic 1989;257: Hlgll-H1877. lS. Dull RO, Davies PF. Flow modulation of agonist (ATP>response (Gas+) coupling in vascular endothelial cells.Am J Php-id 1991;3OzH14~H154. ZO.Tesfamariam B, Cohen RA. Inhibition of adrenergic vasoconstriction by endothetial ceUshear stress.Circ Rer 1988,62:720-72.5. 2L Rem DD, Palmer RMJ, Moncada S. Role of endotheliumderived nitric oxide in the regulation of blood pressure.Aoc NatlAcad Sd US.4 1989;86:33753378. 22. TesfamariamB, Weisbrod RM, Cohen RA. Augmentedadrenergiccontractions of carotid arteries from cholesterol-fed rabbits due to endothelial cell dysfunction.J Canliovasc Phannacol1989;13:82&825. 2S. Cohen RA, Tesfamariam B, Weisbrod RM, Zitnay KM. Arterial denervation in rabbits with diabetesmellitus.Am J physic 1990$.59:I-I55-H61. MMinor RL Jr, Myers PR, Guerra R Jr, Bates JN, Harrison DG. DietMC
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 72
induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Iwe.rr 1990,86:2109-2116. 25. Cohen RA, Tesfamariam B. Diabetes meUitusand the vascular endotheIium. In: Ruderman N, ed. Hyperglycemia, Diabetes and Vascular Disease. New York: Oxford University Press,19924449. 26. Bolotina V, OmelyanenkoV, Heyes B, Ryan U, BregeatovskiP. Variations of membranecholesterol alter the kinetics of Ca*+dependent K+ channelsand membranefluidity in vascular smooth musclecells. Q?igersArch 1989;415:262268. 27. Bolotina V, Gericke M, BregestovskiP. Kinetic differences between Ca*+dependent KC channels in smooth muscle cells isolated from normal and atherosclerotic human aorta. Pmc R Sot Lmd [Biol] 1992;90:~7-732. 28. Gupta S, SussmanI, McArthur CS, Cohen RA, Ruderman NB. Endotheiiurn-dependentinhlbition of NA+-K+ ATPase activity in rabbit aorta by hypergiy cemhx possible role of endotheliumderived nitric oxide. J C/in Invesr 1992,90: 727-732.
DISCUSSION Dr. Thomas W. Smtth (Do&on,
Massachu-
setts): Dr. Cohen, regarding the experiment in which you measured the sodium pump activity by rubidium uptake: as you know, under most circumstances the rate-limiting step in the process of potassium or rubidium uptake is the availability of sodium on the internal side of the membrane. Were those preparations sodium-loaded so that the availability of intracellular sodium was not a variable? Dr. Cohen: They were not. In fact, the data may imply that the sodium levels in the cells have been altered. That has been described in tissues in diabetic animals. Dr. Sm?th: So, it may be that the sodium pump is behaving normally, but there is a change in the sodium homeostasis? Dr. Cohen: That is true. Dr. Phlllp D. Henry (Houston, Texas): Dr. Cohen, in one of your comments, you indicated that the stimulation of endothelial cells with bradykinin did not result in the release of calcium. How do you explain that? In endothelial cells in culture (bovine, rabbit, and rat aortic), there is an increase in calcium activity (fura- method) on stimulation with bradykinin. Dr. Cohen: I did not discuss that in detail. The endothelium was producing nitric oxide (NO). In fact, we have been able to show that we can eliminate the action of endothelium-derived relaxing factor under special circumstances, and we have shown endothelium-dependent relaxation caused by bradykinin, which is inhibited by the NO synthase blockers. In the pig coronary endothelium, it appears that both vasodilator systems are active. It is evident, however, that if the production of NO is blocked, the vessel can still relax adequately by another mechanism. Dr. Peter Danz (Boston, Massachusetts): Dr. Cohen, I would like you to comment on the potassium channel recordings from human athero-
SEPTEMBER 9, 1993
sclerotic arteries. Were they disassociated cells, or was endothelium present in the areas of the recorded smooth muscle cells? Dr. Cohen: The recording was of a single channel in a patch of membrane, and so there were no endothelial cells in the vicinity. Dr. Ganz: Was there an effect of cholesterol on the smooth muscle membrane? Dr. Cohen: In the recordings there was a difference between the channel obtained from the atherosclerotic smooth muscle and that obtained from normal smooth muscle. The data showed that when cholesterol was donated to the membrane, the channel activity was inhibited in a manner similar to that in the channel derived from atherosclerotic smooth muscle. In fact, when cholesterol is removed from the membrane, one obtains the opposite effect. Dr. Ganz: What is the relative role of prostacyclin (PG12) versus NO? Some investigators have been emphasizing the importance of PGIz in the microcirculation. Also, what is the role of bradykinin in flow-mediated dilation? Endothelium releases bradykinin, which acts on the endothelium to release endothelium-derived relaxing factor and PG12,which has obvious implications for angiotensin-converting enzyme (ACE) inhibition therapy. One of the mechanisms of ACE inhibitors could be their effect on bradykinin release during shear stress-mediated dilation. Do you have any comments on that? Dr. Cohen: The size of the vesseland the species from which the vessel is obtained determine which mediator is important. Thus, under certain circumstances NO participates in the response, whereas under other circumstances it may be PGI2. I agree with you that endogenous bradykinin may activate the endothelium under physiologic conditions. Dr. Marc D. Thames (Cleveland, Ohlo): I would like to comment on the modulatory effects of sympathetic activation mediated through NO. Dr. Cohen, your data indicate that we do not see much of an impact on the vasoconstrictor responses to sympathetic activation until the frequency of the electrical stimulation reaches approximately 4 Hz. I mention that because I think it is important to recognize that in normal, resting humans the level of sympathetic activity is extremely low. Even during dynamic exercise the increases in sympa-
thetic nerve activity are very small. The conditions in which large increases in sympathetic nerve activity occur are usually pathologic states in which the endothelium does not work anyway, such as heart failure, atherosclerosis, chronic liver disease, and chronic renal disease. We need to be more cautious about how we interpret the relative roles of these different mechanismsin regulatingvasomotor tone, not only in the coronary circulation but in all circulations. Dr. Cohen: Your comments are well taken. I do not quite agree with your interpretation of the data, however. The data in Figures 3,4,7,8, and 9 show that, from OS-32 Hz, arteries with endothelium contract less than those without endothelium. It is important to realize that sympathetic activity does not occur at a constant rate in any vascular bed. It often occurs in bursts so that there are periods of silence and periods of activity. During periods of activity, the rates can be high. Therefore, it is difficult to make a firm correlation between studies involving isolated blood vessels and those that are physiologically regulated, where actual nerve activity regulates the smooth muscle. Nevertheless, the endothelium inhibits syrnpathetic vasoconstriction. Dr. l’hames: When one records from sympathetic nerves in humans under basal conditions, there is probably less than 1 Hz of resting activity. When a subject does an isometric hand grip or even dynamic muscle exercises, such as a leg exercise, the increases are very modest. That stands in contrast to pathologic states, of course. It is fine to talk about in vitro preparations and about what happens in patients when acetylcholine is injected down coronary arteries. However, we need to be careful about trying to evaluate the relative roles of the different mechanisms that regulate vascular tone because sometimes the answers we get are based on the way the experiments are designed. Dr. Cohen: That is true. As a final comment: when we inject a NO synthase inhibitor, we are preventing the production of NO. Endothelial inhibition is working under physiologic conditions because when we block its effects, sympathetic vasoconstriction increases. Therefore, regardless of the rate at which the sympathetic nerves are functioning, the endothelium is having an inhibitory effect on their action in vivo.
A SYMPOSIUM: MANAGING MYOCARDIAL ISCHEMIA
47c