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1167 lA CC Vol. 9. NO. 5 May 1987:1167- 75 BASIC CONCEPTS IN CARDIOLOGY Arnold M. Katz, MD, FACC, Gu est Editor Regulation of Coronary Artery Tone in Relation to the Activation of Signal Transductors That Regulate Calcium Homeostasis TOSHIYUKI SASAGURI, MD, TAKEO ITOH, PHD ,* MASATO HIRATA, DO, KENJI KITAMURA, DO, HIROSI KURIYAMA, MD Fukuoka, Japan Muscle tone of coronary arteries is regulated by free calcium concentration in the myoplasm. Various agonists, autacoids and putative peptides modify the calcium concentration directl y or through actions of second messengers (signal transductors), such as cyclic guanosine monophosphate (GMP), cyclic adenosine monophosphate (AMP), inositol 1,4,5·trisphosphate, diacylglycerol or calmodulin. For example , acetylcholine (in the presence of intact endothelium cells), alpha-human natriuretic peptide or nitrate compounds increase the amount of cyclic GMP and isoproterenol, prostacyclin (prostaglandin 12 ) or vasoactive intestinal polypeptide increases cyclic AMP. Both cyclic nucleotides reduce free calcium Calcium Homeostasis in Vascular Smooth Muscles in Relation to the ContractionRelaxation Cycle The contraction-relaxation cycle in vasc ular smooth muscle is regulated by changes in free myoplasmic ca lcium concentration . Increa sing calciu m concentration up to 0 . 1 J-tM activa tes the contrac tile mach inary whereas lower ing conce ntration relaxes the tissue ( 1,2). Th e exact level of myoplasm ic calcium needed to activa te vasc ular smoo th muscle depends on the calci um sensitivity of the co ntrac tile protein s (3) . M ech anisms of increased intracellular calcium. Free myoplasm ic ca lcium con cent ration ca n be increased by calcium influx across the plasma membrane or by release of Fro m the Depart ment of Pharmacology, Faculty of Medici ne and the *Departmenl of Bioche mistry , Facu lty of Dent istry, Kyushu University . Fuku oka. Japan . Manuscript received August 4. 1986 : revised manusc ript rece ived Septembe r 30 . 1986. accepted October 7 . 1986. Address for reprints: Hiro si Kuriyama . MD. Departme nt of Pharmaco logy . Facu lty of Medicine. Kyushu Univers ity . Fukuoka 812. Jap an . © 1987 by the American College of Cardiology concentration. On the other hand , acetylcholine (in the presence or absence of endothelium cells), norepinephrine or thromboxane A2 increases inositol 1,4,5-tris phosphate and diacylglycerol, thus causing the increase in the free calcium concentration, whereas vasoactive intestinal peptide and alpha-human natriuretic peptid e reduce them. Calmodulin acts as an internal calcium receptor for regulation of the contractile machinary. Regulation of calcium homeostasis in relation to the muscle tone in the coronary arteries including other vascular tissues is discussed together with the role of second messengers. (J Am Coil Cardiol1987,'9:Jl67-75) calcium fro m intracellular storage sites, mainly the sarcoplasmic reticulum (4), or both . At present, there appear to be four basic mechanisms by which intracellular calcium concentration can be increased : 1) Passive calcium influx . which involves simple diffusion of calci um across the plasma membrane throu gh a conce ntratio n gradient; the extracellular ca lcium concentration of I to 2 mM is much higher than the intracellul ar calcium co ncentration, which is in the micromolar range . 2) Calcium influx through voltage-dependent plasma membrane channels that open in respon se to membrane This article is part of a series of inf ormal teaching reviews devoted to subjects in basic cardiology that are of particular interest because oftheir high potential f or clinical application . The series is edited by Arnold M . Katz. MD. FACC. a leading proponent of the view that basic science can be presented in a clear and stimulating fa shion. The intent of the series is to help the clinician keep abreast of important advances in our understanding ofthe basic mechanisms underlying normal and abnormal cardiac function. 0735-109 7/87/$3.50 1168 lACC Vol. 9. No.5 SASAGURI ET AL. CORONARY TONE AND SIGNALTRANSDUCTORS depolarization or generation of an action potential. In some vascular tissues (for example, the myogenic and resistance vessels), action potentials resulting from calcium influx can be evoked by nerve stimulationunder physiologic conditions (5,6). 3) Calcium influx through receptor-operated plasma membrane channels that open in response to the interaction of agonists with specific receptors. Such interactions may also increase membrane sodium permeability, therebycausing membrane depolarization, which initiates a voltage-dependent calcium influx. 4) Intracellular calcium release. Agonist-induced receptor activation may also trigger calcium release from the sarcoplasmic reticulum. In skeletal and cardiac muscles, calcium release from the sarcoplasmic reticulum is thought to be initiated by activation of voltage-dependent calcium release and calcium-induced calcium release mechanisms (7,8). A voltage-dependent calcium release in smooth muscle has not been clearly shown, but receptor-operated and calcium-induced calcium release mechanisms seem to play a definite role (4,9). Reduction in free myoplasmic calcium. Smooth muscle relaxation is brought about by a reduction in free myoplasmic calcium concentrationcaused largely by an active, adenosine triphosphate (ATP)-dependent,calciumextrusion mechanism (calcium pump) associated with the activation of a calcium-adenosine triphosphatase (ATPase) (10). This is in contrast to the sodium-calcium exchange diffusion mechanism thought to be more important than the calcium pump for reducing free myoplasmic calcium concentration in cardiac muscle. The sodium-calcium exchanger utilizes the potential energy produced by sodium influx down its concentration gradient rather than energy produced by ATP hydrolysis (11,12). In cardiac and smooth muscles, myoplasmic calcium is also reduced by the reuptakeof calcium intocellular stores, mainly the sarcoplasmic reticulum, through an ATP-dependent calcium transport mechanism (13). Drugs that influence calcium movements. A wide variety of drugs influence the calcium movements that regulate the contraction-relaxation cycle in smooth muscle. For example, voltage-dependent calcium influx can be increased by the calcium "agonists" Bay K-8644, YC-170 or CGP 28392 and can be inhibited by calcium channel blockers like nifedipine, verapamil and diltiazem (14,15). Receptoroperated calcium influx can also be activated by agonists that bind to several receptors, such as adrenergic agonists, and inhibited by the corresponding antagonists. The release of calcium from smooth muscle sarcoplasmic reticulum is accelerated by a newly discovered intracellular messenger, inositol I ,4,5-trisphosphate (16) as well as by caffeine, and can be inhibited by procaine. A number of signal transductors (second messengers) are involved in calcium homeostasis, including calcium, cyclic nucleotides and inositol trisphosphate, which mediate the actions of coronary May 1987:1167-75 vasoconstrictors and vasodilators through actions of, for example, alpha- and beta-adrenoceptor agonists and nitrates. This article describes the regulation of coronary artery tone through the actions of signal transductors that regulate calcium homeostasis. Regulation of Coronary Artery Tone What triggers contraction in vascular smooth muscle? (Fig. 1). Activation of the contractile proteins in vascular smooth muscle is thought to begin when calcium binds to the regulator protein, calmodulin. The binding of four calcium ions to calmodulin induces a conformational change in the calmodulin molecule that allows the calcium-calmodulin complex to interact with the inactive apoenzyme of an enzyme called myosin light chain kinase. The active ternary complex: calcium.-calmodulin-myosin light chain kinase formed in this reaction then catalyzes the phosphorylation of the smooth muscle myosin light chain. Phosphorylation of myosin light chain enables the myosin magnesium-A'TPase enzyme to interact with actin, resulting in cross-bridge formation and contraction. Reduction in myoplasmic free calcium concentration by any of the mechanisms described here dissociates the calcium-calmodulin complex, which in turn dissociates calmodulinfrom myosin light chain kinase, thereby regenerating the inactive apoenzyme of the latter enzyme. As a result, myosin phosphorylation ceases, myosin light chain is dephosphorylated by a myosin light chain phosphatase and the muscle relaxes (3,17-19). Although most investigations support the view that myosin phosporylation is the first step in smooth musclecontraction, Figure 1. Regulation of contractile proteins in vascular smooth muscles. ADP = adenosine diphosphate; ATP = adenosine triphosphate; Ca = calcium; MLCK = myosin light chain kinase; myosin-® = phosphorylated myosin; Pi = inorganic phosphate. calmodulin + !f Ca2 + 4 - MLCK (inactive) - calmodulin .0.. MLCK (active) {} ATP actin + ADP >-< myosin myosin-® + actin P~ if I n actin - myosin- ® phosphatase relaxation c- - - - - - - - - - - - -~ contraction JACC Vol. 'I. NO.5 May l'Ig7:1I67-75 some unsolved problems remain. For example, when potassium chloride or an agonist is applied to smooth muscle for long periods of time, sustained contractions are seen despite marked reduction of myosin light chain phosphorylation. This finding may reflect a second calcium-dependent regulatory mechanism that enables nonphosphorylated myosin cross bridges to maintain the interactions between myosin and actin, and so sustain contraction. These nonphosphorylated myosin cross bridges exhibit slow crossbridge cyclings that have been termed" latch bridges" ( 17). These findings, therefore, suggest the existence of a dual activation mechanism in which increased free calcium concentration first activates myosin light chain kinase, whieh phosphorylates myosin and leads to rapid cross-bridge cycling. Myoplasmic calcium concentration then declines to a value intermediate between the peak and rest levels, at which myosin light chain kinase is inactivated and myosin light chain-phosphatase dephosphorylates myosin. However, contraction is maintained, possibly by a second calcium-dependent regulatory mechanism having a higher calcium sensitivity than that of the myosin light chain kinase system. Recent measurements of myoplasmic free calcium concentration indicate that calcium levels remain high during a potassium-induced contraction, but decline rapidly after the appearance of a transient peak in contraction induced by agonists or caffeine (20,21). However, the detailed relation between free calcium concentration and the force of contraction remains to be clarified. How do nitrates relax coronary vascular tissue? Nitroglycerin, sodium nitroprusside and isosorbide dinitrate are well known to dilate vascular smooth muscle. Nitrates are more potent in relaxing venous than arterial strips (22); these observations have been confirmed using blood flow measurements in vivo (23). Angiographic observations support the view that nitroglycerin also causes vasodilation of spastic regions in experimental animals and patients (24,25). Nitrates relax coronary artery strips precontracted by voltage-dependent (high potassium concentrations) or receptor-operated calcium influx (norepinephrine or acetylcholine) (5,26,27). Because the causes of angina pectoris are multifactorial, such nonselective relaxation may be of more value in alleviating anginal pain than that caused by more selective drugs such as calcium channel blockers that act principally on the voltage-dependentcalcium influx. The nitrates apparently do not act on receptors or calcium channels at the surface of the myoplasmic membrane, and they do not act directly on the sarcoplasmic reticulum of "skinned" smooth muscles prepared by disrupting the myoplasmic membrane with detergents (4). Nitrates do not appear to act directly on the myoplasmic membrane, but instead may modify the production of substances that regulate myoplasmic calcium homeostasis. One such substance is cyclic guanosine monophosphate (GMP), whose concentration in vascular tissues is increased by nitrates. Exogenously ap- SASAGURI ET AL. CORONARY TONE AND SIGNALTRANSDUCTORS 1169 plied dibutyryl cyclic GMP, a permeable derivative of cyclic GMP, has much the same action on intact muscle tissues as does nitroglycerin (27). However, nitrates do not modify the amount of cyclic adenosine monophosphate(AMP) (27), the metabolism of phosphatidyl inositol 4,5-bisphosphate or the amounts of diacylglycerol and inositol trisphosphate in vascular smooth muscles (27-29). Role of cyclic GMP in relation to smooth muscle relaxation. Increasedcyclic GMP concentrations in such vascular smooth muscles as coronary arteries activates a cyclic GMP-dependent protein kinase (G-kinase) that catalyzes the phosphorylation of several proteins. This, in turn, leads to relaxation of precontracted smooth muscle by the following mechanisms: I) Cyclic GMP activates a G-kinase that phosphorylates myosin light chain kinase, thereby inhibiting the phosphorylation of myosin light chain induced by the calcium-calmodulin-myosin light chain kinase complex (27,30,31). 2) The G-kinase activated by cyclic GMP through another phosphorylation reaction accelerates the calcium pump of the smooth muscle plasma membrane, thereby leading to a reduction in myoplasmic free calcium concentration (32).3) Although G-kinase activated by cyclic GMP does not appear to modify calcium accumulation into sarcoplasmic reticulum storage sites, it may slightly inhibit calcium release from them (1,27). These observations indicate that the activation of G-kinase by cyclic GMP produced in response to the nitrates reduces myoplasmic free calcium concentration, directly inhibits the contractile proteins and, perhaps most important, accelerates active calcium extrusion from the myoplasm. Cyclic GMP levels in smooth muscle can also be increased by atrial natriureticpeptide and endothelium-derived (dependent) relaxing factor (33-35). Nicorandil (2-nicotineamideethylnitrate), a new antianginal agent that possesses actions similar to those of nitrates (36,37), also increases cyclic GMP levels (38). Unlike nitroglycerin and isosorbide dinitrate, this agent hyperpolarizes the plasma membrane by opening calcium-independent potassium channels (36,37,39). Therefore, this drug acts on coronary vascular smooth muscle in a mannerdifferent from that of the calcium antagonists and other nitrates. Why do beta-adrenoceptor agonists have a cardiotonic action on cardiac muscle but a vasodilating action on the coronary artery? Activation of the betayadrenoceptors of visceral smooth muscles by catecholamines such as isoproterenol, epinephrine and norepinephrine leads to relaxation by a response that is thought to be mediated by cyclic AMP. After treatment with alpha-adrenoceptorblocking agents, the beta--adrenoceptor agonists block the generation of spike potentials and may hyperpolarize the myoplasmic membrane in various visceral muscle cells (5,40); however, these catecholamines consistently cause smooth muscle relaxation in the presence of alpha-adrenoceptor blocking agents. This relaxing effect is accompanied by SASAGURI ET AL. CORONARY TONE AND SIGNAL TRANSDUCTORS 1170 JACC Vol. 9. No.5 May 1987:1167-75 increased levels of cyclic AMP with no significant change in cylic GMP levels (27). The relaxing effects of cyclic AMP are believed to be mediated by activation of a cyclic AMP dependent protein kinase (A-kinase) by the following mechanisms (Fig. 2): I) Activated A-kinase phosphorylates myosin light chain kinase in a manner similar to that of G-kinase, thereby causing the muscle to relax by inhibiting the phosphorylation of myosin (3,17-19,27). 2) Activated A-kinase enhances calcium accumulation by the sarcoplasmic reticulum (27 ,32). 3) Activated A-kinase accelerates calcium extrusion by the plasma membrane calcium pump (32). These effects of activated A-kinase are thus similar to those of activated G-kinase except that these two cyclic nucleotide-dependent protein kinases differ with regard to their effects on calcium accumulation into the sarcoplasmic reticulum. Calcium accumulation is stimulated by A-kinase but not by G-kinase, so that nitroglycerin reduces the amount of calcium stored in the sarcoplasmic reticulum but increases calcium storage in these intracellular membranes (27). Exogenously applied dibutyryl cyclic AMP, a permeable derivative of cyclic AMP that has essentially the same effects as isoproterenol (27), and intracellularly applied cyclic AMP do not modify the amplitude of the inward calcium current in visceral smooth muscles (41). These effects differ from the well known effects of beta-adrenocepror activation, intracellularly applied cyclic AMP and extracellularly applied dibutyryl cyclic AMP to increase voltage-dependent calcium influx in cardiac muscle. These findings indicate that the beta-adrenoceptor blockers (which indirectly inhibit the effects of A-kinase de- scribed) do not prevent angina pectoris by acting directly as coronary vasodilators; rather, they inhibit vasodilation. Thus, their antianginal effects arise from their negative inotropic and chronotropic actions, which reduce cardiac oxygen consumption, their ability to reduce renin secretion and angiotensin II synthesis (42) and their inhibitory actions on the vasomotor center in the central nervous system (43). Relation of Inositol Phospholipid to ReceptorOperated Calcium Release From the Sarcoplasmic Reticulum Inositol trisphosphate and calcium release (Fig. 2). Smooth muscle contraction can occur without a significant change in membrane electrical properties and in the absence of extracellular calcium. Such contractions are generated by release of calcium from the sarcoplasmic reticulum by a process known as pharmacomechanical coupling (1,2,44). The manner in which the activated receptors trigger calcium release has recently been explained by the discovery of a new second messenger, inositol trisphosphate (16,45,46) (Fig. 2). This substance is derived from inositol phospholipids in the membrane; these include phosphatidylinositol and its phosphorylated derivatives phosphatidylinositol 4phosphate and phosphatidylinositoI4,5-bisphosphate. In response to receptor activation, phosphatidyl inositol bisphosphate (PI-P z) is rapidly hydrolyzed to form inositol trisphosphate and diacylglycerol. When a receptor binds to its specific agonist, a phosphodiesterase (phospholipase C), is stimulated to hydrolyze phosphatidyl inositol bisphos- PMA,OAG extracellular signal A23187 extracellular space PI PIP OG PA plasma membrane cytoaol Ca2 + ATPase sarcoplasmic reticulum Figure 2. Transmembrane signaling by way of the turnover of inositol phospholipids. A23187 = a calcium ionophore; ATPase = adenosine triphosphatase; Ca = calcium; CaM = calmodulin; CDP = cytidine diphosphate; C-kinase = cyclic AMP-dependent protein kinase C; CMP = cytidine 5' -monophosphate; DG = diacylglycerol; GTP = guanosine 5'triphosphate; InsP, InsPz, InsP 3 = inositol 1- or 4-monophosphate, inositol 4-bisphosphate, inositol 1,4,5trisphosphate, respectively; OAG = l-oleyl-2-acetyl glycerol; OH = hydroxy residue; ® = energy rich phosphate; PA = phosphatidic acid; PDE = phosphodiesterase (phospholipase C); PI, PIP, PIPz = phosphatidylinositol, phosphatidylinositol 4-monophosphate, phosphatidylinositol 4,5bisphosphate, respectively; PMA phorbol 12-myristate 13-acetate. SASAGURI ET AL. CORONARY TONE AND SIGNAL TRANSDUCTORS lACC Vol. 9, No.5 May 1987:1167-75 phate to form inositol trisphosphate and diacylglycerol (Fig, 2). Subsequently, inositol trisphosphate is convertedto inositol through inositol bisphosphate and inositol phosphate by stepwise dephosphorylations induced by activation of individual phosphatases. The breakdown of inositol trisphosphate is accelerated by calcium concentrations up to I p.,M, the maximal free calcium concentration found in the myoplasm (47). In a parallel reaction, diacylglycerol kinase forms phosphatidic acid from diacylglycerol and ATP, and phosphatidic acid can then be converted to cytidine diphosphate-diacyl glycerate in the presence of cytidine triphosphate. Activation of the enzyme cytidine diphosphatediacylglycerol-inositol phosphatidate transferase allows phosphatidyl inositol to be synthesized from cytidine diphosphate-diacylglycerol and inositol. Phosphatidyl inositol is then phosphorylated to produce phosphatidyl inositol monophsphate and phosphatidyl inositol bisphosphate by the actions of specific kinase (Fig. 2). Our present knowledge of the effects of agonist-induced phosphatidyl inositol bisphosphate hydrolysis on calcium homeostasis in vascular smooth muscles can be summarized as follows .. I) In the porcine coronary artery and in rabbit and dog mesenteric arteries, both acetylcholine and norepinephrine reduce phosphatidyl inositol bisphosphate and increase phosphatidic acid and 3H-inositol-labeled inositol trisphosphate. These actions of acetylcholine and norepinephrineare inhibitedby atropineand prazosin, respectively (48). 2) In these vascular smooth muscles, both acetylcholine and norepinephrine can produce a contraction in calcium-free solution (2). 3) Inositol trisphosphate causes calcium release from isolated sarcoplasmic reticulum fraction, but only a small calcium release from porcine coronary artery smooth muscle sarcolemma (49).4) In porcine coronary arteries inositol trisphosphate releases calcium from skinned muscles and dispersed single muscle cells. 5) Application of inositol trisphosphate produces contractions in skinned porcine coronary and rabbit mesenteric arteries. 6) Breakdown of inositoltrisphosphate to inositol bisphosphate by inositol trisphosphatase is stimulated by increasing calcium concentration (47), and inositol trisphosphatase activity is enhanced by the activation of C-kinase in porcine coronary arteries. These observations indicate that inositol trisphosphate, hydrolyzed from phosphatidyl inositol bisphosphate by the activation of phosphodiesterase, is a physiologic second messenger for the agonist-receptor-induced release of calcium from sarcoplasmic reticulum in vascular smooth muscle. Diacylglycerol and protein kinase C. The other product of phosphatidyl inositol, diacylglycerol, activates another protein kinase, C-kinase, in the presence of both calcium and phosphatidylserine (50). These effects of diacylglycerol can also be produced by a class of carcinogens called phorbol esters (12-0-tetradecanoylphorbol-13-acetate (TPA) or phorbol 12-myristate-13-acetate) (50). Activation of C-kinase leads to the phosphorylation of various proteins in vascular tissue (51) including a 20 kDa protein of myosin light chain. C-kinase is found mainly in the cytoplasm, but when this enzyme is activated, it is concentrated in the sarcolemma by a translocation process (50,52,53); the importance of this translocation in vascular smooth muscles to inhibit the contraction has not been established. Whereas TPA in high concentrations has inhibitory actions on the contractile proteins (51), acetylcholine and norepinephrine have excitatory actions on the mechanextracellular .--- Figure3. Transmembrane signaling by way of the adenylate cyclase system. ADP = adenosine 5/-diphosphate; Akinase = cyclic AMP-dependent protein kinase A; AMP = adenosine monophosphate; ATP = adenosine triphosphate; cAMP = cyclic AMP; GOP = guanosine diphosphate; Gi, Gs = inhibitory, stimulatory GTP binding protein, respectively; GTP = guanosine triphosphate; lAP = islet activating protein (pertussis toxin); NAD = nicotinamide adenine dinucleotide; Ns = Gs; Ni = Gi; Pi = inorganic phosphate. / signals ~ .--- _ "'0 ~~o cholera t-Q'< toxiny NAD 1171 + + Pi Pi AlP t cAMPphosphodiesterase A kinase I + cellular + response protein phosphorylation 1172 lACC Vol. 9, No.5 May 1987:1167-75 SASAGURI ET AL. CORONARY TONE AND SIGNALTRANSDUCTORS agonist_ cAMP -I A kinase ~ agonist __ cellular response response ical properties in porcine coronary and rabbit mesenteric arteries, respectively. Therefore, inositol trisphosphate, rather than the C-kinase system, seems to playa major role in the initiation of contraction (54). Inositol trisphosphate- and calcium-induced calcium release mechanism in vascular tissues. In vascularsmooth muscle, depolarization of the plasma membrane directly increases free myoplasmic calcium concentration by activation of voltage-dependent calcium influx. Although the amount of calcium influx calculated from measurements of the calcium inward current is enough to fully activate the contractile proteins, the calcium that enters the cell by this mechanism does not directly activate the contractile proteins. Instead, this calcium is sequestered in a storage site of the sarcoplasmic reticulum from which it can be released when a calcium-induced calcium release mechanism is triggered by inositol trisphosphate. The previously described mechanism appears to activate the contractile proteins (4, JJ ,26,31) for the following rea- Figure 4. Interaction between main pathways of signal transductions. A-, C- andG-kinases = cyclic AMP-dependent protein kinase A, protein kinase C and cyclic GMP dependent protein kinase G, respectively; cGMP = cyclic guanosine monophosphate; RA = adenylate cyclase activating receptor; Rc = phosphodiesterase (phospholipase C) activating receptor; RG = guanylate cyclase activating receptor; other abbreviations as in Figures 2 and 3. Bidirectional ( - ) and nondirectional (+ ) control systems. sons: I) Procaine inhibits both contractions and calcium releasefrom smoothmusclesarcoplasmic reticulumbut does not inhibit the action potential. Thus, procaine uncouples electricalfrom mechanical activity. 2) Procainehas no direct effect on contractile proteins as estimated from calciuminduced contraction in skinned smooth muscle tissue. 3) After depletion of calcium stores in depolarized vascular smoothmuscle tissue, additionof calcium initially increases calcium accumulation into sarcoplasmic reticulum and only subsequently causes contraction. 4) In skinned vascular smooth muscle tissues, calcium accumulation into the sarcoplasmic reticulum is increased in direct proportion to appliedcalcium below 1.0 JLM; further increases in calcium concentration reduce the amount of calcium stored because of calciuminduced calcium release. Only one-third of the stored calcium is released from fragmented sarcoplasmic reticulum by high concentrations of inositol trisphosphate (10 to 20 JLM). However, in intact vascularsmoothmuscletissues, nearlyall the storedcalcium Table 1. Effects of Various Agents on Contraction and Relaxation of Visceral Smooth Muscles in Relation to Second Messengers Membrane Potential Contraction Relaxation ACh Depo., Hyper.. No change Contraction NAd(a!l NAd (a,) ISOP a-hANP Depo., No change Depo. Hyper., No change No change Contraction Contraction Relaxation Relaxation VIP No change Relaxation TXA, EDRF No change. Depo. Hyper.. No change Contraction Relaxation Secondary Messenger PI-P, (cGMP PI-P, cAMP cAMP cGMP PI-P, cAMP PI-P, PI-P, cGMP (pl-P, + +) + + + + + + -) In the case of acetylcholine, cyclic guanosine monophosphate (GMP) is increased in the presence of endothelium; in the case of endothelium-derived relaxing factors (EDRF), phosphat idyl inositol bisphosphate (PI-P 2 ) is reduced after amounts of cyclic GMP are increased. ACh = acetylcholine; cAMP = cyclic adenosine monophosphate; depo. = depolarization; hANP = human atrial natriuretic peptide; hyper. = hyperpolarization of myoplasmic membrane; ISOP = isoprenaline; TXA 2 = thromboxane A2 ; VIP = vasoactive intestinal peptide. JACC Vol. 9. No.5 May 1987:11 67- 75 seems to be released by high concentrations of agonists (26.54,55). Thus, the calcium released by inositol trisphosphate may accelerate or trigger a larger calcium-induced calcium release from sarcoplasmic reticulum to produce a large contraction in vascular smooth muscle. Do individual signal transductors (second messengers) act independently or do they interact in the regulation of cellular vascular smooth muscle activity? We have seen how neurotransmitters can have multiple actions on vascular smooth muscles by promoting the synthesis of such signal transductors as cyclic AMP, cyclic GMP and inositol trisphosphate. Acetylcholine applied exogenously to excised coronary arteries can generate a variety of responses in smooth muscle from different species; in the guinea pig coronary artery. acetylcholine hyperpolarizes the myoplasmic membrane and increases calcium-dependent potassium permeability. In the rabbit coronary artery. acetylcholine depolarizes the myoplasmic membrane and increases sodium permeability (55), but in the porcine coronary artery, it has no effect on membrane potential and resistance (5,9). However, in all these tissues, acetylcholine produces a contraction that is blocked by atropine. Therefore, the mechanisms of action of the same agonist differ in different species (2). Acetylcholin e accelerates PI-P 2 breakdown and increases production o] inos itol trisphosphate . Stimulation of the alpha.vadrenoceptor by norepinephrine also accelerates PI-P1 breakdown with or without depolarization of the myoplasmic membrane, whereas alpha--adrenoceptor agonists inhibit synthesis of cylic AMP and have no effect on PI-P, turnover. Previously, the alpha- adrenoceptor was though; to be distributed on prejunctional nerve terminals and to act as a negative feedback controller on transmitter release at nerve terminals. However, the alphayadrenoceptor is also distributed on the plasma membranes of both arteries and veins (56). Stimulation of both beta,- and beta-adrenoceptors increases cyclic AMP. Cyclic AMP production is inhibited by cholera toxin and stimulated by pertussis toxin (Fig. 3) through adenosine diphosphate-ribosylation of guanosine triphosphate-binding regulatory proteins that are found in the plasma membranes (57.58). In general, the A- , G- and Cikinase systems (including inositol trisphosphate ) play an important role in the tran smembrane control process of calcium homeostasis in man v types of vasc ular smooth muscle. However, these systems do not seem to act independently of each other. One system potentiates the effect of the other in pancreatic islet cells (monodirectional control system), whereas one inhibits the other in lymphocytes and neutrophils (bidirectional control system) (50) (Fig. 4). The A-, G- and C-kinases are distinctly different in their catalytic functions, but may interact at the level of their target proteins in vascular tissues; for example, activation of peptide receptors modulate second messenger systems to modify the contraction-relaxation cycle. SASAGURI ET AL. CORONA RY TONE AND SIGN AL TRANSDUCTORS 1173 Clinical Implications Contraction and relaxation of visceral smooth muscle are mediated by second messengers that are generated in response to various neurotransmitters and autacoids (Table 1). The actions of vasodilators. such as nitrates, alpha- and beta-adrenoceptor agonists and blocking agents. and some peptide hormones have been briefly described in terms of the actions of signal transductors like cyclic nucleotides, inositol trisphosphate and diacylglycerol on calcium mobilization and homeostasis. Muscle tone in coronary arteries can be regulated by many factors. so that the causes of vasospastic angina are probably multifactorial. Neurogenic , myogenic, humoral and endothelium-derived factors can generate a variety of messengers that may contribute differently from one patient to another. Advances in these areas have been rapid and remarkable. but even though calcium homeostasis in smooth muscle is now better understood, most of the results described in this review introduce more complications than clarifications. More detailed and intense investigations at cellular or molecular levels are needed to explain the physiologic and pathophysiologic states of the coronary artery smooth muscle tone. We thank A. H. Weston. PhD for critical reading of the manuscript. References I. Bolton TB. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Rev 1979;59:606-7IX . 2. Kuriyama H. Ito Y. Suzuki H. Kitamura K. ltoh T. Factors modifying contraction-relaxation cycle in vascular smooth muscles. Am J Physiol 1982:243:HMI-62. 3. Adelstein RS: Eisenberg E. Regulation and kinetics of the actin-myosinATP interaction. Annu Rev Biochem 19X0:49:92 I-56. 4. Itoh T. Kuriyama H. Suzuki H. Excitation-contraction coupling in smooth muscle cells of the guinea-pig mesenteric artery. J Physiol 1981:32 1:513- 35. 5. Ito Y. Kitamura K. Kuriyama H. Effects of acetylcholine am! carecholarnines on the smooth muscle cell of the porcine coronary artery. J Physiol 1979;294:595- 611. 6. Casteels R. Kitamura K. Kuriyama H. Suzuki H. Excitation-contraction coupling in the smooth muscle cells of the rabbit main pulmonary artery. J Physiol 1977:271:63- 79. . 7. Endo M. Calcium release from the sarcoplasmic reticulum. Physiol Rev 1977:57:71-IOX. X. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol 19X3:245:CI -1 4. 9. Itoh T. Kajiwara M. Kitamura K. Kuriyama H. Roles of stored calcium on the mechanical response evoked in smooth muscle cells of the porcine coronary artery. J Physiol 1982:322: 107- 25. 10. Casteel- R. Membrane potential in smooth muscle cells. In: Bulbring E. et al.. cds, Smooth Muscle. London: Arnold, 19X I: 105- 26. II . 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