Gen. Pharrnac. Vol. 21. No. 5, pp. 575-587, 1990

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REVIEW ROLE OF ENDOTHELIUM-FORMED NITRIC OXIDE ON VASCULAR RESPONSES JES~S MARiN and CARLOS F. SANCHEZ-FERRER Departamento de Farmacologia y Terap~utica, Facultad de Medicina, Universidad Aut6noma, C/Arzobispo Morcillo 4, 28029-Madrid, Spain

(Received 8 November 1989)

Abstract--l. Endothelial cells of blood vessels generate factors which can modulate underlying smooth muscle tone, inducing vasorelaxation, (endothelium-derived relaxing factor, EDRF, and endotheliumderived hyperpolarizing factor) and/or vasoconstriction (endothelium-derived contracting factors, EDCFs, including the peptide endothelin). 2. EDRF is nitric oxide (NO) or a RNO compound from which this oxide is released. Its half-life is very short (6-50 sec), and it produces rapid vasodilations and inhibits platelet aggregation. 3. NO is formed from the terminal guanidino of L-arginine,but not of o-arginine. NO effects and NO formation are inhibited by NG-monomethyl-L-arginine(L-NMMA), but not by D-NMMA. These inhibitory effects are blocked by L-arginine. 4. Removal of endothelium or pathological situations that can induce endothelial dysfunction (atherosclerosis, diabetes, hypertension or subarachnoid hemorrhage) cause increases on the vascular contractility elicited by agonists (noradrenaline, serotonin, EDCFs, etc.). These findings suggest that EDRF produces a physiologicalinhibitory modulation of vascular smooth muscle tone and its alteration produces or facilitates the development of diseases such as hypertension or coronary and cerebral vasospasm.

INTRODUCTION Endothelial cells have a wide range of physiological activities, such as uptake and metabolism of circulating noradrenaline (NA) or serotonin, conversion of angiotensin I to angiotensin II, metabolism of bradykinin (BK), biosynthesis of prostacyclin (PGI 2), and modulation of vascular permeability, coagulation of blood, and platelet reactivity (Vanhoutte, 1986, 1988a), as well as the inhibition of NA release from adrenergic nerve endings (Cohen and Weisbrod, 1988). In addition to these activities, Furchgott and Zawadzki (1980) demonstrated the obligatory role of endothelium in the vasodilation caused by acetylcholine (Ach). These authors also demonstrated the capacity of endothelial cells to release a potent relaxing factor(s), named endothelium-derived relaxing factor (EDRF), which modulates the vascular responses. Recently, this factor was chemically identified as nitric oxide (NO) (Palmer et al., 1987), which appears to be originated from L-arginine (Palmer et al., 1988a). EDRF or "endogenous nitrite" will be considered throughout this review as NO or as a substance (RNO) from which NO can be easily released (Angus and Cocks, 1989; Shikano et al., 1988). This factor is a very diffusable compound which easily pass the cellular membranes of smooth muscle cells, modulating their contractile activity (Furchgott, 1984; Angus and Cocks, 1989; lgnarro, 1989). The release of EDRF (NO) is mainly abluminal (F6rsterman et al., 1984; Greenberg and Diecke, 1988; Angus and Cocks, 1989). Ach was the first agent studied producing endothelium-dependent GP:,~--A

vasodilation. Later, it was observed that other agents also elicited this type of response (Table 1), although their ability to induce endothelium-dependent relaxations is different according the type of vessel and the animal species (Peach et al., 1985a; Angus and Cocks, 1989; Ignarro, 1989). In the present work, we will review the most important findings of the last years on the biology of endothelial NO, as well as the current state of our knowledge in this field.

EVOLUTION IN THE CONCEPT OF CHEMICAL STRUCTURE OF EDRF

From the discovery of EDRF (Furchgott and Zawadzki, 1980) until the demonstration that this factor is NO (Palmer et al., 1987), the attempts to characterize EDRF have been centered on analyzing whether or not this factor were a metabolite of arachidonic acid (AA). The involvement of cyclooxygenase in the endothelium-mediated relaxations was initially discarded, because these responses were not modified by inhibitors of this enzyme (aspirin, indomethacin) (Furehgott and Zawadzki, 1980; Furchgott, 1983, 1984; Angus and Cocks, 1989). The initial hypothesis of Furchgott and colleagues was that EDRF were probably a hydroperoxy arachidonate derivative, i.e. the endothelium-dependent relaxation caused by agonists releases AA, which is metabolized by lipoxygenases to EDRF (Furchgott, 1983, 1984; Furchgott and Zawadzki, 1980). This assumption was supported by the following facts: (1) the metabolites of lipoxygenases increase the levels of

575

576

JF.SI.'JSMARiNand CARLOSF. SANCHEZ-FERRER

Table I. Classification of vasodilators in function of their endothelium dependence

Emlothelium -dependent: Acetylcholine. Ca ionophore A-23187. bradykinin, substance P, thrombin, arachidonic acid, eledoisin, histamine, hydralazine, acetylglyceryl ether of phosphorylcholine (PAF, at low doses), calcitonin, vasoactive intestinal polypeptide (VIP).

Endothelium -imlependent: cAMP, PGI~, Beta-adrenergic agonists, forskolin, cholera toxin, adenosine, Y-AMP, PAF (sustained relaxation), papaverine. Ca antagonists, diazoxide, minoxidil, cGMP, nitric oxide, atrial natriuretic factor, sodium nitroprusside, glyceryl trinitrate and other nitrates, sodium azide.

Note: Some agents can produce relaxation dependent or not on endothelium according to animal species and the type of the vessel, mainlywith PAF, bradykinin,histamine,calcitoninand VIP.

cyclic GMP (cGMP) in vascular smooth muscle cells by activation of guanylate cyclase (Furchgott, 1984; Greenberg and Diecke, 1988); (2) quinacrine (mepacrine, an inhibitor of phospholipase A 2), 5, 8, il, 14-eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA) (inhibitors of lipoxygenases, but also cyclooxigenase) inhibit the vasodilation induced by Ach in segments of rabbit aorta (Furchgott, 1983, 1984), and (3) melittin, a polypeptide toxin that activates phospholipase A2, produced endothelium-dependent relaxation (F6rstermann and Neufang, 1985) (see Fig. I). In addition to cyclooxygenase, which generate PGI~ and thromboxane A 2, and lipoxygenases, which biosynthesize hydroperoxydes (HETE, HPETE, etc.) and leukotrienes, the endothelium possess also cytochrome P-450 monooxygenases (Mullane and Pinto, 1987). These enzymes metabolize the AA to

5-HETE and 5,6 epoxide, which is rapidly transformed to 5,6 diol, and produce w and w - 1 hydroxylation (Mullane and Pinto, 1987). Cytochrome P-450 was implied in the endothelium-dependent relaxations by the following facts: (I) the cytochrome induction by 3-methylcholantrene and fl-naphthoflavone increases, whereas its depletion by CoCI, inhibits the responses evoked by AA (Pinto et aL, 1985), and (2) the blockers of cytochrome P-450, metyrapone and SKF 525A, reduce the relaxations induced by Ach and A-23187 in rabbit aorta and abolish those produced by AA (Singer et al., 1984). Other investigators have also proposed the involvement of these enzymes in the formation of EDRF (Angus and Cocks, 1989). In spite of these facts, it is necessary to bear in mind that ETYA and NDGA inhibit not only lipoxygenases but monooxygenases as well (Ferreri et al., 1984). Furthermore, the drugs which inhibit lipoxygenases and cytochrome P-450 are scarcely selectives and can generate superoxide anions (O~) (Moncada et al., 1988; Angus and Cocks, 1989), which destroy EDRF (Fig. 2). This indicates that the effects of these drugs could be produced by properties other than inhibition of such enzymes (Moncada et al., 1986; F6rstermann et al., 1988a). Therefore, most of the pharmacological approaches about the involvement of these enzymes in the formation of EDRF needed to be reviewed. The pathways whereby the AA is metabolized, the enzymes involved and the agents interfering with them arc shown in Fig. 3. The possibility that EDRF were a free oxygen radical derived from fatty acids metabolism by means of cyclooxygenase, lipoxygenases or cytochrome P-450 was also considered (Figs. I and 2). Indeed, these enzymes generate O, or other free oxygen

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Fig. I. Schematic representation of the influence of endothelium removal (E-, e.g. by incubation of vascular segments with saponin, S), Hb (which inhibits soluble guanylate cyclase and binds EDRF), methylene blue (inhibitor of soluble guanylate cyclase), lipoxygenases inhibitors (nordihydroguaiaretic acid [NDGA]; 5, 8, l l, 14-eicosatetraynoic acid [ETYA] which also block ciclooxygenase; phenidone; gossipol), antioxidants generators of O;- (phenidone; gossipol; dithiothreitol, DTT; pyrogallol; NDGA: ETYA; hydroquinone) and N°-monomethyl L-arginine (L-NMMA) on the rapid and transient endothelium-dependent relaxations induced by Ach, Ca ionophore A-23187 and bradykinin (BK) on arterial segments precontracted with noradrenaline (NA) or prostaglandin F2,(PGF2=). These types of responses, which are not affected by inhibitors of ciclooxigenase,show the lability of the released substance, EDRF, whose half-life oscillated from 6 to 50 sec. For more details see the text. W = washed.

Endothelium and vascular responses BK

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Fig. 2. Scheme showing the mechanism of EDRF inactivation by Of (generated by lipoxygenases, cytochrome P-450, pyrogallo], Fe2+, hydroquinone, phenidone, ETYA, NGDA, dithiothreitol and methylene blue) and protection by 02 scavengers(Scar.). Likewise,the inhibitory effect of methylene blue and Hb on soluble guanylate cyclase(SGC), as well as the inhibition of EDRF by binding to Hb is also depicted. For more details see the text. R = Receptor; EC = endothelial cell; SMC = Smooth muscle cell. radicals, which activate guanylate cyclase (Greenberg and Diecke, 1988; Rubanyi, 1988a). However, the fact that the Of scavengers cytochrome c and superoxide dismutase (SOD) increase the half-life of EDRF (Rubanyi, 1988a) rules out this assumption. The rapid and transient vasodilation caused by EDRF and the above mentioned facts suggest that it is a labile factor which participates in redox reaction at physiologic pH (Rubanyi et al., 1985a; Rubanyi and Vanhoutte, 1986). Other agents widely used which inhibit EDRF, and so endothelium-dependent relaxations, are oxyhemoglobin (Hb) and methylene blue (Moncada et al., 1988; Ignarro, 1989) (Fig. 2). Hb has an elevated volume and probably does not cross the membrane of

smooth muscle ceils (Angus and Cocks, 1989; Hongo et al., 1988a). Its inhibitory effects are complex since it is able: (1) to bind EDRF (NO) (Martin et al.,

1986), forming the nitrosyl-hemoprotein complex (Ignarro et al., 1987; Ignarro, 1989), which prevents its passage to the above mentioned cells; (2) to block soluble guanylate cyclase (Murad et al., 1978; Martin et al., 1985), and (3) to generate Of during its auto-oxidation in some conditions (Misra and Fridovich, 1972). Frequently, the effects of Hb are not reduced by SOD (Hutchinson et al., 1987), suggesting that its main action is to bind EDRF (Moncada et al., 1988). Hb by itself or hemolysate induces contraction of cerebral arteries, indicating that it may be related to the vasospasm following

Phospholiplds

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Ciclooxygenase Lip°xygenasesLJ HETE,HPETE (-) Arachidonic Acid (_) Leukotrienes Indomethacine ETYA, NDGA Gossipol .Phenidone

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Monooxygenases t Cimetidine Cit. P-450 (- Metyrapone SKF 525 A

.6i° .d..." 5-6,Ep6xide

5-HETE w y w-1 Hydroxylation

Fig. 3. Schematic representation of the formation of arachidonic acid, its metabolism by different enzymes and the pharmacologic interference (activation +, inhibition - ) with the enzymes involved in the distinct steps. For more details see the text.

578

JEsUS MARiN and CARLOS F. SANCHEZ-FERRER

subarachnoid hemorrhage (Tanishima, 1980; Toda et 1980; Wellum et al., 1982; Fujiwara and Kuriyama, 1984; Fujiwara et al., 1986; Kim et al., 1988a). In addition, ultrastructural changes have been observed with Hb in cultured arterial smooth muscle cells that resemble myonecrosis (Fujii and Fujitsu, 1988). It is interesting to comment on the detection in human plasma of a potent inhibitory activity of EDRF-induced relaxation in isolated vessels (Edwards et al., 1986). They isolated a haptoglobin-hemoglobin complex in sufficient concentrations to produce this inhibition. Methylene blue also inhibits the effects of E D R F by several mechanisms, the most important among them are the ability: (I) to produce free oxygen radicals by its redox properties (Moncada et al., 1988), and (2) to inhibit soluble guanylate cyclase (Martin et al., 1985: Mulsch et al., 1987). al.,

EDRF AND NO, PRESENT SITUATION

The orientation of the investigation about the identity of E D R F had a marked change when Furchgott (1988) proposed that E D R F were NO. Indeed, this author demonstrated that both E D R F and NO (obtained by acidification of NaNO 2) possessed striking similarities. Thus, NO produces transient relaxations of rabbit aorta rings, mimicking those caused by EDRF; the relaxations caused by NO and E D R F were analogously inhibited by Hb and potentiated by SOD. Previous works had demonstrated that relaxations induced by NO are always accompanied by an elevation of c G M P in vascular smooth muscle cells (Arnold et al., 1977; Gruetter et al., 1981), as occurs with EDRF. due to the stimulation of soluble guanylate cyclase (Diamond and Chou, 1983: Rapoport and Murad, 1983; Furchgott, 1984; Ignarro and Kadowitz. 1985; Vanhoutte, 1987a; Ignarro, 1989). Furthermore, both compounds also inhibit platelet aggregation (Furlong et al., 1987; Radomski et al.. 1987c; Moncada et al., 1988). The proposal of Furchgott was elegantly confirmed by Palmer et al. (1987), who demonstrated that cultured endothelial cells from pig aorta released NO, detected by chemiluminescence, in a concentration sufficient to produce vasodilation. Moreover, they also demonstrated that the relaxations elicited by E D R F and NO were inhibited by Hb and increased by SOD in a similar degree; i.e. both compounds were pharmacologically indistinguishable with analogous half-life. In bovine pulmonary endothelial cells, lgnarro et al. (1987) also demonstrated a similar behavior between EDRF, released by both A-23187 and Ach, and NO in terms of Hb binding. The stability of E D R F and NO was similarly enhanced by infusion of SOD or cytochrome c. Their relaxations were analogously reduced by Fe -'~, hydroquinone and pyrogallol, and this reduction was blocked by SOD and cytochrome c (Radomski et al., 1987c; Hutchinson et al., 1987). Moncada and colleagues showed that both NO and E D R F inhibited the platelet aggregation, with similar half-lives (Radomski et al., 1987c) and platelet adhesion (Radomski et al., 1987a, b), and elicited disaggregation when platelets were previously aggregated (Radomski et al., 1987c). The antiaggregatory activ-

ity of NO was increased by SOD and reduced by Hb and Fe -'~, as occurs with E D R F (Radomski et al., 1987c). Moreover, experiments using specific phosphodiesterase inhibitor o f c G M P (MY 5445 and M & B 22948) and direct measurement of this cyclic compound, indicate that E D R F and NO produce their effects on vascular smooth muscle cells and platelets by stimulation of soluble guanylate cyclase increasing the intracellular levels of cGMP (Moncada et al., 1988). There exists some properties of E D R F released from cultured endothelial cells that are not explained if this compound were simply NO. Indeed, E D R F is negatively charged (Cocks et al., 1985; Long et al., 1987), its stability is enhanced at 0°C, it can be lyophylized and the product of this lyophylization can be reconstituted maintaining its properties (Angus and Cocks, 1989). These findings are difficult to explain if E D R F were a dissolved gas (Shikano et al., 1988; Angus and Cocks, 1989). In addition, comparative bioassay studies have shown that E D R F only relaxes vascular smooth muscle, whereas NO also caused relaxation of nonvascular smooth muscle preparations (e.g. guinea-pig taenia coli and trachea) (Shikano et al., 1987, 1988; Dusting et al., 1988). Nevertheless, recently, it has been reported that E D R F and NO relax both types of preparations, although the vascular one is the most sensitive to these substances (Buga et al., 1989). A direct evidence indicates that E D R F is not NO, but a related compound (RNO) that easily releases NO; this was suggested by the studies of Feelisch and Noack (1987). They observed that the nitrovasodilators sodium nitroprusside and SIN-I (the active metabolite of the organic nitrate molsidomine) may spontaneously release NO and also glyceryl trinitrate with the addition of thiol reagents. These authors concluded that all these compounds, including EDRF, may simply be pro-drugs of NO, and E D R F could be a RNO compound which can rapidly breakdown releasing NO. The latter compound in solution is quickly oxidized by O: or O,- to NO; and/or NO 3 (Moncada et al., 1988). BIOSYNTHESIS OF NO

Once demonstrated by Moncada and co-workers that E D R F is NO, they treated to investigate the origin of this NO. Thus, Palmer et al. (1988a, b), using cultured endothelial cells of porcine aorta as donor and spiral strips of rabbit aorta as receptor of the effluent, demonstrated either by bioassay, chemiluminiscence and mass spectrometry that the stimulation of endothelial cells with A-23187 released NO, which is originated from terminal guanidino nitrogen atom(s) of L-arginine, but not D-arginine. The NO released was reversibly increased by L-arginine and L-citrulline, but not D-arginine or other related compounds. The precise mechanism by which NO is formed is not known. It is probable that deimination leds to generation of NH~ which is subsequently oxidized to NO, as appears to occur in the macrophages (Hibbs et al., 1987b). NO may be also formed from the terminal guanidino group of Larginine through N-oxidation by oxygen radicals produced in the phospholipases activation (Palmer

Endothelium and vascular responses et al., 1988a) or from an intermediate compound

(organic nitroso compound, nitrite or ammonia) generated in the metabolism of this terminal (Ignarro, 1989). Likewise, it was demonstrated that an analog of L-arginine, NG-monomethyl-L-arginine (L-NMMA), but not its enantiomer D-NMMA, inhibited the formation of NO by endothelial cells in culture stimulated by BK or Ach, as well as the endotheliumdependent relaxations induced by Ach, A-23187, substance P or L-arginine in rings of rabbit aorta. Nevertheless, the endothelium-independent relaxations elicted by glyceryl trinitrate or sodium nitroprusside were unaffected by L-NMMA. L-NMMA inhibition of both the endothelium-dependent relaxations and NO release by endothelial cells were antagonized by L-arginine, but not by D-arginine (Palmer et al., 1988a, b; Rees et al., 1989). These authors also observed that L-arginine, but not Darginine, L-homoarginine, L-citrulline, urea, etc., produces small, but significant, endothelium-dependent vasodilation in rings of rabbit aorta (Rees et al., 1989). L-arginine, but not D-arginine, increases Achevoked NO release as well (Rees et al., 1989). This suggests the existence of a L-arginine deiminase-like enzyme (Fig. 4), although its existence in mammalian cells has not been clearly demonstrated (lgnarro, 1989). In conclusion, L-arginine appears to be the physiological precursor for the basal and stimulated NO formation involved in endothelium-dependent vasodilation (Rees et al., 1989). Synthesis and release of NO have been described in other cells. Thus, the activated macrophages also generate NO from the terminal guanidino nitrogen atom(s) of L-arginine (Hibbs et al., 1987a, b) and probably peritoneal neutrophils produce this oxide too (Rimele et al., 1988). The physiological role of this non-vascular NO is not yet known, but it could be related with inflammatory processes or local regulation of microcirculation. It has been observed that neither L-arginine nor D-arginine induced endothelium-dependent relaxations in precontracted rat aortic rings, but benzoyl derivatives of L-arginine did so (Thomas and Ramwell, 1988). These relaxations were markedly reduced by endothelium removal and abolished by methylene blue. They suggest the presence in arterial endothelial cells of a peptidyl arginine deiminase activity with possible formation of citrulline (nonvasodilatory) and ammonia, as occurs in macrophages, which may be transformed in NO by oxygenases (Hibbs et al., 1987b). Thomas and Ramwell (1988) proposed the existence in the endothelial cells of a peptidyl arginine deiminase which had no activity for L-arginine; they also suggest the existence of intermediate peptides of short half-life, which contain L-arginine and are released from endothelium by the agonists (e.g. Ach). However, Bhardwaj and Moore 0989) reported that salts of L-arginine produced rapid endothelium-dependent relaxation, which are ditficuit to explain by the formation of these intermediate peptides, suggesting that the relaxation induced by these salts is similar to that caused by Ach, and so produced by NO release. The doubt now is if L-arginine release NO, is transformed in NO or both processes occur.

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Fig 4 Schematic representation of the formation of EDRF and the hyperpolarizing factor (EDHF) by the endothelial cell (EC) and their stimulatory effects (+) on soluble guanylate cyclase (SGC) and Na, K-ATPase, respectively, present in the smooth muscle cell (SMC). The activation of receptors (R) of EC by the agonists (Ach, BK, ADP, etc.) induces Ca entry the cell producing the chain of events, which causes the synthesis of EDRF (NO) from L-arginine by means of L-arginine deiminase. This synthesis is blocked by N°-monomethyl L-arginine (L-NMMA). The generated cGMP induces relaxation by several mechanisms, such as inhibition of Ca entry through receptor-dependent Ca channels (RDC), stimulation of membrane Ca ATPase or the cGMP-dependent protein kinase (cGMP-kinase). The latter enzyme can phosphorylate the myosin light chain kinase (MLCK). All these mechanisms produce vasodilation. MECHANISMS INVOLVED ON THE RELEASE OF EDRF

The release of EDRF is a Ca-dependent process. This assumption is mainly based on the following findings: (I) the dose-dependent relaxations induced by the Ca ionophore A-23187 and methacholine are inhibited by Ca depletion (Singer and Peach, 1982); (2) Ca-antagonists also inhibit these responses (Singer and Peach, 1982; Long and Stone, 1985; Grifl~th et al., 1986); (3) Ca and dihydropyridine Ca agonists release EDRF (Carvalho and Furchgott, 1982; Rubanyi et al., 1985b; Robertson, 1986), and (4) A-23187 produces endothelium-dependent relaxation and release of EDRF from cultured endothelial cells (Furchgott 1984; Cocks et al., 1985). The blocking effect of Ca antagonists on the release of EDRF, above mentioned, was not observed by other investigators (Miller et al., 1985; Jayakody et al., 1987), having suggested that endothelial cells do not contain functional voltage-dependent Ca channels (Morgan-Boyd et al., 1987; Colden-Standfield et al., 1987). In conclusion, the type of Ca channel or the pathway by which Ca enters into these cells is unknown. In addition to Ca, oxygen is also needed for the release of EDRF (Furchgott and Zawadzki,

580

JESI3SMARiN and CARLOSF. SANCHEZ-FERRER

1980: Furchgott, 1984), indicating that it is needed for NO synthesis and/or for the coupling agonist-recepfor that induces NO generation (Ignarro, 1989); NO might be stored in the endothelial cells from which could be released by agonists in an oxygen- and Ca-dependent way (Ignarro, 1989). Contradictory results have been reported concerning the role of Na/Ca exchange in E D R F release. Thus, amiloride and dichlorbenzamil, Na/Ca exchange inhibitors, inhibit E D R F release indicating the participation of this exchange in the secretor process (Winquist et al., 1986; Schoeffter and Miller, 1986), whereas Cocks et al. (1988) have proposed that this exchange operates to extrude Ca from endothelial cells. The agonists that release E D R F stimulate phospholipase C in cultured endothelial cells, releasing the intracellular second messenger inositol trisphosphate (Lambert et al., 1986; Derian and Moskowitz, 1986; Ganz et al., 1986). Phorbol esters release E D R F indicating a participation of protein-kinase C in this secretion (Greenberg and Diecke, 1988). This finding along with the necessity of extracellular Ca for E D R F release suggests that secretion from the cndothelium is similar to other secretory cells. The released E D R F has a short, but variable, half-life (6-50sec) (Angus and Cocks, 1989). This factor is a labile compound, which is easily inactivated by free oxygen radical and its inactivation is diminished by reduction of the 02 content of Krebs' solution or by using scavengers of O, (Rubanyi and Vanhoutte, 1986; Angus and Cocks, 1989). The fact that the half-lives of E D R F and NO, under the same conditions, were similar (30 sec, Palmer et al., 1987) further supports the assumption that E D R F and NO are the same substance. MECHANISM OF ACTION OF EDRF

It has been demonstrated that E D R F (NO) increases the intraccllular levels of cGMP by stimulation of soluble guanylate cyclase in smooth muscle cells (Holzmann, 1982; Furchgott, 1984; Ignarro and Kadowitz, 1985; Vanhoutte, 1987a; Cocks and Angus, 1989). In addition, lgnarro (1989) suggested that the endogenous receptor for NO is probably the heme moiety of this enzyme. This cGMP increase produces relaxation presumably by reduction of the free Ca in those cells (Collins et al., 1986; Schini et al., 1987). Several mechanisms have been proposed to explain cGMP-evoked relaxation: (1) inhibition of inositol triphosphate (IP~) generation (Rapoport and Muard 1986); (2) stimulation of the intracellular Ca sequestration (Lincoln, 1983); (3) increase of the dephosphorylation of the myosin light chain (Rapoport et al., 1983); (4) inhibition of receptoroperated Ca channels (Godfraind, 1986); (5) activation of cGMP-dependent protein kinases (Rapoport et al.. 1983; Popescu et al., 1985; Fiscus, 1988); (6) stimulation of membrane Ca-ATPase (Fiscus, 1988); and (7) increase of the K permeability through K channels causing membrane hyperpolarization (Komori and Suzuki, 1987a). It is not known whether the relaxation is only produced by cGMP increase or also other actions of E D R F could contribute to vasodilatation (Fig. 4).

NO also activates soluble guanylate cyclase of platelets, increasing platelet cGMP levels (Melliom et al., 1981). The mechanism involved in the platelet aggregation inhibition by NO is the same one whereby NO relaxes vascular smooth muscle, i.e. by reduction of intracellular free Ca concentration (lgnarro, 1989). Depolarization and ouabain inhibit EDRFdependent vasorelaxation (Rapoport et al., 1985a, b; Greenberg et al., 1986), indicating an involvement of Na pump in the effect and/or the secretion of EDRF. The suppression of endothelium increases the vasoconstriction induced by different agents (Martin et al., 1986; Angus and Cocks, 1989). This effect could be due to an inhibitory effect on the contractile mechanisms of vascular smooth muscle cell exerted by E D R F or to an inhibition of the release of E D R F by these contractile agents. In any case, E D R F appears to interfere with development of the contraction (Fig. 5).

EDRF OF VEINS

The first studies about E D R F in veins show that this factor fails to induce endothelium-dependent relaxation (Furchgott, 1983; 1984; Vanhoutte and Houston, 1985; Griffith et al., 1986). However, veins from some animal species have the ability to generate endothelium-dependent relaxation to AA and cholinergic agents (Greenberg and Tanaka, 1982; Gruetter and Lemkc, 1986; Miller et al., 1986), although the response is generally less in veins than arteries. It has also been reported that E D R F from arteries relaxes veins, suggesting the existence of receptors for E D R F in veins, even in the absence of endogenous E D R F (Greenberg and Diecke, 1988). These findings suggest that endothelial factor of arteries and veins may be different, but some type of E D R F present in arteries exists also in veins (Angus and Cocks, 1989). Recently, it has been demonstrated in a in t~ivo study that E D R F is an endogenous venodilator in man (Collier and Vallance, 1989). They observed that the removal of venous endothelium of large veins on the back of the hand produces vasoconstriction and loss of relaxation induced by Ach, but not by glyceryl trinitrate. E(-)

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Fig. 5. Scheme showing the potentiator (a) and inhibitor (b) effect of endothelium removal (E-) on the response induced by agonists (A) [noradrenaline, serotonin, clonidine, etc. in the case (a) A-23187, Ach, arachidonic acid, AA and ADP in case (b)]. These different actions are dependent on the studied vessel and can be reflecting the absence of a relaxant (EDRF) (a) or a contractile factor (EDCF) (b). For more details see the text.

Endothelium and vascular responses OTHER ENDOTHELIAL

VASOACTIVEFACTORS

Discarding PGI2 released from the endothelium, recent work indicates that NO is not the only vasoactire substance secreted; other EDRFs different to NO may be synthesized by endothelial cells (Myers et al., 1989). Thus, Ach is able to induce endotheliumdependent hyperpolarization of vascular smooth muscle (Bolton et al., 1984; Komori and Suzuki, 1987a; Feletou and Vanhoutte, 1988). This hyperpolarization cannot be mimicked by NO (Huang et al., 1988; Komori et al., 1988), suggesting the existence of a new endothelial factor, different from NO, which was called "endothelium-derived hyperpolarizing factor" (EDHF) (Komori and Suzuki, 1987a, b; Feletou and Vanhoutte, 1988; B6ny and Brunet, 1988). E D H F seems to be released by specific stimulation of endothelial muscarinic M~ receptors, whereas stimulation of M2 secretes E D R F (Komori et al., 1988) (Fig. 4). E D H F is neither PGI 2 nor NO, because they do not modify the membrane potential and Hb blocks the relaxation more effectively than the hyperpolarization (Komori and Suzuki, 1987a, b; Komori et al., 1988; Chen et al., 1988; Nishiye et al., 1989). These authors suggest the existence of both endothelium-derived factors E D R F and E D H F involved in the sustained and initial transient relaxations, respectively (Fig. 6). Two different mechanisms of action have been proposed for EDHF: the activation of ATPase Na-K of vascular smooth muscle (Feletou and Vanhoutte, 1988) or the opening of K-channels and the increase of potassium conductance through muscle cell membrane (Chen et al., 1988; Kauser et al., 1989). The characterization of this factor, however, is not yet established. In addition to the relaxant factors released by the vascular endothelium, there is evidence that these cells can also mediate vasoconstrictor responses through the secretion of one or more substances which are able to contract the underlying smooth muscle. This concept was introduced by the observations that the removal of endothelium reduced or abolished the contractions induced by different stimuli, such as thrombin, AA, or hypoxia in isolated blood vessels (De Mey and Vanhoutte, 1982; Miller and Vanhoutte, 1985), as well as by the demonstration of a bioassayable vasoconstrictor factor in response to hypoxia (Rubanyi and Vanhoutte, 1985). Simultaneously, some authors observed contractile properties in the supernatant of endothelial cultured cells (O'Brien and McMurtry, 1984; Hickey et al., 1985). Further studies have indicated the existence of clear differences in the endothelium-dependent contractions, concerning the releasing stimuli and, essentially, the chemical nature of the involved factors. Thus, at least three distinct endotheliumderived contractile factors (EDCFs) (Fig. 6) have been proposed (Rubanyi, 1988b; Greenberg and Diecke, 1988): EDCF~, a metabolite of arachidonic acid (Miller and Vanhoutte, 1985; Vanhoutte, 1987b; 1988b); EDCF2, a polypeptide (O'Brien and McMurtry, 1984; Hickey et al., 1985; Gillespie et al., 1986) that has been isolated and identified as a 21 aminoacid peptide named endothelin (Yanagisawa et al., 1988); and EDCF3, unidentified substance(s), released in response to anoxia (Rubanyi and

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Vanhoutte, 1985) or increases in transmural pressure (Harder, 1987; Rubanyi, 1988c; Harder et al., 1989) (Fig. 6). The nature and mechanisms of action of these contractile factors need further determination.

PHYSIOLOGICAL ROLE OF EDRF

Since the first publication about E D R F (Furchgott and Zawadzki, 1980), there has been an exponential growth in our knowledge about the ability of vascular endothelium to modulate the tone of underlying smooth muscle in response to physiological or pharmacological stimuli. Nevertheless, the exact physiological role of endothelium in the vascular reactivity is not yet well understood. It is clear that the ability of endothelium to modulate smooth muscle tone arose early during evolution (Vanhoutte, 1988c). However, Ach, the most classical agent that releases E D R F from endothelium, does not circulate in the blood and seems unlikely, although is not impossible, that it could reach the endothelium when released from cholinergic nerves (Vanhoutte, 1988c). Furthermore, the ability of Ach and other agents to release E D R F (NO) and/or other endothelial vasoactive factors is dependent on the animal and the specific vascular bed studied (Peach et al., 1985b; Angus and Cocks, 1989). On the basis of these facts, one can speculate that endothelium may be involved in local mechanisms to autoregulate blood flow of different organs. Thus, endothelial cells can be acting as transducers of several physical or chemical stimuli that are able to modify vascular tone, and consequently blood flow, through the release of relaxant or contractile endothelial factors. The variability in the observed endothelial responses could be reflecting the different metabolic needs of distinct organs, which would require a predominant relaxant or contractile activity, or a balance between both kinds of responses. At the moment, of course, these are only speculations, and no demonstration of this hypothesis is available. However, there is some evidence that could support this idea: the activation of glutamate

Agonists

Ca

2+

F 1 (AA.Metld~.)

R

F 2 (Po~ypeptlde) F 3 (Dllfusable f~ctor)Anoxie

Pressure

[~RF (NO) Fig. 6. Scheme showing the different factors produced by endothelial cells (EC): hyperpolarizing (EDHF), relaxant (EDRF) and contracting (EDCF=). The latter factor are of three types: EDCF I, a metabolite of arachidonic acid (AA) via lipoxygenases or ciclooxygenase, EDCF 2, a polypeptide, and EDCF 3, a diffusable factor, formed in anoxia and hypoxia situations or in response to increases in perfusion pressure. EDCF 2 may be the peptide endothelin (ET), which is the most potent vasoconstrictor agent known to date. For more details see the text.

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J~6s MARiN and CARLOSF. SANCHEZ-FERRER

receptors in the brain is able to generate EDRF, which produces relaxation of aortic strips and increases cGMP levels in the brain cells, and its effects are blocked by Hb and mimicked by NO (Garthwaite et al., 1988). Although the meaning of this finding is unknown, it is tempting to suppose this cerebral NO may work relaxing intracerebral microvessels as an autoregulatory mechanism; in such case, the vasoactive substance would be synthesized not in the endothelium but in the same perfused tissue. In addition to the actions previously indicated for E D R F (NO) another physiological effect has been proposed for this compound. Indeed, NO can react with O{ removing the oxygen free radical and protecting vascular smooth muscle and other cells outside vascular endothelium (Feigl, 1988). PHYSIOPATHOLOG1CAL IMPLICATIONS OF ENDOTHELIUM-DERIVED FACTORS

A great part of the evidence supporting the important role ofendothelium on vascular physiology came from the discovery that in some important cardiovascular diseases there exist endothelial alterations, which will be briefly summarized. A therosclerosis

It has been reported in vessels with atherosclerotic lesions from human and animals (fed with a high cholesterol concentration or other appropriate diets), that endothelium-dependent vasodilator responses are reduced (Freiman et al., 1986; Sreeharan et al., 1986; Verbeurcn et al., 1986; F6rstermann et al., 1988b; Osborne et al., 1989: Shimokawa and Vanhouttc, 1989a). This effect may be due to the following mechanisms: (1) E D R F penetrates with difficulty the atherosclerotic lesions (acting as a barrier and so preventing the endothelial factor reaching the vascular smooth muscle cells): (2) the generation of E D R F is reduced in atherosclerotic vessels; (3) the receptors for E D R F in vascular smooth muscle or those present in endothelial cells are impaired or down regulated in these vessels, and (4) the mechanisms involved between receptor activation and vascular response are altered (Greenberg and Diecke, 1988; Angus and Cocks, 1989; Shimokawa and Vanhoutte, 1989b). The reduction in the generation or release of E D R F favours the platelet aggregation, in part due to platelet cGMP reduction. These vascular alterations produce increases in the vasoconstrictor responses to different agents, such as catecholamines (released from adrenergic nerve endings), serotonin, prostaglandins (both released from platelets) or contractile factors (EDCFs) (Cocks and Angus, 1983; Greenberg and Diecke, 1988; Katusic et al., 1988; Angus and Cocks, 1989). These increases in contractile activity favours the coronary vasospasm. Thus, Ludmer et al. (1986) observed that intracoronary administration of Ach produces a marked vasoconstriction in atherosclerotic vessels in comparison with the small vasodilation obtained in normal vessels. In some cases, the coronary vasospasm could occur previously to develop visible atherosclerotic lesions. On the other hand, it has been proposed that vascular diseases developed during diabetes may

also be related with an impairment of endotheliumdependent relaxations (Durante et al., 1988; Pieper and Gross, 1988; Kamata et al., 1989). Hypertension

Hypertension produces changes in endothelium of blood vessels, which cause a reduction in endothelium-dependent relaxations (Konishi and Su, 1983; Lockette et al., 1986; Van de Voorde and Leusen, 1986), and an enhancement of endothelium-dependent contractions (Liischer and Vanhoutte, 1986a, b; Lfischer et al., 1988). Loss of endothelial relaxation seems to be secondary to elevation in blood pressure, because it appears both in genetic and experimentally induced hypertension, and recovers when pressure returns to normality (Lockette et al., 1986; Lamping and Dole, 1987; Van de Voorde et al., 1988). The endothelium-independent vasodilation, however, is not modified, suggesting that the mechanisms involved in the relaxation are not affected (Angus and Cocks, 1989). The reduction in the endotheliumdependent vasodilation appears to be associated to diminution of E D R F production and the generation of cGMP in these vessels (Otsuka et al., 1988; Angus and Cocks, 1989). The endothelium-dependent vasoconstriction seems to be mediated by metabolites of arachidonic acid (EDCF0, which are not thromboxane A 2 (L/ischer and Vanhoutte, 1986a, b; Liischer et al., 1988). The renal vasculature of spontaneously hypertensive rats seems to be more sensitive to the vasoconstrictor endothelial peptide endothelin than that of normotensive animals, also suggesting a possible role for this peptide in the maintenance of hypertension (Tomobe et al., 1988). An association has also been reported between aging and hypertension to induce alterations in the endothelial modulation of vascular tone, which indicates that old-age is an additional risk factor for endothelium function (Lee et al., 1987; Hongo et al., 1988b). Cerebral vasospasm

Cerebral vasospasm is a clinical event that accounts for a major portion of the morbidity and mortality following subarachnoid hemorrhage (Powers and Grubb, 1987). The cause of cerebral vasospasm is unknown, although different hypothesis involving circulating or cerebrospinal vasoconstrictors, cerebrovascular innervation or smooth muscle changes have been proposed (Kassell et al., 1985). It has been demonstrated that endotheliumdependent relaxations of cerebral vessels are reduced after subarachnoid hemorrhage; this inhibition has been attributed to a damage in endothelial cells or to the presence of Hb in cerebrospinal fluid, which binds and inactivates E D R F (Nakagomi et al., 1987a, b; Kim et al., 1988a; Hongo et al., 1988c). Loss of endothelial relaxations is accompanied by maintenance of endothelium-dependent contractions (Kim et al., 1988a, b). Both effects can account for developing of cerebral vasospasm, facilitating contractile activity of cerebrovascular smooth muscle and reducing its relaxant ability. It has been suggested that subarachnoid hemorrhage may decrease the secretion of E D R F from the endothelium (Hongo et al., 1988c, d); however, Kim et al. (1989), in bioassay experiments, found that the release of

Endothelium and vascular responses E D R F is not impaired after subarachnoid hemorrhage, indicating that the loss of endotheliumdependent relaxation is due to a decreased transfer of E D R F or by a reduced responsiveness of the underlying smooth muscle to the factor. Additional work is needed to further clarify this important question. SUMMARY

Endothelial cells of blood vessels generate factors which can modulate underlying smooth muscle tone, inducing vasorelaxation, (endothelium-derived relaxing factor, E D R F , and endothelium-derived hyperpolarizing factor, E D H F ) and/or vasoconstriction (endothelium-derived contracting factors, EDCFs, including the peptide endothelin). Initially, it was suggested that E D R F is derived from arachidonic acid by the action of lipoxygenases. Nevertheless, recently it has been demonstrated that this factor is nitric oxide (NO) or perhaps a compound (RNO) from which this oxide may be easily released. E D R F (NO) is a labile substance, which has a short halflife (6-50 sec) and is able to produce vasodilation and inhibition of platelet aggregation. Acetylcholine induces endothelium-dependent relaxation by the release of E D R F and by other factor, E D H F , which produces hyperpolarization of smooth muscle cells. N O is biosynthesized from the terminal guanidino group of L-arginine, but not D-arginine. The vasodilator effects of N O and its formation by endothelial cells are inhibited in a concentration-dependent manner by NC'-monomethyl-L-arginine (L-NMMA), but not D - N M M A . This inhibition is antagonized by L-arginine, suggesting that this amino acid and L - N M M A are competing for the same active locus of the enzyme generator of NO. Removal of endothelium or pathological situations that can induce endothelial dysfunction, such as atherosclerosis, diabetes, hypertension or subarachnoid hemorrhage, cause increases of the vascular contractility elicited by agonists (noradrenaline, serotonin, E D C F s , etc.). These findings suggest that E D R F produces a physiological inhibitory modulation of vascular smooth muscle tone and its alteration produces or facilitates the development of diseases such as hypertension or coronary and cerebral vasospasm. Acknowledgements--This work has been supported by grants from D.G.C.I.Y.T. (PB87/0117), F.I.S.S. (86/622; 87,;1666: 89/0072) and Universidad Aut6noma de Madrid. REFERENCES

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