Lung (1991) 169:185-202 034120409t 00015V New York Inc. 1991

Review

Endothelium-Derived Relaxing Factor and the Pulmonary Circulation G. Cremona, A. T. Dinh Xuan, and T. W. Higenbottam Department of Respiratory Physiology, Papworth Hospital, Cambridge, CB3 8RE, UK

Abstract. Endothelium-derived relaxing factor (EDRF) is probably identical

to nitric oxide (NO) and is released by the vascular endothelium both in the basal unstimulated state and in response to a wide range of physical and chemical stimuli. Since it was first described 10 years ago, evidence is accumulating that it is an important modulator of vascular smooth muscle tone. EDRF acts on the pulmonary vascular bed as on the systemic circulation. EDRF release to pharmacologic stimuli is impaired in pulmonary arteries from patients with chronic hypoxemia. This impairment is associated with severity of respiratory failure and of structural change of vessel walls. Disturbance of EDRF activity may be important in the pathophysiology of pulmonary vascular disease. This brief review describes the current status of experimental studies concerning the possible role of EDRF on the pulmonary circulation in normal conditions and in the pathogenesis of pulmonary hypertension. Key words: Pulmonary circulation--Endothelium-derived relaxing factor--Hypoxic pulmonary vasoconstriction--Pulmonary hypertension. Introduction

The pulmonary vasculature exhibits an enormous capacity for adaptation to general and local changes in blood flow. During exercise, pulmonary blood flow may increase fourfold without raising mean pulmonary artery pressure [43]. Local alveolar hypoxia can cause regional pulmonary vasoconstriction resulting in a reduction in pulmonary perfusion [42]. This regulation of pulmonary vascular tone is probably localized and does not necessarily require neural and/or humoral influences [44]. Offprint requests to: T. W. Higenbottam

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Table 1. Agents that act on the pulmonary vascular bed to produce EDRF

Agent

Species

References

Arachidonic acid Histamine Leukotrienes Bradykinin Calcitonin gene-related peptide Substance P Vasoactive intestinal peptide Thrombin ATP and ADP

Dog, rabbit Rat, guinea pig Guinea pig Dog, cattle Rat (aorta) Guinea pig Rat (aorta) Dog Dog, humans

19, 32, 109 1, 20 54, 114 5 12 7 40, 57 24, 32 32, 37

Recent discoveries focus upon the vital role of the endothelium in the local regulation of vascular tone. Prostacyclin (PGI2), a cyclo-oxygenase product with powerful vasodilator properties, is generated by endothelial cells [92]. This provided one of the first clues to the importance of vascular endothelium. Another group of powerful vasodilators, distinct from PGI 2 [48], and called endothelium-derived relaxing factors (EDRF), were identified in 1980 by Furchgott and Zawadzki [52]. A major component of EDRF has now been identified as the gas nitric oxide (NO) [100]. Endothelial cells may also release another factor, endothelium-derived hyperpolarizing factor (EDHF), which causes vascular smooth muscle relaxation through hyperpolarization [21]. The relationship between EDHF and NO remains unclear and recent evidence shows that NO also induces hyperpolarization of vascular smooth muscle [ 119], which suggests that it may account for at least a part of this phenomenon. In addition to producing vasorelaxing substances, the endothelium appears to elaborate a powerful group of vasoconstrictors, one of which has been identified with a peptide called endothelin [130]. Vascular tone in the systemic as well as the pulmonary circulation probably reflects the "balance" between the local "vasorelaxing" and "vasoconstricting" influences [36]. We review here the physiology of EDRF with particular reference to its role in the regulation of pulmonary vascular tone in both health and disease.

Release of EDRF by the Endothelium Acetylcholine (ACh) was the first pharmacologic agent shown to cause the release of EDRF from endothelial cells [52], which it was presumed to do by acting o n M 2 muscarinic receptors [78]. A wide range of compounds has subsequently also been shown to act on endothelium in a similar manner to release EDRF. These include neuropeptides together with products of platelet aggregation and thrombus formation (Table 1). Physical stimuli such as in-

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creased shear stress on endothelial cells can also cause EDRF release [57, 67, 79, 90, 110]. This may be of importance under physiological and/or pathologic conditions in which increased shear stress could occur due to an increase in blood flow or a reduction in the caliber of blood vessels. The presence of endothelial receptors that cause release of EDRF varies according to the anatomical location within the vasculature, and differences exist between animal species. For example, different responses are obtained from arteries and veins in both dog [33, 112] and humans [82, 121]. ACh causes relaxation of coronary arteries in dogs but constricts porcine coronary arteries [54], which probably results from the absence of endothelial muscarinic receptors in the latter species. In addition to responding to variations in blood flow and pharmacologic stimuli, EDRF is released continuously under basal conditions [24]. Not only can basal release blunt pharmacologically induced vasoconstriction [57] but inhibition of basal release can also lead to a rise in vascular resistance [122]. This indicates that EDRF continuously modulates background vascular tone. Some evidence has been presented supporting the argument that different molecular species of EDRF contribute to basal tone as opposed to that influencing changes in vascular tone [65]. Basal vascular tone influences the response of the endothelium to EDRF agonists. For example ACh, histamine, and ATP can cause vasoconstriction in normal lungs [113], while they cause vasoditatation when the pulmonary vasculature is constricted by serotonin.

Identity of EDRF Bioassay studies [25, 49] have indicated that EDRF is a highly diffusible molecule with a half-life in an aqueous buffered solution of only a few seconds. Nitric oxide (NO), a highly diffusible oxidant gas, is known to activate soluble, or cytosolic, guanylate cyclase of smooth muscle cells, thus causing relaxation [55]. Nitrovasodilators such as sodium nitroprusside are thought to emit NO within smooth muscle [58] and so, by a similar mechanism, cause smooth muscle relaxation. Two research groups simultaneously proposed that EDRF and NO were the same [50, 70]. From a series of studies in which NO and EDRF were compared with respect to their pharmacologic properties, it was possible to conclude that NO probably contributed to the main vasorelaxant properties of EDRF [70, 100]. This also includes their ability to inhibit platelet aggregation and adhesion [27, 104]. It was also possible to demonstrate, using a chemiluminescent analysis, that cultured endothelial cells release NO on stimulation with bradykinin [100]. It is still debated whether NO production is sufficient to explain all the properties of EDRF, and some authors have argued that the thiol derivative of NO, S-nitrosocysteine, is more equipotent with EDRF than NO alone [94]. Despite these uncertainties as to the active species of EDRF, NO or an NO-containing compound is now recognised as a major vasodilator product of vascular endothelial cells [123].

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EndothelialCell

t R~?/~/

L-arginine

.~

L-argininc ,,~No-monomcthvi-L-arginine {~,~ ~..--.., (L-NMMA) IEDRI~synthetase I ~ ~) I ~ Citrulline

NO or R-NO

Smooth Muscle C e l l f . . ~

J

~ Nitro-vasodilators

GuanylatecG~MpcyClase/.

[Relaxation 1 Fig. 1. The L-arginine-dependent metabolic pathway giving rise to NO or a thiol derivative (shown as R-NO). The NO activates soluble guanylate cyclase of smooth muscle cells, causing a rise in cyclic Y-5'-guanylate monophosphate (CGMP).

NO is synthesized from the terminal guanidino nitrogen atom of L-arginine [93, 99]. This substrate is specific to the reaction since a number of analogues including its D-enantiomer are inactive. Moreover, an analogue of L-arginine, L-NC-monomethyl arginine (L-NMMA), is a competitive and enantiomorphically specific inhibitor of NO production [106]. Indeed much of the recently defined physiological role of EDRF in vivo has resulted from the use of LNMMA to block NO production, which in turn causes a rise in vascular resistance [122]. The coproduct of NO from L-arginine is citrulline [101]. This metabolic pathway producing NO and citrulline (Fig. 1) depends upon the presence of calcium and calmodulin [102, 127].Magnesium ions seem, on the other hand, to have a dual role of enhancing or inhibiting vasorelaxation depending upon the integrity of the endothelium [4, 80]. Production of NO has been demonstrated not only in endothelial cells but also in macrophages [77] and nervous tissue [15]. Indeed, nonadrenergic and noncholinergic nerves appear to elaborate and release NO [16] and the enzyme nitric oxide synthase has been identified in nervous tissue [14]. This enzyme has now been shown to be extensively distributed in vascular endothelium [14]. The production of NO by a wide variety of cells and the occurrence in reptiles indicates an early evolutionary origin of this metabolic pathway [91].

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NO is a highly diffusible gas [ 10] having water and lipid solubilities comparable to carbon monoxide (CO) and oxygen (O:) [53]. It combines with hemoglobin 280 times faster than CO [4t]. As a result NO inhalation has been used as a measure of lung diffusing capacity [I0]. Studies in dogs [89] and humans [10] show that the major limit to uptake is the rate of diffusion through the red cell. Although it has been argued that thiol derivatives of NO would facilitate diffusion [94], in view of the high diffusibility of the native molecule, it is more likely that such thiol derivatives provide "storage," that is, to prevent NO from diffusing rapidly from the cell. It is possible that thiol derivative molecules of NO not only have a "storage" role but also impede oxidation of EDRF. The half-life of NO depends upon the concentration of associated oxidants and free radicals within the system under study [8]. The pharmacologic effect of EDRF (and NO) can be abolished by superoxide ions, while the addition of superoxide dismutase or ferricytochrome considerably lengthens the half-life of EDRF [59, 111]. These observations suggest that, in vivo, "free" EDRF is probably rapidly oxidized by locally formed free radicals. Exogenous redox molecules such as phenidone, hydroquinone, and methylene blue inhibit endothelium-dependent relaxation [85] by reacting directly with EDRF and, in the case of methylene blue, by also interfering with the activation of soluble guanylate cyclase by NO. EDRF (NO) released from the luminal side of the endothelium is likely to be rapidly inactivated by hemoglobin [85]. This explains why EDRF exerts its effect preferentially at its abluminal site and lacks a systemic effect. In normoxia NO combines with oxyhemoglobin to form methemoglobin [76]. The endogenous production of NO probably accounts for all the measurable amounts of methemoglobin found in the blood of nonsmokers [9] who, unlike smokers, are not continuously exposed to inhaled NO.

Mechanisms of Activation of Soluble Guanylate Cyclase by NO and the Role of cGMP

Nitric oxide, EDRF, and nitrovasodilators, by activating soluble or cytosolic guanylate cyclase in smooth muscle cells, increase the levels of cyclic 3' 5' guanosine monophosphate (cGMP) [62, 75]. Activation of soluble guanylate cyclase depends upon the presence of an associated heme moiety. In the presence of thiols the heme is reduced to its ferrous state, which allows it to form a paramagnetic species with NO, nitrosyl-heme, which, in turn, activates the enzyme moiety of guanylate cyclase. The mechanism for this appears similar to that of protoporphyrin IX [29, 97] and involves electron transfer. The rise in intracellular level of cGMP causes inhibition of calcium release from the sarcoplasmic reticulum and inhibition of calcium entry through receptor-operated channels, perhaps by inhibiting phosphoinositide turnover [28, 105]. Both effects inhibit smooth muscle contraction through a distinct mechanism from that of a rise in cAMP [2]. Activation of guanylate cyclase and

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increase in cGMP are also the mechanisms by which EDRF [NO] causes platelet disaggregation [17]. Much of the evidence that EDRF is continuously released under basal conditions comes from the observations that cGMP levels fall on removal of endothelium from vessels [71, 72]. It is possible to enhance the effects of EDRF [NO] by the use of phosphodiesterase inhibitors [84], which impede the breakdown of cGMP. This may offer a potential therapy for those diseases inwhich EDRF release could be suboptimal.

Other "Relaxing Factors" Derived from Endothelial Cells Not all the effects of EDRF can be explained by NO or its nitrosothiol derivative, but rely on other molecular species. This view has been suggested from studies of vessels under resting or basal conditions [65] and after stimulation with ADP [11]. Their structure remains unknown. It had also been thought that endothelial cells released an agent that causes relaxation by hyperpolarization of vascular smooth muscle [20]. This agent, called endothelium-derived hyperpolarizing factor (EDHF), probably activates one of the ATP-dependent potassium channels [117]. EDHF is released after stimulation of muscarinic Ml receptors on endothelial cells [120]. However, whether EDHF and EDRF (NO) are distinct molecular species is currently being debated. Nitric oxide can cause hyperpolarization of smooth muscle cells from a wide range of blood vessels [118]. Also L-NMMA appears to inhibit both smooth muscle relaxation and hyperpolarization [ 118], which suggests a specific relaxant action of endogenous NO induced by hyperpolarization. Furthermore, since direct physical contact between endothelial cells and smooth muscle cells occurs in vivo, it is possible that hyperpolarization induced in an endothelial cell could be directly transferred to the smooth muscle cell [31]. Such hyperpolarization of the endothelial cell can be caused by, for example, increased shear stress [119].

EDRF and Pulmonary Vasculature Much of the initial research on EDRF involved studies of systemic blood vessels. However, endothelium-dependent relaxation has been demonstrated in pulmonary arteries from animals [18] and from humans (Fig. 2) [37]. Since the vasorelaxant effects of ACh are partly inhibited by pretreatment with L-NMMA, it is likely that NO also contributes at least in part to the endotheliumdependent relaxation of the human pulmonary vascular bed [38]. However, most studies have been of larger conduit pulmonary blood arteries and evidence from porcine pulmonary arteries indicates a different pattern of EDRF release between larger than smaller vessels [131]: the smaller the vessel the larger the release of EDRF. It is particularly difficult to define, as a result of its complex branching

EDRF and the Pulmonary Circulation

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With Endothelium Acelylcholine (-log M)

Without Endothelium

lo 9

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Fig. 2. Typical response of human pulmonary arterial rings, with and without endothelium, to cumulative doses of acetylcholine and adenosine diphosphate. Each ring was precontracted with phenylephrine (PE; 10 -6 M). Sodium nitroprusside (NP; 10 -4 M) was added at the end of the experiment to assess endothelium-independent relaxation (reproduced with kind permission from ref. 37).

structure, where in the pulmonary vascular bed the greatest resistance to flow is sited and where the predominant regulation of resistance to pulmonary blood flow occurs. Estimation of longitudinal resistance using direct measurement of pressures in the pulmonary vessels suggest that the greatest resistance lies in arteries greater than 0.9 mm [61] and these vessels are influenced by vasoactive agents. Hypoxic pulmonary vasoconstriction, however, appears to involve smaller and more distensible arteries [60]. Finally, although the vertical distribution of lung perfusion is mainly governed by the passive relationship among alveolar, pulmonary arterial, and venous pressures, the reduced perfusion of the most dependent regions of the lung is determined by vascular tone [96]. Reduction in this tone increases basal perfusion but only has limited effects upon pulmonary vascular resistance. It is assumed that alveolar hypoxia, through reduced ventilation, is the principal mechanism of this regulatory mechanism. Basal pulmonary vascular tone is uniquely low in the lung and appears important in determining the nature of the response [69] since when PVR is elevated, infusion into the pulmonary artery of ACh causes vasodilatation in both rabbits [22] and cats [95]. This response, although unaffected by cyclo- or lipo-oxygenase inhibitors, is abolished by atropine, hemoglobin, and quinacrine, all known to be inhibitors of the effects of EDRF. ACh therefore probably acts

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192

PVR (mmHg.L-i .mn1 ) 20

15

10

I

BL

I

NO

I

Air

I

NO

I

Air

Fig. 3. The effect of breathing 40 p p m N O in air or air on the m e a n PVR of 8 patients with severe pulmonary hypertension. A significant reduction of PVR was o b s e r v e d during the 2 periods of inhalation of N O (from ref. 67). B L indicates baseline, ** p < 0.01.

as a vasodilator through release of EDRF. There is evidence in the systemic circulation (e.g., mesenteric [51], renal [6], and hindlimb [45] as well as arm in humans [122]) that NO is continuously released from the endothelium, thus regulating basal vascular tone [56]. Despite the difficulties in identifying the major resistance vessels in the lung, studies in isolated perfused lungs [3, 86, 114] have shown an enhanced constrictor response to hypoxia on infusion of methylene blue, although baseline resistance values were unchanged in normoxic conditions. Preliminary studies [30] in blood free isolated perfused lung preparation in humans, the lung having been obtained from patients undergoing heart-lung transplantation (lILT), show that baseline pulmonary vascular resistance rises on infusion of methylene blue. This suggests that EDRF is being released continuously and plays a role in the determination of basal pulmonary vascular resistance (PVR). Similar results are obtained in vivo. ACh infusion during right heart catheterization lowers elevated PVR in pulmonary hypertensive patients but has little effect in individuals with normal PVR values [47]. Similarly inhaled NO (40 ppm in air) lowers PVR in patients with severe pulmonary hypertension (Fig. 3) but not in those with normal values of PVR [64]. EDRF may also be important in the adaptation of the pulmonary vasculature to increased blood flow in exercise. Experimental disruption of pulmonary endothelium causes an increased pulmonary artery pressure response to increased blood flow [88].

E D R F and Vascular Disease

A suggested model for vascular disease is one in which the endothelium is disrupted. Extensive vascular injury would be required for this model to explain disease. Lesser degrees of "injury" may alter the function of the endothelium while not disrupting its integrity. For example, experimentally induced systemic hypertension is associated with a significant impairment of the release of EDRF

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with ACh [83]. In experimental models ofatherosclerosis, hypercholesterolemic rabbits [26, 124] and monkeys [46] show evidence of impaired release of EDRF. When the animal resumes a diet with a normal cholesterol content, EDRF release returns to normal although the intimal thickening of the arteries associated with the atherosclerosis fails to reverse [63]. In humans, reduced basal and stimulated release of NO has been observed in atherosclerotic coronary arteries [23]. These observations support the idea that reversible dysfunction of the endothelium could occur in disease.

Hypoxic Pulmonary Vasoconstriction In addition to the structural changes of the pulmonary vasculature that can occur with chronic alveolar hypoxia, acute hypoxia can cause vasoconstriction and a rise in pulmonary vascular resistance in most mammals [15]. Neural factors are not thought to be involved. Heart-lung transplantation, which leads to irreversible pulmonary denervation [107], does not blunt the appropriate hypoxic vasoconstriction when studied at right heart catheterization [116]. Humoral mediators (e.g., catecholamines, histamine, angiotensin II, serotonin and prostaglandins) all act as modulators of vascular tone but none has emerged as a mediator of the hypoxic response [129]. Despite early promise, leukotrienes have failed to explain the response [74]. Since both alveolar and mixed venous blood hypoxia can cause pulmonary vasoconstriction [87] and isolated pulmonary arteries respond to hypoxia [81], it is likely that the sensor resides in the lung and is in close proximity and linked to the contractile elements of the pulmonary vasculature. Weir [128] suggested in 1978 that suppression of normoxic vasodilatation could be the cause of hypoxic vasoconstriction.

EDRF and Experimental Pulmonary Hypoxia Hypoxia-induced contraction of isolated strips of pulmonary artery is dependent on the presence ofendothelium [66] and does not involve eicosanoids, catecholamines, or ACh. Although there is evidence that EDRF is released acutely following a hypoxic challenge [13, 115], the endothelium-dependent relaxation in response to pharmacologic stimuli is reduced with both acute [73] and chronic hypoxia [98]. Thirty minutes of hypoxia is sufficient to impair the release of EDRF from cultured endothelial cells when stimulated by bradykinin [126]. Part of this may also be explained by impaired activation of guanylate cyclase in pulmonary arteries by EDRF in hypoxic conditions [108]. It is pertinent to remember that soluble guanylate cyclase is associated with, and dependent on, a heme moiety [29] and that its activity is enhanced under hyperoxic conditions [107]. By implication, hypoxia is likely to impair the function of this enzyme. This might explain in part the sigmoid relationship between hypoxic vasoconstriction and oxygen tension. Guanylate cyclase is one of the types of

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enzyme capable of undergoing rapid adaptation. Recovery of its normal activity despite hypoxia may explain the restoration of normal relaxation with prolonged hypoxia. Redox molecules such as methylene blue and hydroquinone [13, 86] potentiate the hypoxic vasopressor response in rat lungs pretreated with cyclo-oxygenase inhibitors. This suggests either impaired EDRF release or reduced efficacy on smooth muscle. We can conclude that acute hypoxia may limit the effect of EDRF. This is probably because hypoxia limits the ability of pulmonary vascular smooth muscle to relax by inhibiting soluble guanylate cyclase. Although it remains possible that EDRF release is reduced with acute hypoxia, however, the question is how such experimental findings relate to the pathophysiological and histopathologic findings (structural changes) of chronic hypoxic lung disease, in particular whether the endothelium function becomes persistently impaired with chronic hypoxia.

Pathophysiology of Secondary Pulmonary Hypertension The success of heart-lung transplantation as a treatment for end-stage chronic lung disease and severe pulmonary vascular disease [681 has provided the opportunity to study in vitro the pathophysiology of pulmonary vascular disease. Studies of conduit pulmonary arteries from patients with cor pulmonale and secondary pulmonary hypertension from congenital heart disease show impaired endothelium-dependent relaxation (Fig. 4) [34, 35]. Since the vascular rings respond normally to nitroprusside, this suggests impaired release of EDRF. In addition, L-NMMA potentiates these effects [38]. When the preoperative arterial blood gas levels were known, as in 18 patients with chronic lung disease, the impairment of release of EDRF with both ACh and ADP was correlated with the degree of hypoxia (Fig. 5) [39]. The lower the oxygen tension, the poorer the endothelium-dependent relaxation. These observations suggest that normoxia necessary prerequisite for the normal release of EDRF. An interesting physiological and morphologic correlation was observed. The degree of intimal thickening of the studied blood vessels correlated with the impaired release of EDRF. It is possible that failure to release EDRF stimulates fibromuscular hyperplasia of the intima. The physiological correlate with the impaired endothelium relaxation in patients with chronic tung disease could be the recognized rise in pulmonary artery pressure, since cardiac output increases with exercise in patients with chronic obstructive lung disease [62]. Their pulmonary vascular bed, unlike that of normal individuals, fails to adapt to situations of increased blood flow such as occur during exercise.

195

EDRF and the Pulmonary Circulation

200 C -.~_. c-' 0...

%. ¢03 cO-

150

o cO 0

100

cO O

E .__q

Fig. 4. The pulmonary arteries from patients with Eisenmenger's syndrome (n = 4) (full squares) show impaired relaxation with acetylcholine compared with controls (n = 4) (full circles). The open symbols represent the mean values of tension in vessels where the endothelium was removed (reproduced with kind permission from ref. 35).

50

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|

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-10

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I

I

I

I

-9

-8

-7

-6

-5

log [Acetylcholine] (M)

1 O0

r = 0.59 p < 0.01

0

~9

80

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0

60

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5

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Pa02 (kPa)

Fig. 5. Correlation between maximal relaxation to acetylcholine of pulmonary arterial rings obtained from 18 patients with chronic obstructive lung disease and their preoperative values of arterial oxygen tension (reproduced with kind permission from ref. 39).

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Aggregating platelets

Hypoxia

Fig. 6. Stimulation, synthesis, and action of nitric oxide (NO) in the vessel wall. Various physical and pharmacologic agents such as adenosine diphosphate (ADP), serotonin (5-HT), and others act through specific receptors on the vascular endothelium and stimulate the enzyme NO synthase to convert L-arginine to NO and L-citrulline. The released NO or its thiol derivative (R-NO) stimulates soluble guanylate cyclase, which in turn produces cyclic 3'-5'-guanylate monophosphate (cGMP), which acts on the smooth muscle causing relaxation. The rise in cGMP has a negative feedback on NO synthase. Nitric oxide also causes hyperpolarization and consequent relaxation of the smooth muscle cells. Some endothelium-dependent vasorelaxants such as acetylcholine (ACh) and arachidonic acid (AA) exert their action also by separate pathways such as the production of endotheliumderived hyperpolarizing factor and prostacyclin (PGI2).

Conclusion E v i d e n c e is a c c u m u l a t i n g to s u p p o r t a p h y s i o l o g i c a l r o l e o f E D R F in r e g u l a t i n g pulmonary vascular resistance. This may be particularly important for the adapt a t i o n o f t h e p u l m o n a r y v a s c u l a r b e d to t h e i n c r e a s e d b l o o d flow d u r i n g e x e r c i s e , as well as r e s p o n s e to h u m o r a l l y r e l e a s e d a g e n t s (Fig. 6). H y p o x i c p u l m o n a r y vasoconstriction may likewise represent impaired release and effect of EDRF o n s m o o t h m u s c l e cells, w h i l e c h r o n i c h y p o x i a m a y i m p a i r E D R F r e l e a s e . M u c h still n e e d s to b e l e a r n e d , p a r t i c u l a r l y w i t h r e s p e c t to t h e d e t e r m i n a n t s o f t h e "balance" between vasorelaxants and vasoconstrictive agents released and elaborated by endothelium.

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Acknowledgments. This work was supported by the British Heart Foundation, Cystic Fibrosis Trust, and the Wellcome Foundation.

References 1. Abacioglu N, Ercan ZS, Kanzik L, Zengil H, Demiryurek T, Turker RK (1987) Endotheliumdependent relaxing effect of histamine on the isolated guinea pig main pulmonary artery strips. Agents Action 22:30-35 2. Adelstein RS, Hathaway DR (1979) Role of calcium and cyclic adenosine 3' 5'-monophosphate in regulatory smooth muscle contraction. Am J Cardiot 44:783-787 3. Adnot S, Raffestin B, Eddahibi S, Braquet P, Chabrier P-E (1991) Loss of endotheliumdependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J Clin Invest 87:155-162 4. Altura BT, Altura BM (1987) Endothelium-dependent relaxation in coronary arteries requires magnesium ions. Br J Pharmacol 91:449-451 5. Altura BM, Chand N (1981) Bradykinin-induced relaxation of renal and pulmonary arteries is dependent on intact endothelial cells. Br J Pharmacol 74:10-11 6. Bhardwaj R, Moore PK (1989) The effect of arginine and nitric oxide on resistance blood vessels of the perfused rat kidney. Br J Pharmacol 97:739-744 7. Bolton TB, Clapp LH (1986) Endothelial-dependent relaxant actions of carbachol and substance P in arterial smooth muscle. Br J Pharmacol 87:713-723 8. Borland CDR, Chamberlain AT, Higenbottam TW, Barber RW, Thrush BA (1985) A comparison between the rates of reaction of nitric oxide in the gas phase and in whole cigarette smoke. Beitr Tabakforsch Int 13:67-73 9. Borland CDR, Harmes K, Cracknell N, Mack D, Higenbottam TW (1985) Methaemoglobin levels in smokers and non-smokers. Arch Environ Health 40:330-333 10. Borland CDR, Higenbottam TW (1989) A simultaneous single breath measurement of pulmonary diffusing capacity with nitric oxide and carbon monoxide. Eur Respir J 2:56-63 11. Boulanger C, Henderikson H, Lorenz RR, Vanhoutte PM (I989) Release of different relaxing factors by cultured porcine endothelial cells. Circ Res 64:1070-1078 12. Brain SD, Williams TJ, Tippins JR, Morris H, Maclntyre I (1985) Calitonin gene-related peptide is a potent vasodilator. Nature 313:54-56 13. Brashers VL, Peach MJ, Rose CE Jr (1988) Augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation. J Clin Invest 82:1495-1502 14. Bredt DS, Hwang PM, Snyder SH (1990) Localization of nitric oxide synthetase indicating a neural role for nitric oxide. Nature 437:768-770 15. Bredt DS, Snyder SH (1990) Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 87:682-685 16. Bult H, Boeckxstaens GE, Pelckmans PA, Jordaens FH, Van Maercke YM, Herman AG (1990) Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter. Nature 345:346-347 17. Busse R (1987) Stimulation of soluble guanylate cyclase activity by endothelium-derived relaxing factor: a general principle of its vasodilator and anti-aggregatory properties. Thromb Res Suppl VII:3-13. 18. Chand N, Altura BM (1981) Acetylcholine and bradykinin relax intrapulmonary arteries by acting on endothelial ceils: role in lung vascular disease. Science 213:1376-1379 19. Chand N, Altura BM (1981) Bradykinin and arachidonic acid, but not beta adrenergic or histaminergic vasorelaxation is mediated by intact endothelial cells. Clin Res 29:443-447 20. Chen GF, Suzuki H (1989) Direct and indirect actions of acetylcholine and histamine on intra pulmonary artery and vein muscles of the rat. Jpn J Physiol 39:51-65 21. Chen GF, Suzuki H (1990) Calcium dependency of the endothelium-dependent hyperpolarization in smooth muscle cells of the rabbit carotid artery. J Physiol (Lond) 421:521-534

198

G. Cremona et al.

22. Cherry PD, Gillis CN (1987) Evidence for the role of endothelium-derived relaxing factor in acetylcholine-induced vasodilatation in the intact lung. J Pharmacol Exp Ther 241:516-520 23. Chester AH, O'Neil GS, Moncada S, Tadjkarimi, Yacoub MH (1990) Low basal and stimulated release of nitric oxide in atherosclerotic epicardial coronary arteries. Lancet 336:897-900. 24. Christie MI, Griffith TM, Lewis MJ (1989) A comparison of basal and agonist-stimulated release of endothelium-derived relaxing factor from different arteries. Br J Pharmacol 98:397--406 25. Cocks TM, Angus JA, Campbell JH, Campbell GR (1985) Release and properties of endothelium-derived relaxing factor (EDRF) from endothelial cells in culture. J Cell Physiol 123:310-320 26. Coene M-C, Herman AG, Jordaens F, Van Hove C, Verbeuren TJ, Zamekeyn LL (1985) Endothelium-dependent relaxations in isolated arteries of control and hypercholesterolemic rabbits. Br J Pharmacol 85:267p 27. Cohen RA, Shepherd JT, Vanhoutte PM (I983) Inhibitory role of the endothet[um in the response of isolated coronary arteries to platelets. Science 22:273-274 28. Collins P, Griffith TM, Henderson AH, Lewis MJ (1986) Endothelium-derived relaxing factor alters calcium fluxes in rabbit aorta: a cyclic guanosine monophosphate-mediated effect. J Physiol (Lond) 381:427-437 29. Craven PA, DeRubertis FR (1978) Restoration of the responsiveness of purified guanylate cyclase to nitroguanidine, nitric oxide and related activators by heine and heme proteins: evidence for the involvement of the paramagnetic nitrosyl-heme complex in enzyme activation. J Biol Chem 253:8433-8443 30. Cremona G, Higenbottam TW, Dinh Xuan AT, Wells FC, Large S, Stewart S, Wallwork J (1991) Influence of endothelium-derived relaxing factor (EDRF) on basal vascular resistance in isolated perfused human lungs. Thorax 46:283P (abstract) 31. Davies PF (1989) How do vascular endothelial cells respond to flow? NIPS 4:22-25. 32. De Mey JG, Claeys M, Vanhoutte PM (1982) Endothelium-dependent inhibitory effects of acetylcholine, adenosine triphosphate, thrombin and arachidonic acid in the canine femoral artery. J Pharmacol Exp Ther 222:166-173 33. De Mey JG, Vanhoutte PM (1982) Heterogenous behaviour of the canine arterial and venous wall. Circ Res 51:439-447 34. Dinh Xuan AT, Higenbottam TW, Pepke-Zaba J, Clelland C, Wallwork J (1989) Reduced endothelium-dependent relaxation of cystic fibrosis pulmonary arteries. Eur J Pharmacol 163:401-403 34. Dinh Xuan AT, Higenbottam TW, Clelland C, Pepke-Zaba J, Cremona G, Wallwork J (1990) Impairment of pulmonary endothelium-dependent relaxation in patients with Eisenmenger's syndrome. Br J Pharmacol 99:9-10 36. Dinh Xuan AT, Higenbottam TW (1989) Non-prostanoid endothelium-derived vasoactive factors. J Int Med Res 17:305-315 37. Dinh Xuan AT, Higenbottam TW, Clelland C, Pepke-Zaba J, Wells FC, Wallwork J (1990) Acetylcholine and adenosine disphosphate cause endothelium-dependent relaxation of isolated human pulmonary arteries. Eur Respir J 3:633-638 38. Dinh Xuan AT, Higenbottam TW, Pepke-Zaba J, Clelland CA, Wells FC, WaUwork J (1990) The L-arginine analogue, NG-monomethyl-L-arginine, inhibits endothelium-dependent relaxation of human pulmonary arteries. Am Rev Respir Dis 141(4):A350 (abstract) 39. Dinh Xuan AT, Higenbottam TW, WaUwork J (1991) Relationship between chronic hypoxia and in vitro relaxation mediated by endothetium-derived relaxing factor in human chronic obstructive lung disease. Angiology 41: (in press) 40. D'Orleans-Juste P, Dione S, Mizrahe J, Regoli D (1985) Effects of peptides and non-peptides on isolated arterial smooth muscles: role of endothelium. Eur J Pharmacol 114:9-21 41. Doyle MP, Hoekstra JW (1981) Oxidation of nitrogen oxides by bound dioxygen in haemoproteins. J Inorg Biochem 14:351-358 42. Durand J, Leroy Ladurie M, Ranson-Bitker B (1970) Effects of hypoxia and hypercapnia on the repartition of pulmonary blood flow in supine subjects. Prog Respir Res 5:156.

EDRF and the Pulmonary Circulation

199

43. Ehrasam RE, Perruchoud A, Oberholzer M, Burkhart F, Herzog H (1983) Influence of age on pulmonary haemodynamics at rest and during supine exercise. Clin Sci 65:653-660. 44. Fishman AP (1976) Hypoxia on the pulmonary circulation: how and where it acts. Circ Res 38:221-231 45. Forstermann U, Dudel C, Frolich JC (1987) Endothelium-derived relaxing factor is likely to modulate the tone of resistance arteries in rabbit hindlimb in vivo. J Pharmacol Exp Ther 243:1055-1061 46. Freiman PC, KitcheU GG, Heistod DD, Armstrong ML, Harrison DG (1986) Atherosclerosis impairs endothelium-dependent vascular relaxation to acetylcholine and thrombin in primates. Circ Res 58:783-789 47. Fritts AW, Harris P, Clauss H, OdeU JE, Cournand A (1958) The effect of acetylcholine on the human pulmonary circulation under normal and hypoxic condition. J Clin Invest 37:99-108 48. Furchgott RF (1983) Role of endothelium in responses of vascular smooth muscle. Circ Res 53:557-573 49. Furchgott RF (1984) The role of the endothelium in the responses of vascular smooth muscle to drugs. Annu Rev Pharmacol Toxicol 24:175-197 50. Furchgott RF (1988) Studies on the relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activatable inhibitory factor from bovine retractor penis factor is inorganic nitric oxide and endothelium-derived relaxing factor is nitric oxide. In: Vanhoutte PM (ed). Vasodilatation, vascular smooth muscle, peptides, autonomic nerves and endothelium. Raven Press, New York, pp 401-404 51. Furchgott RF, Carvalho MH, Khan MT, Matsunaga K (1987) Evidence for endotheliumdependent vasodilatation of resistance vessels by acetylcholine. Blood Vessels 24:145-9 52. Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373-376 53. Gerrard W (1980) In: Gas solubilities: widespread applications. Pergamon Press, Oxford, .Chap. 15, pp. 347-358 54. Grasen T, Leisner H, Tiedt N (1986) Absence of role of endothelium in response of isolated porcine coronary arteries to acetylcholine. Cardiovasc Res 20:299-302 55. Griffith TM, Edwards DH, Lewis MJ, Newby AC, Henderson AH (1984) The nature of endo}helium-derived relaxing factor. Nature 308:645-647 56. Griffith TM, Edwards DH, Davies RL, Harrison TJ, Evans KT (1987) EDRF coordinates the behaviour of vascular resistance vessels. Nature 329:442-445. 57. Griffith TM, Henderson AH, Edwards OH, Lewis MJ (1984) Isolated perfused rabbit coronary artery and aortic strip preparations: the role of endothelium-derived relaxant factor. J Physiol (Lond) 351:13-24 58. Gruetter CA, Barry BK, McNamara DB, Gruetter DY, Kadowitz PJ, Ignarro LJ (1979) Relaxation of bovine coronary artery and activation of coronary arery guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosoamine. J Cyclic Nucleotide Protein Morph Res 5:211-224 59. Gryglewski RJ, Palmer RMJ, Moncada S (1986) S uperoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320:454-456 60. Hakim TS, Michel RP, Minami H, Chang HK (1983) Site of pulmonary hypoxic vasoconstriction studied with arterial and venous occlusion. J Appl Physiol 54:1298-1302 61. Hakim TS, Kelly S (1989) Occlusion vs micropipette pressures in the pulmonary circulation. J Appl Physiol 67:1277-1185 62. Harris P, Heath D (1986) Pulmonary haemodynamics in chronic bronchitis and emphysema. In: The human pulmonary circulation. Churchill Livingstone, Edinburgh, pp 522-544 63. Harrison DG, Armstrong ML, Freiman PC, Heistod DD (1987) Restoration of endotheliumdependent relaxation by dietary treatment of atherosclerosis. J Clin Invest 80:1808-1811 64. Higenbottam TW, Pepke-Zaha J, Scott J, Wallwork J (1988) Inhaled endothelium-derived relaxing factor in primary pulmonary hypertension. Am Rev Respir Dis 137:A107 (abstract) 65. Hoeffner U, Boulanger C, Vanhoutte PM (1989) Proximal and distal dog coronary arteries respond differently to basal EDRF but not to NO. Am J Physiol 256:H828-H831.

200

G. Cremona et al.

66. Holden WE, McCall E (1984) Hypoxia-induced contraction of porcine pulmonary artery strips depend on intact endothelium. Exp Lung Res 7:101-112 67. Holtz J, Forstermann U, Pohl U, Giesler M, Bassenge E (1984) Flow-dependent endotheliummediated dilatation of epicardial coronary arteries in conscious dogs: effects of cyclooxygenase inhibitor. J Cardiovasc Pharmacol 6:1161-1169 68. Hutter JA, Despins P, Higenbottam TW, Stewart S, Wallwork J (1988) Heart-lung transplantation: better use of resources. Am J Med 85:4-11 69. Hyman AL, Kadowitz PJ (1989) Influence of tone on responses to acetylcholine in the rabbit pulmonary vascular bed. J Appl Physiol 67:1388-1394 70. Ignarro LJ, Buga GM, Wood KS, Byrns RE (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 84:9265-9269 71. Ignarro L J, Byrns RE, Buga GM, Wood KS (1987) Mechanisms of endothelium-dependent vascular smooth muscle relaxation elicited by bradykinin and VIP. Am J Physiol 253:H 1074--H 1082 72. Ignarro LJ, Harbison RG, Wood KS, Kadowitz PJ (1986) Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J Pharmacol Exp Ther 237:893-900 73. Johns RA, Linden JM, Peach MJ (1989) Endothelium-dependent relaxation and cyclic GMP accumulation in rabbit pulmonary artery are selectively impaired by moderate hypoxia. Circ Res 65:1508-1515 74. Kadowitz PG, Hyman AL (1984) Analysis of leukotriene D4 in the pulmonary vascular bed. Circ Res 55:707-717 75. Katsuki S, Arnold W, Mittal C, Murad F (1977) Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerine and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. J Cyclic Nucleotide Res 3:23-35. 76. Kiese M (1973) Ferrihemoglobin in normal blood. In: Methaemoglobinemia. A comprehensive test. CRC Press, Cleveland, Chap 3, p 12 77. Knowles RG, Palacios M, Palmer RMJ, Moncada S (1989) Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci USA 86:5159-5162 78. Komori K, Suzuki H (1987) Heterogenous distribution of muscarinic receptors in the rabbit saphenous artery. Br J Pharmacol 92:657-664 79. Komori K, Suzuki H (1987) Electrical responses of smooth muscle cells during cholinergic vasodilatation in the rabbit saphenous artery. Circ Res 61:586-593 80. Ku DD, Ann HS (1987) Magnesium deficiency produces endothelium-dependent vasorelaxation in canine coronary arteries. J Pharmacol Exp Ther 241:961-966 81. Lloyd TC (1968) Hypoxic pulmonary vasoconstriction: role of perivascular tissue. 25:560-565 82. Luscher TF, Diederich D, Siebenmann R, Lehmann K, Stulz P, yon Segesser L, Yang Z, Turina M, Gradel E, Weber E, Buhler FR (1988) Difference between endothetium-dependent relaxation in arterial and in venous coronary bypass grafts. N Engl J Med 319:462-467 83. Luscher TF, Vanhoutte PM (1986) Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension 8:344-348 84. Martin W, Furchgott RF, Villani GM, Jothianandan D (1986) Phosphodiesterase inhibitors induce endothelium-dependent relaxation of rat and rabbit aorta by potentiating the effects of spontaneously released endothelium-derived relaxing factor. J Pharmacol Exp Ther 237:539-547 85. Martin W, Villani GM, Jothianandan D, Furchgott RF (1985) Selective blockade of endotheliurn-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J Pharmacol Exp Ther 232:708-716 86. Mazmanian GM, Baudet B, Brink C, Cerrina J, Kirkiacharian S, Weiss M (1989) Methylene blue potentiates vascular reactivity in isolated rat lungs. J Appl Physiol 66:1040-1045 87. McMurty IF, Petrum MD, Reeves JT (1978) Lungs from chronically hypoxic rats have decreased pressor response to acute hypoxia. Am J Physiol 235:H104-H109

EDRF and the Pulmonary Circulation

201

88. Melkumyants AM, Balashov SA, Khayutin VM (1989) Endothelium dependent control of arterial diameter by blood viscosity. Cardiovasc Res 23:741-747 89. Meyer M, Schuster KD, Schulz H, Mohr M, Piiper J (1990) Pulmonary diffusing capacities for nitric oxide and carbon monoxide determined by rebreathing in dogs. J Appl Physiol 68:2344-2357 90. Miller VM, Aarhus LL, Vanhoutte PM (1986) Modulation of endothelium-dependent responses by chronic alteration of blood flow. Am J Physiol 251:H520-H527 91. Miller VM, Vanhoutte PM (1986) Endothelium-dependent responses in isolated blood vessels of lower vertebrates. Blood Vessels 23:225-235 92. Moncada S, Gryglewski RJ, Bunting S, Vane JR (1976) An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663-665 93. Moncada S, Palmer RMJ, Higgs EA (1989) Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication. Biochem Pharmacol 38:1709-1715 94. Myers PR, Minor RL, Guerra R, Bates JN, Harrison DG (1990) Vasorelaxant properties of the endothelium-derived relaxing factor more closely resembles S-nitrosocysteine than nitric oxide. Nature 345:161-163 95. Nandiwada PA, Hyman AL, Kadowitz PJ (1983) Pulmonary vasodilator response to vagal stimulation and acelylcholine in ¢'e cat. Circ Res 53:86-95 96. Nemery B, Wijns W, Piret L, Cauwe F, Brasseur L, Frans A (t983) Pulmonary vascular tone is a determinant of basal lung peffusion in normal seated subjects. J Appl Physiol 54:262266 97. Ohlstein EH, Wood KS, Ignarro LJ (1982) Purification and properties of heme-deficient hepatic soluble guanylate cyclase: effects of heine and other factors on enzyme activation by NO, HO-heme and protoporphyrin IX. Arch Biochem Biophys 218:187-198 98. Orton EC, Reeves JT, Stenmark KR (1988) Pulmonary vasodilation with structurally altered pulmonary vessels and pulmonary hypertension. J Appl Physiol 65:2459-2467 99. Palmer RMJ, Ashton DS, Moncada S (1988) Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333:664-666 100. Palmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524-526 101. Palmer RMJ, Moncada S (1989) A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun 158:348-352 102. Peach MJ, Singer HA, Izzo N J, Loeb AL (1987) Role of calcium in endothelium-dependent relaxation of arterial smooth muscle. Am J Cardiol 59:35A-43A 103. Pinto A, Abraham NG, McKane KM (1986) Cytochrome P450-dependent mono-oxygenase activity and endothelial-dependent relaxations induced by arachidonic acid. J Pharmacol Exp Ther 236:445-451 104. Radomski MW, Palmer RMJ, Moncada S (1987) Endogenous nitric oxide inhibits human platelets adhesion to vascular endothelium. Lancet 2:1057-1058 105. Rapoport RM (1986) Cyclic guanosine monophosphate inhibition of contraction may be mediated through inhibition of phosphatidyl-inositol hydrolysis in rat aorta. Circ Res 58:407-410 106. Rees DD, Palmer RMJ, Hodson HF, Moncada S (1989) A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol 96:418-424 107. Robin ED, Theodore J, Burke CM, Oesterle SN, Fowler MB, Jamieson SW, Baldwin JL, Morris AJ, Hunt SA, Vankessel A, Stimson EB, Shumway NE (1987). Hypoxic pulmonary vasoconstriction persists in the human transplanted lungs. Clin Sci 72:283-287 108. Rodman DM, Yamaguchi T, Hasunuma K, O'Brien RF, McMurtry IF (1990) Effects of hypoxia on endothelium-dependent relaxation of rat pulmonary artery. Am J Physiol 258:L207-L214 109. Rodman DM, Yamaguchi T, O'Brien RF, McMurtry I (1988) Methylene blue enhances hypoxic contraction in isolated rat pulmonary arteries. Chest 93:93s-94s (abst): 1278

202

G. Cremona et al.

110. Rubanyi GM, Romero JC, Vanhoutte PM (1986) Flow-induced release of endothelium-derived relaxing factor. Am J Physiol 250:H1145-H1149 111. Rubanyi GM, Vanhoutte PM (1986) Superoxide anion and hyperoxia inactivate endothelium derived relaxing factor. Am J Physiol 250:H222-H227 112. Rubanyi GM, Vanhoutte PM (1988) Heterogeneity of endothelium-dependent responses to acetycholine in canine femoral arteries and veins. Separation of the role played by endothelial and smooth muscle cells. Blood Vessels 25:75-81 113. Rudolph AM, Kurland MD, Auld PAM, Paul MH (1959) Effects of vasodilator drugs on normal and serotonin-constricted pulmonary vessels of the dog. Am J Physiol 197:617-623 114. Sakuma I, Gross SS, Levi R (1987) Peptidoleukotrienes induce an endothelium-dependent relaxation of guinea pig main pulmonary artery and thoracic aorta. Prostaglandins 34:685-696 115. Liu S, Crawley DE, Barnes P J, Evans TW ( 199 I) Endothelium-derived relaxing factor inhibits hypoxic vasoconstriction in rats. Am Rev Respir Dis 143:32-37 116. Springall DR, Polak JM, Howard L, Power RF, Krausz T, Manickom S, Bamien NR, Khagani A, Rose M, Yacoub MH (1990) Persistence of intrinsic neurones and possible phenotypic changes after extrinsic denervation of human respiratory tract by heart-lung transplantation. 141:1538-1546 117. Standen NB, Quayle JM, Davies NW, Brayden JE, Huang T, Nelson MT (1989) Hyperpolarizing vasodilators activate ATP-sensitive K + channels in arterial smooth muscle. Science 245:177-180 118. Tare M, Parkington HC, Coleman HA, Neild TO, Dusting GJ (1990) Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium. Nature 346:69-71 119. Taugner R, Kirckheim H, Forssmann WG (1984) Myoendothelial contacts in glomerular arterioles and in renal interlobular arteries of rat, mouse and Tupaia belangeri. Cell Tissue Res 235:319-325. 120. Taylor SG, Southerthon JS, Weston AH, Baker JRJ (1988) Endothelium-dependent effects of acetylcholine in rat aorta: a comparison with sodium nitroprusside and cromakalim. Br J Pharmacol 94:853-863. 121. Thulesius O, Ugaily-Thulesius L, Neglen P, Shuhaiber H (1988) The role of the endothelium in the control of venous tone: studies on isolated human veins. Clin Physiol 8:359-366 122. Vallance P, Collier J, Moncada S (1989) Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 2:997-1000 123. Vane JR, Angaard EE, Botting RM (1990) Regulatory functions of the vascular endothelium N Engl J Med 323:27-36 124. Verbeuren TJ, Jordaens FH, Zammeheyn LL, Van Hove CE, Coene M-C, Herman AG (1986) Effect of hypercholesterolemia on vascular reactivity in the rabbit: endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits. Circ Res 58:552-564 125. Von Euler U, Liljestrand G (i946) Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand 12:301-320 126. Warren JB, Maltby NH, McCormack D, Barnes PJ (1989) Pulmonary endothelium-derived relaxing factor is impaired in hypoxia. Clin Sci 77:671-676 127. Weinheimer G, Oswald H (1986) Inhibition of endothelium-dependent smooth muscle relaxation by calmodulin antagonists. Arch Pharmacol 332:391-397 128. Weir EK (1978) Does normoxic pulmonary vasodilatation rather than hypoxic vasoconstriction account for the pulmonary pressor response to hypoxia. Lancet 1:476-477 129. Weir EK (1984) In: Weir EK, Reeves JT (eds) Pulmonary hypertension. Futura, New York, p 251 130. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki "Y, Goto K, Masaki T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-415 131. Zellers TM, VanHoutte PM (1989) Heterogeneity of endothelium-dependent and independent responses among large and small pulmonary arteries. Pulmon Pharmacol 2:201-208 Accepted for publication: 2 March 1991