PHYSIOLOGICAL SOCIETY SYMPOSIUM: CONTROL OF THE PULMONARY CIRCULATION LUNG DISEASE AND PULMONARY ENDOTHELIAL NITRIC OXIDE

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1 Experimental Physiology (1995), 80, Printed in Great Britain PHYSIOLOGICAL SOCIETY SYMPOSIUM: CONTROL OF THE PULMONARY CIRCULATION LUNG DISEASE AND PULMONARY ENDOTHELIAL NITRIC OXIDE TIM HIGENBOTTAM Department ofrespiratory Physiology, Papworth and Addenbrookes Hospitals, Cambridge CB3 8RE, UK (MANUSCRIPT RECEIVED 22 JUNE 1995, ACCEPTED 11 JULY 1995) BACKGROUND The uncovering of nitric oxide (NO) and the L-arginine-citrulline metabolic pathway by which it is formed represent remarkable and unexpected discoveries in biological science. The discovery of this whole new system of physiological and immunological regulation began with the finding of endothelium-derived relaxing factor (EDRF) by Robert Furchgott (Furchgott & Zawadzki, 1980). It soon became evident that EDRF was the gas NO (Palmer, Ferrige & Moncada, 1987; Feelisch, te Poel, Zamora, Deussen & Moncada, 1994) and that its production was not limited to the endothelium. The enzyme nitric oxide synthase (NOS), which elaborates NO fromn L-arginine, is found in most cells of the body (Nathan & Xie, 1994) and arose early in evolution (Gelperin, 1994). NOS has three distinct isoforms, each with a separate gene located on different chromosomes (Nathan & Xie, 1994). The major functional difference between the isoforms of NOS is in their need for Ca2+. The Ca2+-independent isoform, called previously inducible or inos, has been found in most cells. Its expression can be induced by cytokines. A Ca2+-dependent isoform is ncnos, which was first identified in nerve cells but has since been found in epithelial, skeletal and smooth muscle cells. The endothelial isoform is also Ca2' dependent and is called ecnos. It is also found in other cell types. The lung has a complex network of NO-producing cells. We are only just beginning to differentiate the individual functions of NO in the different systems and know little of its effects in organs. These studies require an integreted approach, involving molecular and cellular biology together with whole organ physiology. Endothelial NO diffuses to the smooth muscle cells, where it combines with the haem moiety of the soluble guanylate cyclase enzyme (Ignarro, Harbison, Wood & Kadowitz, 1986). This guanylate cyclase is activated by NO and increases the intracellular concentration of cyclic guanosine monophosphate (cgmp). The second messenger cgmp causes relaxation and reduction of tone in the smooth muscle cell (Ignarro, 1990). This review considers the role of endothelial NO in the control of the pulmonary circulation. Presented at the Oxford Meeting of the Society in July 1995.

2 856 T. HIGENBOTTAM EXCHANGE OF NITRIC OXIDE BETWEEN LUNG AND AIR Nitric oxide is present in the exhaled breath of animals, including man (Gustafsson, Leone, Persson, Wiklund & Moncada, 1991; Borland, Cox & Higenbottam, 1993; Leone, Francis, Rhodes & Moncada, 1994). This phenomenon depends on the large surface area of the air-liquid interface of the lungs. The partitioning of the gaseous NO between the liquid and gas phase depends upon the physical properties of both NO and the liquid. The solubility of NO in water is similar to that of oxygen (02), which is fourfold less soluble than carbon dioxide (CO2) (Steward, Allott, Cowles & Mapleson, 1973). In order to dissolve all the gaseous NO in water, a pressure of over 33 atmospheres is required (Gerrard, 1980). At the usual one atmosphere of pressure experienced in the lungs only 3 % of the NO is likely to remain in solution; the remaining 97 % partitions to the gas phase. A further property of NO that affects its exchange within the lungs is its high affinity for haemoglobin (Hb). Compared with 02 it has a sixfold higher affinity for Hb (Olsen, 1981). When NO is inhaled, it is only taken up in the alveolar region (Borland & Higenbottam, 1989), like carbon monoxide (CO), which is another ligand of Hb. Indeed, NO has been used with CO to measure lung diffusing capacity. It is possible to detect NO in the exhaled air. Any NO formed close to the air-liquid interface of the lungs is likely to enter the air space or combine with the haemoglobin of circulating red blood cells. Only a small amount will remain in the tissue or in solution in the aqueous liquid that lines the surfaces of the lungs. It has proved possible to measure the rate of production of NO in the lungs. Nitric oxide in air can be measured using the chemiluminescent analyser (Borland & Higenbottam, 1989). This is specific for NO and is sensitive down to picomolar concentrations. By combining these measurements of exhaled NO with studies of the effects of stimulation and inhibition of NO release by the pulmonary vascular bed, it has proved possible to define the role of NO in the control of the pulmonary circulation. ENDOTHELIAL NITRIC OXIDE IN THE LUNGS Pulmonary NO is in part derived from the pulmonary vascular endothelium. In studies of isolated, perfused and ventilated lungs of large mammals (Cremona, Wood, Hall, Bower & Higenbottam, 1994), including humans, sheep, pigs and dogs, it has proved possible to demonstrate this endothelial origin and the role of NO in the control of the circulation. By the addition of vasoactive agents to solution perfusing the isolated lungs, it is possible to study the regulation of vascular tone. The thromboxane analogue U46689 elevates pulmonary vascular resistance (PVR) in most mammalian species. Inhibition of endothelial NOS can be achieved by adding NW-nitro-L-arginine methyl ester (L-NAME; 10-7 M) to the perfusate. The endothelial, Ca2+-dependent, NOS can be stimulated by acetylcholine (ACh; 10-7 M), which acts on the muscarinic Ml receptors found on the endothelial cell membrane. In isolated perfused lungs, addition of these agents to the perfusate allows selective study of the regulation of vascular tone of pulmonary arteries, capillaries and veins. Inhibition of pulmonary endothelial NOS in porcine lungs results in a reduction in exhaled NO and a rise in PVR (Table 1) (Cremona, Higenbottam, Takao, Hall & Bower, 1995 a). The thromboxane analogue U46689 also increases PVR but fails to alter exhaled NO. Subsequent

3 LUNG DISEASE AND ENDOTHELIAL NITRIC OXIDE 857 Table 1. Exhaled NO concentration and molar rate ofproduction, and pulmonary vascular resistance of isolated procine lung Nitric oxide Molar rate of Pulmonary vascular concentration production of NO resistance (p.p.b.) (nmol min-1) (mmhg ml-1 kg-' min-') Series 1 (n = 8) Baseline Hypoxia (F1 O2, 5 %) Baseline ± 0.1 U46619 (10 9 M) ACh(10-6M) Series 2 (n = 4) Baseline ± 0.1 L-NAME (10-5 M) 1-5 ± ± Baseline Erythrocytes (Hct, %) ± 0.1 Values shown are means + S.D. All lungs were studied under normoxic conditions except for those in the hypoxia experiment. addition of ACh (10'7 M) to the perfusate dramatically increases exhaled NO and lowers PVR. These results show that the concentration of NO in the exhaled air is closely correlated with its production by the pulmonary endothelium. Changes in production alter PVR, but changes in PVR do not affect NO release. PULMONARY ENDOTHELIAL NITRIC OXIDE AND ALVEOLAR HYPOXIA Ventilation of the isolated lung preparation with a low fractional inspired 02 (Fj10) of 10 %, compared with air, causes exhaled NO to fall and PVR to rise (see Table 1). Exchanging the colloidal lung perfusate for one containing blood to give a haematocrit of 9 % reduces the exhaled NO concentration but leaves PVR unchanged (Cremona et al a). Nitric oxide is a powerful ligand of Hb. The fall in exhaled NO when Hb is present in the vascular compartment confirms that NO originates from the endothelium. The reduction of exhaled NO by a low Fi 02 suggests that its rate of release from the pulmonary endothelial cells is regulated by the alveolar 02 concentration. Hypoxia also reduces NO production by cultured endothelial cells and this does not apply only to the pulmonary endothelium (Shaul & Wells, 1994). In the corpus cavernosum also, hypoxia causes a marked fall in NO release (Kim, Vardi, Padma-Nathan, Daley, Goldstein & Saenz de Tejada, 1993). Penile erection depends upon an increased release of NO, which leads to the blood filling of the corpus cavernosum and consequent increased turgor (Rajfer, Aronson, Bush, Dorey & Ignarro, 1992). To prevent sustained erection and priapism the increased release of NO needs to be reduced. Hypoxia of the static blood in the corpus cavernosum terminates the erection; the 02 tension falls as a result of tissue extraction. The hypoxic regulation of pulmonary endothelial NO release could offer a mechanism parallel to that controlling pulmonary perfusion by direct hypoxic vasoconstriction. The fall in 02 tension in the smooth muscle cells of small pulmonary arteries can inhibit the Ca2+-

4 858 A to E 45 A 30 1A 0.15 ~~~v. T. HIGENBOTTAM B E 60 Ȧ Flow (ml min-'kg-1) Flow (ml min-' kg-1) C 50 D F S 5) F V) fn (Iv Flow (ml min-' kg-') 200 ()I~~~~~~~~~~~~~~~~~~~~~~~~~~ Flow (ml min-' kg-') Fig. 1. Changes in pressure-flow relationships after blockage of NO production by L-NAME in isolated lungs perfused with Krebs-dextran solution. Mean pressure-flow lines in isolated lungs are shown before (continuous lines) and after (dashed lines) L-NAME (10-5 M). Dotted lines show 95 % confidence limits. A, lungs from human donors (n = 4); B, pig lungs (n = 6); C, sheep lungs (n = 4); and D, dog lungs (n = 3). Redrawn from Cremona et al. (1995 b). dependent K+ channel with associated contraction (Post, Hume, Archer & Weir, 1992). A fall in the endothelial NO release with hypoxia would have a similar effect on vascular tone. SPECIES DIFFERENCES IN THE ROLE OF PULMONARY ENDOTHELIAL NITRIC OXIDE It is unlikely that the role of NO in the pulmonary vascular bed is the same in different mammalian species. Inhibition of NOS in the pulmonary vasculature has varying effects in different species (Cremona, Higenbottam, Wood, Bower, Stewart, Large, Wells & Wallwork, 1995 b). In humans (Fig. 1 A), pigs (Fig. 1 B) and sheep (Fig. 1 C) addition of L-NAME to the perfusate of isolated normoxic lungs increases PVR, as shown by a change in the relationship between pressure and flow. In the dog lung (Fig. 1 D) this is not the case, the relationship between pulmonary blood flow and perfusion pressure being unchanged. The same has also been reported in the rat. At present, there is no physiological explanation for these species differences in the role of pulmonary endothelial NO. One idea is that it results from differences between animals in the need of the pulmonary circulation to adapt to increased blood flow.

5 LUNG DISEASE AND ENDOTHELIAL NITRIC OXIDE 859 PULMONARY ENDOTHELIAL NITRIC OXIDE RELEASE IN PULMONARY HYPERTENSION It is known that, in the systemic circulation, certain diseases alter endothelial cell function. This may also be true in the pulmonary circulation. Dissected pulmonary arteries from patients with hypoxic lung disease, studied in an organ bath, have shown an impaired stimulated release of endothelial NO (Dinh-Xuan, Higenbottam, Clelland, Pepke-Zaba, Cremona, Butt, Large, Wells & Wallwork, 1991). This defect is not the result of substrate deficiency, nor does it result from dysfunction of specific endothelial cell membrane receptors (Dinh-Xuan, Pepke-Zaba, Butt, Cremona & Higenbottam, 1993). Explanted lungs from patients undergoing heart-lung transplantation can be studied with the isolated perfused and ventilated lung assembly. Using this preparation it has proved possible to show that the same impairment of stimulated release of endothelial NO is present not only in isolated vessels but also in the whole lung (Cremona et al b). In these studies, PVR was first elevated with the addition of the thromboxane analogue A46689 to the perfusate. A dose-response curve with acetylcholine was then performed until maximal vasodilatation occurred. The maximum fall in PVR was less in lungs from patients with chronic obstructive lung disease and hypoxia than in lungs from normal donors. Lungs from patients with severe pulmonary hypertension responded less well to ACh than normal lungs but better than the hypoxic lungs (Fig. 2A). At present there is no known mechanism to account for the impaired stimulated release of endothelial NO in these diseased lungs. We suspect that chronic hypoxia may interfere with the phosphorylation of NOS, which is an important process in the activation of endothelial NOS (Michel, Li & Busconi, 1993). In contrast, the basal release of NO appears to be normal in pulmonary hypertension. Addition of L-NAME (10-5 M) to the perfusate of the isolated lungs under basal conditions causes PVR to rise by comparable amounts in lungs from patients with hypoxaemic chronic lung disease, from those with severe pulmonary hypertension and also from normal patients (Fig. 2B). REGIONAL DIFFERENCES IN THE RELEASE OF PULMONARY ENDOTHELIAL NITRIC OXIDE AND THE SITE OF ACTION OF INHALED NITRIC OXIDE It has proved possible to show that regional differences in the production of endothelial NO exist in the lungs. The arterial, venous and double occlusion technique, which was first described by Hakim in the dog (Hakim, Michel & Chang, 1982), has been applied to answer this question. From records of the changes in pressure after occlusion, it is possible to determine the resistance of different segments of the pulmonary circulation. Inhibition of endothelial NOS with L-NAME causes a rise in the resistance of only the 'muscular' arterial and precapillary arteries (see Fig. 3A; Cremona, Takao, Bower, Hall & Higenbottam, 1995 c). Little or no change occurs in the resistance to flow through the venous segments. These effects could be abolished by addition of sodium nitroprusside (SNP; 10-7 M) to the perfusate. Inhalation of a gas mixture containing 40 p.p.m. of NO reverses the effects of inhibition of NOS in pulmonary arteries. This supports the notion that inhaled NO is taken up not only into the alveolar capillaries but that it also enters the vascular smooth muscle cells of resistance blood vessels. The overall view is that only the endothelium of the arterial segments of the pulmonary circulation produce NO in physiologically active concentrations.

6 860 T. HIGENBOTTAM A -10 r ; -40 * Mtl Donor CF-bronchiectasis ES-PPH B I-1 b4._ -E T~~~~~~ * Donor CF-bronchiectasis ES-PPH Fig. 2. A, the maximal change in pulmonary vascular resistance after treatment with acetylcholine (10-9 to 10-5 M) in lungs from donors and in lungs from CF (cystic fibrosis)-bronchiectasis and ES (Eisenmenger syndrome)-pph (primary pulmonary hypertension) patients perfused with Krebs-dextran solution and preconstricted with U Results are presented as means (dots), with the standard error shown by the large open rectangles and the standard deviation shown by the error bars. B, the mean pulmonary vascular resistance in lungs from donors, CF-bronchiectasis and ES-PPH patients perfused with Krebs-dextran solution. These were measured before (O), then after blockage of NO production with L-NAME (10-5 M; 3) and then after sodium nitroprusside (10-5 M; *). Asterisk indicates significant difference from initial level (P < 0 05). The distribution of perfusion of the capillary bed of the lungs is determined not only by the resistance to flow in the arteries but also by the compliance of the alveolar capillaries (Overholser, Lomongino, Parker, Pou & Harris, 1994). The occlusion technique of measurement can be applied to study the compliance of arteries, veins and the 'central segment' of the pulmonary circulation, which is made up principally of the alveolar capillaries, the precapillary arteries and the postcapillary veins (Linehan, Dawson & Rickaby,

7 LIUNGN )ISEASE AND ENDoTHELAIA NITRIC OXIDF 861 0(),a,,4 043 u 001 Control L-NAME No Post-NO SNP 3 () Cointt ol L -NAME Fig. 3. A, eilect of A"'-I111S1-ostntne ( 1() tncl). obseqlcit inlhtled NO (8() p.p.m.) and socdiuim inlti-opiusstde (SNP) Oll thec SCeImCenltall plilmohi V ISClatM tststance tnlle.lststcd yn OCclUStOin techniqle., \VC11OUS, *, postcapillary DO precaltilll;try, aiid 0, atrteria VaSClatIresistance. Altei- trmitnation o' v entitlatito xith NO, resist.ance retlir-ned to the elevated lev els causedi by Vanit o L atgtnand thi.s w as taikeni as a seconid contol valuo. 1, cifcct of A'nt`-ltr- L-Magtinne ( 11) M) onl the coiiplianilce of the atrterital (U). capillatvr (C) and venous (Q) compartments estimated by nonllittear regression acreetding to the 2R 3C miodel. Asteci-isk indicattes signiitficant difiet enees from control (P < (0)5). Amendcieid from Cremona eiel. ( 19 c). 1982). Addition of the NOS inhibitor N`-mnitro-L-arginine agalin shows inhomogeneity in the changes in compliance. Strikingly, the compliancc of the ccietr-al segment, including the alveolar capillaries, falls to a greater degree than that of- the other segmcnts (Fig. 3 B). This offers a further insight into the potential rolc of endothelial NO in the luing. Pulmonary nitric oxide production falls with alveolar hypoxia (see Table 1). The change in PVR is principally achieved in the resistance blood vcssels, muscular pulmonary arteries and precapillary arteries. These vessels are concerned with the distribution of pulmonary

8 862 T. HIGENBOTTAM 20 r 20 r r- -E bon 15 H * * 15F 10 lo0 r Control 4 8 PGI2 (ml h-') Is 12 Control NO Air NO NO (40 p.p.m.) Air 1-1 r_e -E on 30 r 20 H 4-._ -E 30 r = 20 :> Ez (I A I',- Control Control NO Air NO PGI2 (ml h-') NO (40 p.p.m.) II,u Air Fig. 4. The haemodynamic effects of infused prostacyclin (PG12) and inhaled NO. The figure shows a comparison of the effects on pulmonary (PVR) and systemic (SVR) vascular resistance of an intravenous infusion of prostacyclin (PG12) (0-5 mg in 250 ml) at rates of 4, 8 and 12 ml h-', and the inhalation of NO (40 p.p.m. in air) with control values in 8 patients with pulmonary hypertension. Means and S.E.M. are shown. * P < 0-05; ** P < Redrawn from Pepke-Zaba et al. (1991). perfusion. In addition, the fall in NO production is also likely to reduce the compliance of the capillaries. A powerful mechanism is therefore provided, which responds to hypoxia and which can divert blood flow away from underventilated regions of the lungs. In other words, the endothelial NO system of the lungs offers a complementary system to hypoxic vasoconstriction in ensuring the matching of the distribution of ventilation and perfusion. Inhalation of NO might therefore offer a means of reducing pulmonary hypertension. In primary pulmonary hypertension, when NO is inhaled in concentrations of 40 p.p.m., it acts as a selective pulmonary vasodilator (see Fig. 4) (Pepke-Zaba, Higenbottam, Dinh-Xuan, Stone & Wallwork, 1991). This has proved to be a highly effective treatment for persistent pulmonary hypertension of the newborn (PPHN) (Kinsella, Neish, Shaffer & Abman, 1992), although inhaled NO enters ventilated regions of the lungs. In these regions of the lung perfusion is increased in patients with acute respiratory distress syndrome (ARDS), with a resulting reduction of right-to-left shunting of blood and an improvement of oxygenation (Rossaint, Falke, Lopez, Slama, Pison & Zapol, 1993). There is also the potential to treat other lung diseases with inhaled NO; results of controlled studies are awaited.

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