Endogenous nitric oxide modulates adrenergic neural vasoconstriction in guinea-pig pulmonary artery

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1 Br. J. Pharmaol. (1991), 14, ,'-. Mamillan Press Ltd, 1991 ndogenous nitri oxide modulates adrenergi neural vasoonstrition in guinea-pig pulmonary artery S.F. Liu, D.. Crawley, T.W. vans & 1P.J. Barnes Department of Thorai Mediine, National Heart & Lung Institute, Dovehouse Street, London SW3 6LY 1 letrial field stimulation (FS) of guinea-pig isolated pulmonary artery indued a frequenydependent ontration. This was abolished by tetrodotoxin (1,UM) and prevented by phentolamine and prazosin (both 1,UM), indiating a role for ax-adrenoeptors ativated by noradrenaline (NA) released from perivasular adrenergi nerves. 2 L-N-monomethyl arginine (L-NMMA,.3-1OOpM) aused a onentration-dependent enhanement of the FS-indued ontration with a fold inrease at 1pM (n = 6). The augmenting effet of 3,UM L-NMMA on the ontration to FS was ompletely reversed by 1-3PM L-arginine, but not by an idential onentration of D-arginine. 3 The ontratile response to exogenous NA was similarly enhaned by 34UM L-NMMA ( fold inrease, n = 5). 4 The ontratile responses to exogenous phenylephrine and prostaglandin F2. whih mathed the ontration to FS (4Hz) were equally augmented by 34uM L-NMMA. 5 In vessel rings submaximally ontrated with the thromboxane analogue U4469 (2pM), the seletive a2-adrenoeptor agonist UK1434 indued onentration-dependent relaxation, whih was abolished by removal of endothelium. NA had little relaxant effet on these preontrated vessel rings unless in the presene of prazosin (1 um). 6 Indomethain had no signifiant effet on the ontratile response to FS or NA, indiating that vasodilator ylo-oxygenase produts suh as prostaylin are not involved in modulating these responses. 7 Our results suggest that endogenous nitri oxide inhibits the ontratile response to adrenergi nerve stimulation in the guinea-pig pulmonary artery by a postjuntional mehanism, but release of prostaylin does not modulate these responses. Basal release of nitri oxide from endothelial ells may aount for this inhibition. Keywords: Pulmonary artery; innervation; adrenoeptor; nitri oxide; prostaylin; endothelium; DRF Introdution ndothelium plays an important modulatory role in the response of vasular smooth musle to a variety of stimuli (De Mey et al., 1982; Furhgott, 1984; Bullok et al., 1986). ndothelium has an inhibitory effet on the ontratile responses to a-adrenoeptor stimulation either by exogenous administration of noradrenaline (ks & Angus, 1983) and other a- adrenoeptor agonists (gleme et al., 1984; Lues & Shumann, 1984; Martin et al., 1986) or by adrenergi nerve stimulation in rabbit arotid artery (Tesfamariam et al., 1987; hen & Weisbrod, 1988) and rat audal artery (Hynes et al., 1988). This inhibitory effet of endothelium on the ontratile response to adrenergi nerve stimulation is believed to be due to the release of relaxing fator(s) from endothelial ells (Tesfamariam et al., 1987; Tesfamariam & Halpern, 1987; hen & Weisbrod, 1988). The fator(s) responsible for the inhibition of neurogeni adrenergi vasoonstrition has not yet been identified. The pulmonary irulation differs markedly from the systemi irulation in several aspets; the different response to aute hypoxia, whih auses ontration in pulmonary, but relaxation in systemi vessels (Staub, 1985) is well known. There are also differenes in other ontrol mehanisms (Fishman, 199), the distribution (Hyman et al., 1989) and affinities (Shaul et al., 199) of adrenoeptors, and in the response to some peptides, suh as vasoative intestinal polypeptide (Sata et al., 1986). Although the presene of endothelial a2-adrenoeptors has been demonstrated in the systemi vessels of several regions (Angus et al., 1986; Bullok et al., 1986; Vanhoutte & Miller, 1989), the presene of these ' Author for orrespondene. reeptors on pulmonary artery is still unertain (Vanhoutte & Miller, 1989). The two major relaxing fators released from vasular endothelium are prostaylin (PGI2) and endothelium-derived relaxing fator (DRF). DRF has been haraterized as nitri oxide (NO) or a related ompound (Palmer et al., 1987; Ignarro et al., 1987; Monada et al., 1988; 1989), whih is synthesized from L-arginine (Palmer et al., 1988a,b; Shmidt et al., 1988; Monada et al., 1989) in vasular endothelial ells. The L-arginine analogue L-NG-monomethyl arginine (L- NMMA) is a speifi inhibitor of NO prodution (Palmer et al., 1988b; Monada et al., 1989; Johns et al., 199). Using the inhibitor and preursor of NO formation and the PGI2 synthesis inhibitor indomethain, we have explored the modulatory role of endogenous NO and PGI2 on the ontratile response to adrenergi nerve stimulation in guinea-pig pulmonary artery. We also investigated the possible presene of endothelial (2-adrenoeptors. Methods Tissue preparation Male Dunkin-Hartley guinea-pigs (3-35g) were killed by ervial disloation and pulmonary arteries were rapidly removed. The two branhes of the vessels were arefully disseted, ut into rings 2 mm in length, mounted over a pair of rigid wires and suspended in an organ bath ontaining 2ml Krebs-Henseleit (KH) solution of the following omposition (mm): NaCl 8, KCl 5.9, MgSO4 1.2, CaCl2 2.5, NaH2PO4 1.2, gluose 5.6, NaHCO3 25.5, DTA.27 and asorbi aid.3. One wire was fixed and the other attahed to a fore transduer (FT.3 Grass Instruments, Quiny, U.S.A.).

2 566 S.F. LIU et al. Changes in isometri fore were reorded on a polygraph (Grass Model 7). The KH solution was maintained at 37C and bubbled with a mixture of 95% 2 and 5% CO2. Initially, the vessel rings were repeatedly stimulated by FS (5 V,.2 ms duration, 16Hz, 15 s) starting from zero resting tension, until the maximum response to FS was ahieved at an inrement of 1 mg, and the optimal resting tension determined. Rings were then allowed to equilibrate at this tension (7 mg) in the bath for at least 6 min and washed with fresh KH solution every 2 min during the equilibration period. Nerve stimulation letrial field stimulation (FS) was applied by two platinum wire eletrodes positioned at eah end of the vessel ring and onneted to a Grass S88 stimulator (Grass Instruments, Quiny, U.S.A.). To ativate the intramural nerves without induing a myogeni response, voltage-duration urves were performed in the presene and absene of.3pm tetrodotoxin by reording the smallest detetable response (< 2% of maximal response) and optimal parameters (5 V,.2 ms duration) determined in a preliminary study. Frequenyresponse relationships were onstruted in a frequeny range of 1-16 Hz, eah stimulation being applied for 15 s every 4 min. To assess the nature of the ontratile response to FS, the vessel rings were inubated with tetrodotoxin (1 um), phentolamine (1 gm) or prazosin (1 pm) for 15 min and stimulated at 1-16Hz or 8-24Hz in separate experiments. To assess the effets of endogenous NO and PGI2 on the adrenergi ontration, vessel rings were stimulated with fixed eletrial stimuli (5 V,.2 ms, 4Hz) at 4min intervals. When responses were onstant, the vessel rings were inubated with inhibitory agents for 1 min and a further 3-4 stimulations performed. In the reversibility study, L- or D-arginine were added to the organ bath after the maximal effet of a dose of L-NMMA was ahieved. To larify the relative pre- or postjuntional ation of endogenous NO and PGI2, the effet of 31uM L-NMMA and 1 1UM indomethain on the mathed ontrations indued by FS (4Hz) and exogenous noradrenaline (NA) were ompared in paired rings from the same vessel. mparisons were also made among the mathed ontrations indued by FS, NA, phenylephrine (P) and prostaglandin F2, (PGF2a) in paired vessel rings. To math the FS-indued ontration, variable onentrations of NA (.1-.3pM), P (.8-.1 pm) and PGF2,, (.3-.6pM) were used. nentration-response urves To determine the effet of L-NMMA on ontration to exogenous NA, onentration-response urves to NA were onstruted in the presene and absene of 3pM L-NMMA. In the relaxation study, paired vessel rings were preontrated with 2 UM U4469 and onentration-relaxation urves to NA in the absene and presene of prazosin (1 pm), prazosin plus propranolol (both 1 M) and prazosin plus yohimbine (both 1 M), and to UK1434 in the presene and absene of an intat endothelium or 3AM L-NMMA were obtained. Drugs The following drugs were used: noradrenaline hydrohloride, prazosin hydrohloride, yohimbine hydrohloride, indomethain, U4469 (9,-dideoxy-1 la,9a-epoxymethano-prostaglandin F2.), tetrodotoxin, phentolamine hydrohloride, propranolol hydrohloride, L-arginine hydrohloride, D- arginine hydrohloride, sodium nitroprusside (Sigma, Poole, Dorset), prostaglandin F2. solution (Upjohn, Crawley, Sussex), UK1434 (Pfizer, Sandwih, Kent) and L-N-monomethyl arginine (a generous gift from Dr S. Monada, Wellome Researh Laboratory, Bekenham, Kent). Analysis of results ntration is presented in absolute tension or expressed as a perentage of its maximum. An individual onentrationresponse urve to NA was fitted and the C5 value estimated by use of a omputer programme (Graph PAD InPlot, Graph PAD Software, San Diego, CA, U.S.A.). Relaxation was expressed as a perentage of the U4469-indued ontration or the maximum relaxation to nitroprusside (in the endothelial denudation study). The ontratile responses to FS and other vasoonstritors in the presene of the inhibitors or their vehiles were ompared with the responses before adding these inhibitors or vehiles, and expressed as perentage augmentation. Values were presented as mean + s.e.mean and n indiates the number of animals in eah group. Statistial analysis of results was performed by use of Student's t test or one way analysis of variane following by t test with Bonferroni orretion, when multiple omparisons were made. For data whih were abnormally distributed or with unequal variane, a Mann-Whitney U test was used. A P value <.5 was onsidered to be signifiant. Results Adrenergi response to eletrial field stimulation FS (SOV,.2ms duration, 1-16Hz, 15s) indued a frequeny-dependent ontration of the guinea-pig pulmonary artery rings at resting tension. This ontration was abolished by 1 M tetrodotoxin and antagonized by phentolamine and was due to the ativa- prazosin (both 1 gm), indiating that it tion of ax-adrenoeptors by neurally released NA from perivasular sympatheti nerves. The FS-eliited ontration was , , , and mg at 1, 2, 4, 8 and 16 Hz respetively (n = 6). ffets of L-NG-monomethyl arginine and L-arginine on eletrialfield stimulation-indued ontration L-NMMA (.3-1 pm) enhaned the FS-indued ontratile responses in a onentration-dependent manner (Figure 1) and inreased basal tone, espeially at high onentrations. The inrease in basal tone aused by L-NMMA was , and mg at onentrations of 1, 3 and 1,UM respetively (P <.2, ompared with ontrol, n = 6). The augmentation of FS-indued ontration m 21 D 18 : log M [L-NMMAI Figure 1 ffet of L-NG-monomethyl arginine (L-NMMA) on the ontratile response to adrenergi nerve stimulation (5 V,.2 ms duration, 4 Hz for 15 s) in guinea-pig pulmonary artery rings, showing onentration-dependent augmentation of the adrenergi neurogeni vasoonstrition. Mean of 6 animals with s.e.mean shown by vertial bars.

3 NO MODULATS ADRNRGIC VASOCONSTRICTION 567 by 3AM L-NMMA was ompletely reversed by 1-3pM L-arginine, although an idential onentration of D-arginine was ineffetive (Figure 2). ffets of L-NG-monomethyl arginine on noradrenaline-indued ontration NA aused a onentration-dependent ontration of pulmonary vessel rings at resting tension. Rings pretreated with 3pUM L-NMMA had a lower threshold onentration of NA than the ontrol rings ( vs nm, P <.5, n = 5). The pd2 values for ontrol and L-NMMA-treated rings was and , respetively (P <.3, n = 5), representing a 4.9 fold inrease in sensitivity to NA in L-NMMA pretreated rings. The maximal tension generation was and mg for ontrol and L-NMMA group, whih was not signifiantly different (P >.5, n = 5). mparison of the effets of L-NG-monomethyl arginine on eletrial field stimulation- and noradrenaline-indued ontrations To investigate the relative pre- and postjuntional effet of L-NMMA, we ompared the augmenting effets of 3pM L-NMMA on the ontratile response to FS (4Hz) and a mathed ontratile response to NA (.1-.3uM), whih was and mg respetively (P >.5, n = 6) before L-NMMA treatment. L-NMMA augmented FS- and NA-indued ontration equally (Figure 3), suggesting that L-NMMA enhaned adrenergi responses via a postjuntional mehanism. Vasorelaxant response to UK1434 and noradrenaline To determine the presene and the possible role of a2-adrenoeptors in this vessel, vasorelaxant responses of U4469-preontrated vessel rings with and without endothelium to the seletive CX2-adrenoeptor agonist UK1434 and endothelium intat rings to NA in the absene and presene of various blokers, were determined. U4469 raised the vasular tone to , and mg in the rings with and without endothelium, and in the presene of 3OpM L-NMMA respetively (n = 5). UK1434 indued a ) 4 25r- 2 F H 5~ -rn Itr ntrol **... 1,... *:-::-:--:.::-: A -_ L-NMMA L-Arg ** L-NMMA + D-Arg Figure 2 Reversibility of 3jUM L-NG-monomethyl arginine (L- NMMA)-indued augmentations of adrenergi ontration to eletrial field stimulation (FS, 5V,.2 ms, 4Hz for 15 s) by L- and D- arginine. L-arginine (L-Arg) 1-3pM, but not idential onentrations of D-arginine (D-Arg) ompletely reversed the L-NMMA effet. **: P <.1, ompared with ontrol. ) - 4 r 3k 25k 15k :..:..: :-:--:-::-:--: ( 1 I 5 l FS NA P PGF2o Figure 3 mparison of the effet of L-NG-monomethyl arginine (L- NMMA) on mathed ontratile responses to adrenergi nerve stimulation (FS, 5 V,.2 ms, 4Hz for 15 s), exogenous noradrenaline (NA), phenylephrine (P) and prostaglandin F2. (PGF2a). ntrols are shown in stippled olumns and L-NMMA (3,UM) in open olumns. ** P <.1, ompared with ontrol. onentration-dependent relaxation of the U4469- preontrated rings (Figure 4). This relaxation was abolished by endothelial denudation. Pretreatment of the vessel rings with 3YM L-NMMA mimiked the effet of endothelium denudation (Figure 4), indiating that a2-adrenoeptors loated on endothelial ells mediate vasorelaxation by stimulation of NO release. The U4469-produed ontration was , , and mg in ontrol, prazosin, prazosin plus propranolol and prazosin plus yohimbine groups, respetively (P >.5, n = 5). NA had little relaxant effet even on the preontrated rings. The maximal relaxation ahieved at the onentration of 3,UM was a % redution of the U4469-eliited tension. In the presene of prazosin (1 UM), NA indued a onentration-._ xo z C,) x i LO) C: CZ ** ** log M [UK 1434] Figure 4 ffets of endothelium removal and L-N-monomethyl arginine (L-NMMA) on the relaxant response of the U4469- preontrated vessel rings to UK1434. Relaxation was expressed as perentage of nitroprusside (SNP) maximum relaxation: (A) with (ontrol), () without intat endothelium and (A) in the presene of 3OPM L-NMMA. * P <.5 and ** P <.1, ompared with ontrol rings. Mean + s.e.mean of 5 animals with s.e.mean shown by vertial bars. X

4 568 S.F. LIU et al. 6- 'r. 5 C.rJ. 1* 4-3- ~~2 C x 1 -i log M [Noradrenaline] Figure 5 Relaxation of U4469 preontrated vessel rings to noradrenaline (NA) in the absene (A, ontrol) and presene of prazosin (A, 1 pm), prazosin plus propranolol (, both 1 pm) and prazosin plus yohimbine (*, both 1 pm). Relaxation was expressed as perentage of U4469 (2pM)-indued ontration. NA aused little relaxant effet (A) exept in the presene of prazosin (A). * P <.5 and ** P <.1, ompared with ontrol. t P <.1, ompared with prazosin pretreated rings, n = 5 in all groups. dependent redution of the U4469-indued vasular tone. This relaxation was signifiantly antagonized by yohimbine (1 pm), but unaffeted by propranolol (1,M, Figure 5), suggesting that although x2-adrenoeptors are present on these vessels, they play little role in mediating the response to NA. ffets of L-NG-monomethyl arginine on the ontrations to other vasoonstritors The ontration indued by a single onentration of P and PGF2e was respetively and mg before treatment with L-NMMA (P >.5, n = 5). These ontrations, whih were similar to the ontratile response to FS, were enhaned to the same extent by 3,M L-NMMA (Figure 3). ffets of indomethain Indomethain slightly, but not signifiantly enhaned the ontratile responses to FS at onentrations of 1 and 3 pm but had no effet at 1gM. At a onentration of 3,M, indomethain slightly enhaned FS-indued ontration in 4 of the 6 tested vessel rings from 6 guinea-pigs, and slightly inhibited the FS-indued ontration in 2 rings. Indomethain (1 gm) slightly enhaned the ontration to NA. These results suggest that endogenous ylo-oxygenase produts are not important in modulating response to adrenergi nerve stimulation. Disussion Removal of endothelium inreases vasoonstritor responses to x-adrenoeptor agonists (ks & Angus, 1983; gleme et al., 1984; Lues & Shumann, 1984; Carrier & White, 1985; Martin et al., 1986) and to adrenergi nerve stimulation (Tesfamariam et al., 1987; hen & Weisbrod, 1988; Hynes et al., 1988). However, it has not yet been established whih fator is responsible for this inhibitory ation of endothelium. We now report that the NO synthase inhibitor L-NMMA enhanes the ontratile response to adrenergi nerve stimulation onentration-dependently in guinea-pig pulmonary t artery. This augmentation is ompletely reversed by L-arginine but not D-arginine. By ontrast, this neurogeni ontration is unaffeted by indomethain. These results indiate that endogenous NO but not PGI2 modulates the adrenergi neurogeni vasoonstrition. It has been reported that NO an be released from inhibitory non-adrenergi, non-holinergi nerve (i-nanc) endings of several tissues (Gillespie et al., 1989; Tuker et al., 199; Li & Rand, 1991), inluding erebral arteries (Toda & Okamura, 199). It is possible that stimulation by FS of i-nanc nerves, whih have been demonstrated in this vessel (Liu et al., 1991b), indues NO release, or alternatively, stimulation of i-nanc nerves releases mediator(s), whih at on endothelial ells to stimulate NO release (Liu et al., 1991b). However, release of NO from i-nanc is unlikely. Firstly, we found, in our previous study, that NO mediating the i-nanc relaxation is released from endothelium (Liu et al., 1991b). Seondly, we demonstrated in the present study that FS- and exogenous NA-indued ontrations were augmented by L-NMMA to an equal extent. It is possible that different mehanisms in uptake and aess to reeptors of endogenous and exogenous NA and the presene of a negative feedbak mehanism in endogenous NA release in response to FS may make a strit omparison of FS- and exogenous NAindued ontration diffiult. However, the equal augmentation of FS- and NA-indued ontrations by L-NMMA at least indiates that prejuntional ation of NO is not a major mehanism. Taken together with the equal augmentation of mathed ontrations to P and PGF2a, our results suggest L-NMMA augments adrenergi responses via a ommon mehanism at a postjuntional level. The possibility that a mediator from i-nanc nerves stimulates NO release annot would exert a be totally exluded in the present study, but it postjuntional effet. Thus, our results suggest that L-NMMA augments the ontratile response to adrenergi nerve stimulation by a postjuntional mehanism. The presene of endothelial z2-adrenoeptors in guinea-pig pulmonary artery, as in systemi vessels (Vanhoutte & Miller, 1989), is indiated by the demonstration that the seletive x2-adrenoeptor agonist UK1434 and nonseletive agonist NA, in the presene of an al-adrenoeptor antagonist, indued a onentration-dependent relaxation of preontrated vessel rings. This relaxation was unaffeted by the f6-adrenoeptor antagonist, propranolol, but was signifiantly antagonized by the a2-adrenoeptor antagonist yohimbine. We also provide evidene to indiate that ativation of these reeptors indue vasorelaxation by stimulating endothelium-derived NO release. Release of DRF an offset NA-indued ontration (ks & Angus, 1983; Angus et al., 1986; Vanhoutte & Miller, 1989). It is possible that NA from adrenergi nerves stimulates X2-adrenoeptors to indue NO release, whih then inhibits the ontratile response to FS. However, there is evidene against this possibility. Firstly, NA relaxed the preontrated vessel rings only in the presene of a1-adrenoeptor blokade and NA had little relaxant effet even when the vasular tone was raised, whereas FS was applied when the vessel rings were at resting tension. This implies that the weak Lx2-adrenoeptor ation of NA is profoundly masked by its a-adrenoeptor ation. Seondly, a similar magnitude of ontratile response to P and PGF2. was augmented by L-NMMA to the same extent as in vessels ontrated with NA, although neither had any relaxant effet on the preontrated vessel rings. It is more likely that there is a ontinuous basal release of NO from vasular endothelium whih gives a toni inhibition of the ontratile responses to NA and adrenergi nerve stimulation. Inhibition of this NO release by L-NMMA enhanes ontration to adrenergi stimulation. This explanation is supported by our observation that L-NMMA inreased basal smooth musle tone in a onentration-related manner. Basal release of NO has previously been reported both in systemi (Rees et al., 1989) and pulmonary (Wiklund et al., 199; Liu et al., 1991a) irulations.

5 NO MODULATS ADRNRGIC VASOCONSTRICTION 569 An inrease in endothelial shear stress by hanging the perfusate veloity and visosity has been demonstrated to inhibit adrenergi ontration to eletrial field stimulation in perfused arotid artery (Tesfamariam & hen, 1988). Whether smooth musle ontration also imposes shear stress on endothelial ells and inreases NO release remains to be determined. In summary, our results demonstrate that endogenous nitri oxide inhibits adrenergi neurogeni vasoonstrition via a Referenes ANGUS, J.A., COCKS, T.M. & SATOH, K. (1986). a2-adrenoeptors and endothelium-dependent relaxation in anine large arteries. Br. J. Pharmaol., 88, BULLOCK, G.R., TAYLOR, S.G. & WSTON, A.H. (1986). Influene of the vasular endothelium on agonist-indued ontrations and relaxations in rat aorta. Br. J. Pharmaol., 89, CARRIR, G.O. & WHIT, R.. (1985). nhanement of alpha-1 and alpha-2 adrenergi agonist-indued vasoonstrition by removal of endothelium in rat aorta. J. Pharmaol. xp. 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