General Pharmacology 32 (1999) 583–589

Modulation of adrenergic contraction of dog pulmonary arteriesby nitric oxide and prostacyclin

Gloria Segarra, Pascual Medina, Francisco Revert, Salvador Masia, Jose M. Vila,Luis Such, Martın Aldasoro *

Department of Physiology, University of Valencia, Blasco Ibanez 17, 46010 Valencia, Spain

Manuscript received September 3, 1998; manuscript accepted November 6, 1998

Abstract

The aim of this work was to investigate the influence of endothelium-derived nitric oxide and prostaglandins on the contractileresponses of isolated dog pulmonary arteries to electrical field stimulation and noradrenaline. Electrical field stimulation (1–8 Hz,20 v, 0.25 ms duration, for 30 s) produced frequency-dependent contractions that were abolished by tetrodotoxin, guanethidineand, prazosin (all at 1026 M). Noradrenaline induced concentration-dependent contractions with an EC50 of 1.85 3 1026 M. Theincreases in tension induced by electrical stimulation and noradrenaline were of greater magnitude in arteries denuded of endothe-lium. In segments with endothelium, NG-nitro-l-arginine methyl ester (1024 M) or indomethacin (1025 M) had no effects on thebasal tone, but significantly enhanced the neurogenic and noradrenaline-induced contractions. The potentiation by NG-nitro-l-arginine methyl ester of electrical stimulation-induced contractile responses was partially reversed by l-arginine (1024 M). In thepresence of NG-nitro-l-arginine methyl ester together with indomethacin the electrical stimulation-induced contractile responseswere higher than those obtained when only NG-nitro-l-arginine methyl ester or indomethacin was used. NG-nitro-l-argininemethyl ester and indomethacin did not influence neurogenic-induced contractile responses of endothelium-denuded arteries. Theresults suggest that endothelial cells of isolated dog pulmonary arteries depress the contractile response to electrical field stimula-tion of intramural nerves and that endothelium-derived dilator prostaglandins and nitric oxide may interact to inhibit contractileeffects of adrenergic stimulation. 1999 Elsevier Science Inc. All rights reserved.

Keywords: Dog pulmonary artery; Adrenergic nerves; Endothelium; Vasodilator prostaglandins; Nitric oxide

The two major relaxing factors released from vascu- (Liu et al., 1991; Gonzalez and Estrada, 1991; Aldasorolar endothelium are prostacyclin (Moncada et al., 1976) et al., 1996). A direct release of nitric oxide from peri-and endothelium-derived relaxing factor (Furchgott vascular nerve endings or from smooth muscle cells hasand Zawadzki, 1980). Endothelium-derived relaxing also been considered (Toda and Okamura, 1990; Mac-factor has been characterized as nitric oxide (or a sub- Lean et al., 1993). With regard to systemic arteries, re-stance containing nitric oxide) and plays a decisive role cent experiments suggest that the presence of intact en-in the responses of vascular smooth muscle to a variety dothelial cells is a prerequisite for the release of nitricof stimuli (Ignarro et al., 1987; Palmer et al., 1987; Mon- oxide during adrenergic nerve-induced contractionscada et al., 1988). Removal of the endothelium pro- (Martınez et al., 1994; Aldasoro et al., 1996).duces a significant increase in the contractile responses Control mechanisms of pulmonary circulation differto adrenergic stimulation of arteries from various ani- in several aspects from those present in the systemic cir-mal species (Tesfamariam et al., 1985; Hynes et al., culation (Hyman et al., 1989; Fishman, 1990). Studies in1988), including man (Aldasoro et al., 1993, 1996). The the rat indicate that locally-produced prostacyclin is im-inhibitory effect of endothelium on the contractile re- portant in the regulation of vasomotor tone and vascu-sponses to adrenergic nerve stimulation seems to be lar cell differentiation and growth in the pulmonary cir-due to the release of nitric oxide from endothelial cells culation (Rabinovitch et al., 1988; Shaul et al., 1991)

and cyclooxygenase products mediate the low basal vas-cular resistance in the dog lung (Barnard et al., 1993).* Corresponding author. Tel.: 34 96 3864644; Fax: 34 96 3864642;

E-mail: aldasoro@uv.es. In addition, prostacyclin production in pulmonary ar-

0306-3623/99/$–see front matter 1999 Elsevier Science Inc. All rights reserved.PII : S0306-3623(98)00285-7

584 G. Segarra et al./General Pharmacology 32 (1999) 583–589

teries is greater than in systemic arteries (Samuelsson et Electrical field stimulation was provided by a Grassal., 1978). There are interactions between endothelium- S88 stimulator (Grass Instruments, Quincy, MA, U.S.A.)derived mediators (Shimokawa et al., 1988; Maurice et via two platinum electrodes positioned on each side andal., 1991). Especially interesting is the recently demon- parallel to the axis of the vascular ring. To obtain neu-strated synergistic interaction between endothelium- rogenic contractile responses and to avoid direct stimu-derived nitric oxide and prostacyclin during endothe- lation of smooth muscle, frequency-response relation-lium-dependent relaxation evoked by bradykinin and ships were determined on a group of arteries in thecalcium ionophore A23187 in dog pulmonary arteries presence and absence of 1026 M tetrodotoxin, following(Gambone et al., 1997). However, the intervention of a procedure previously described (Duckworth et al.,these two endothelial-relaxing factors during adrener- 1989; Aldasoro et al., 1993). In summary, the protocolgic constriction is not known. Hence, the present study was designed to find the optimal stimulation parame-was designed to evaluate the modulatory role of endog- ters (15 V, 0.25 ms duration) for causing a contractileenous nitric oxide and prostaglandins on the contractile response that was completely eliminated by 1026 M te-responses of isolated dog pulmonary arteries to electri- trodotoxin. Frequency-response relationships were de-cal field stimulation and noradrenaline. Observations termined using 30-s trains of pulses at 1, 2, 4, and 8 Hz.were made in the presence and absence of endothelium A period of 10 min was allowed between stimulations.and after exposure to NG-nitro-l-arginine methyl ester At the end of each series, the stimulation sequence was(an inhibitor of nitric oxide synthase) and/or indometh- repeated 15 min after the addition of tetrodotoxinacin (an inhibitor of cyclooxygenase). (1026 M), prazosin (1026 M), or guanethidine (1026 M)

to confirm the neurogenic and adrenergic nature of thecontraction. As a control, a second set of stimulations1. Materials and methodswas recorded on artery rings in the absence of antag-

1.1. Recording of isometric tension onists.After an initial set of stimulations, the vessel ringsLungs were obtained from 21 mongrel dogs (9–17

were incubated with NG-nitro-l-arginine methyl esterkg), of either sex, that had been anesthetized with so-(1024 M) for 15 min and a second set of stimulations wasdium pentobarbital (30 mg/kg, IV) and killed by bleed-given. The third set of stimulations was given after a 10ing from the common carotid arteries. Segmental pul-min incubation with l-arginine (1024 M) or d-argininemonary arteries were dissected free and placed in(1024 M) of rings previously exposed to NG-nitro-l-argi-chilled Krebs-Henseleit solution. Arterial rings (3 mmnine methyl ester. As a control, three consecutive setsin length, 2–3 mm in outside diameter) were cut for iso-of stimulations were given at identical intervals. Lessmetric recording of tension. In z50% of the artery ringsthan 10% variability in the magnitude of electrical stim-the endothelium was removed mechanically by insertingulation-induced contractions was observed for a givena roughened stainless-steel wire into the lumen andring during three consecutive sets of control stimula-gently rolling the rings on a wet filter paper.tions. To assess the possible intervention of endogenousTwo stainless steel pins, 200 mm in diameter, were in-prostaglandins on the neurogenic contraction, electricaltroduced through the arterial lumen. One pin was fixedstimulation was applied before and 20 min after the ad-to the wall of the organ bath, while the other was con-dition of 1025 M indomethacin in the organ bath. Tonected to a force-displacement transducer (Grass FT03,evaluate the possibility of synergistic interaction be-Grass Instruments, Quincy, MA, U.S.A.). Changes intween nitric oxide and endogenous prostaglandins,isometric force were recorded on a Grass polygrapharterial segments were incubated with both NG-nitro-(model 7, Grass Instruments, Quincy, MA, U.S.A.).l-arginine methyl ester (1024 M) and indomethacinEach artery ring was set up in a 4-ml bath containing(1025 M) and electrical stimulation was repeated.modified Krebs-Henseleit solution of the following milli-

Concentration-response curves for noradrenalinemolar composition: NaCl, 115; KCl, 4.6; MgCl2·6H2O,were determined in the absence and presence of NG-1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 5; and disodiumnitro-l-arginine methyl ester (1024 M) or/and indo-EDTA, 0.01. The solution was equilibrated with 95%methacin (1025 M) from separate artery preparationsO2 and 5% CO2 to give a pH of 7.3–7.4. Temperaturewith and without endothelium.was held at 378C. To establish the resting tension for

After each experiment the arteries were carefullymaximal force development, a series of preliminary ex-opened flat and stained with AgNO3 to visualize the en-periments was performed on artery rings of similardothelium (Caplan and Schwartz, 1973). Only resultslength and outer diameter which were exposed repeat-from vessels with more than 70% of the endotheliumedly to 60 mM KCl. The basal tension was increasedwere considered as control segments. Vessels in whichgradually until the contractions were maximal. The op-the endothelium had been rubbed never showed moretimal resting tension was 2 g. The vessels were allowedthan 5% of their intima covered with endothelium ei-to attain the steady level of tension during a 2-h accom-

modation period before testing. ther before or after the experiment. Functional integrity

G. Segarra et al./General Pharmacology 32 (1999) 583–589 585

of the endothelium was confirmed by the presence ofrelaxation induced by acetylcholine (1027–1026 M) dur-ing contraction obtained with noradrenaline (1026–1025

M) (Furchgott and Zawadzki, 1980).

1.2. Drugs and solutions

The following drugs were used: noradrenaline hy-drochloride, acetylcholine chloride, guanethidine, indo-methacin, tetrodotoxin, NG-nitro-l-arginine methyl es-ter, l-arginine hydrochloride, d-arginine hydrochloride(Sigma Chemical Co, St. Louis, MO, U.S.A.), and pra-zosin (Pfizer, Sandwich, Kent, U.K.). Drugs were pre-pared and diluted in distilled water except for indo-methacin, which was dissolved in absolute ethanol andsodium bicarbonate solution (150 mM) and readjustedto pH 7.4 with HCl prior to use. Stock solutions of thedrugs were freshly prepared every day.

1.3. Statistical analysisFig. 1. Sections of experimental records showing contractile effects ofelectrical field stimulation in dog pulmonary arteries in the absenceThe contractile response is expressed as a percen-and in the presence of tetrodotoxin (1026 M), guanethidine (1026 M),tage of the maximal tension developed by 60 mM KClor prazosin (1026 M). Tracings are representative of various arteryby each preparation. EC50 values (concentrations ofrings from different animals.

noradrenaline producing half-maximal contractions)were determined from individual concentration-response

ing tension as compared to arteries incubated in Krebs-curves by non linear regression analysis, and the geo-Henseleit solution (p . 0.05). However, NG-nitro-l-metric means were calculated from these values (Flem-arginine methyl ester (1024 M) significantly enhanceding et al., 1972). Data are expressed as means 6 SEM;(p , 0.05) the electrical field stimulation-induced con-n indicates the number of animals in each group. Thetractile responses of artery rings with endothelium, butresults were evaluated statistically by means of paireddid not influence the contractile responses of endothe-or unpaired Student’s t-test or one-way analysis of vari-lium-denuded arteries (Fig. 2). The augmentation ofance. A probability value (p) of ,0.05 was consideredelectrical field stimulation-induced contraction in arter-to be significant.ies with endothelium was partially reversed by l-argi-nine (1026 M), but not by d-arginine (Fig. 2).

2. Results2.3. Effects of indomethacin

2.1. Electrical field stimulationArteries exposed to indomethacin (1027–1025 M) did

Electrical field stimulation produced frequency- not show significant changes in resting tension as com-dependent contractions of artery rings at resting ten- pared to arteries incubated in Krebs-Henseleit solutionsion. Because the increases in tension induced by elec- (p . 0.05). However, indomethacin (1025 M) signifi-trical field stimulation were abolished by tetrodotoxin cantly enhanced (p , 0.05) the electrical field stimula-(1026 M), guanethidine (1026 M), and prazosin (1026 M) tion-induced contractile responses of artery rings with(Fig. 1) it is assumed that the effect was due to the re- endothelium, but did not influence the contractile re-lease of noradrenaline from adrenergic nerves acting on sponses of endothelium-denuded arteries (Fig. 3).a1-adrenoceptors.

2.4. Effects of NG-nitro-L-arginine methyl esterThe contractile responses were of greater magnitudeand indomethacinin arteries denuded of endothelium (Fig. 2). The in-

creased contraction observed in vessels without endo- The presence of NG-nitro-l-arginine methyl esterthelium does not reflect a nonspecific effect on smooth (1024 M) together with indomethacin (1025 M) signifi-muscle since the contractile response to KCl (60 mM) cantly enhanced (p , 0.05) the electrical field stimula-was similar in intact and denuded vessels (4720 6 380 tion-induced contractile responses of artery rings withmg vs. 4301 6 524 mg) (p . 0.05). endothelium with respect to the control rings. The

combined inhibition of nitric oxide synthase and cyclo-2.2. Effects of NG-nitro-L-arginine methyl esteroxygenase also significantly enhanced (p , 0.05) thecontractile responses with respect to indomethacin orArteries exposed to NG-nitro-l-arginine methyl ester

(1026–1024 M) did not show significant changes in rest- NG-nitro-l-arginine methyl ester treated arteries. (Fig. 3).

586 G. Segarra et al./General Pharmacology 32 (1999) 583–589

Fig. 2. Contractile effects of electrical field stimulation in artery rings Fig. 3. Contractile effects of electrical field stimulation in artery ringswith (1E) (n 5 7) and without (2E) (n 5 7) endothelium. After anwith (1E) (n 5 7) and without (2E) (n 5 7) endothelium. After an

initial set of stimulations, rings were stimulated in the presence of NG- initial set of stimulations, rings were stimulated in the presence of NG-nitro-l-arginine methyl ester (1024 M) (n 5 7), indomethacin (1025nitro-l-arginine methyl ester (1024 M) (n 5 7). In arteries with endo-

thelium l-arginine (1024 M) (n 5 6) or d-arginine (1024 M) (n 5 6) M) (n 5 7), and NG-nitro-l-arginine methyl ester 1 indomethacin(n 5 6). *Significant difference from control rings with endotheliumwas applied and the stimulation was repeated. *Significant difference

from control rings with endothelium (p , 0.05). Results are expressed (p , 0.05). **Significant difference from rings incubated with NG-ni-tro-l-arginine methyl ester or indomethacin (p , 0.05). Results areas percentage of the contraction developed by 60 mM KCl by each

preparation. Values are presented as mean 6 SEM shown by: control expressed as percentage of the contraction developed by 60 mM KClby each preparation. Values are presented as mean 6 SEM shown by:(solid bars); NG-nitro-l-arginine methyl ester (open bars); NG-nitro-l-

arginine methyl ester 1 l-arginine (diagonally striped bars); NG-nitro- control (solid bars); NG-nitro-l-arginine methyl ester (open bars); in-domethacin (diagonally striped bars); NG-nitro-l-arginine methyl es-l-arginine methyl ester 1 d-arginine (vertically striped bars); n 5

number of animals. ter 1 indomethacin (vertically striped bars); n 5 number of animals.

2.5. Effects of noradrenalineated responses in dog pulmonary arteries. Treatment

The mean concentration-response curves for nor- with tetrodotoxin, guanethidine, and prazosin abol-adrenaline in arteries with and without endothelium are ished the increases in tension induced by electrical fieldillustrated in Fig. 4. Noradrenaline produced concentra- stimulation. These results suggest that the observedtion-dependent contraction in all cases. In arteries with- neurogenic effects are mainly mediated by the adrener-out endothelium, the concentration-response curves for gic neurotransmitter release from adrenergic nervesnoradrenaline were shifted 3.4 fold to the left (p , 0.05) acting on a1-adrenoceptors.as compared with intact arteries and the maximal con- We found that removal of endothelium produced atractions were greater in arteries denuded of endothe- significant increase in the contractile response of doglium (Table 1). pulmonary arteries to electrical field stimulation. This

Treatment with NG-nitro-l-arginine methyl ester may be attributed to removal of endothelium derived(1024 M) or indomethacin (1025 M) induced a parallel relaxant factors which oppose the adrenergic vasocon-leftward shift (about 4.9 and 7.4 times, respectively) striction mediated by a-adrenoceptors of smooth muscle.(p , 0.05) (Fig. 4) of the response to noradrenaline in In guinea-pig, rabbit, and human pulmonary arteriesarteries with endothelium. Treatment with NG-nitro-l- the main factor responsible for the inhibitory action ofarginine methyl ester (1024 M) together with indometh- the endothelium seems to be nitric oxide (Liu et al.,acin 1025 M) induced a parallel leftward shift of 11.2 1991; MacLean et al., 1993; Martınez et al., 1995). Wetimes and the maximal contraction induced by nor- now report that both nitric oxide and vasodilator pros-adrenaline when nitric oxide synthase and cyclooxygen- taglandins released from endothelial cells modulate thease were inhibited was increased with respect to the neurogenic induced contraction, because the nitric oxidecontrol segments and with respect to NG-nitro-l-argi-

inhibitor NG-nitro-l-arginine methyl ester and the cyclo-nine methyl ester or indomethacin-treated arteries. Ta-oxygenase inhibitor indomethacin augmented contrac-ble 1 summarizes the geometric mean EC50 and maxi-tile responses in pulmonary arteries with endothelium,mal contraction values for noradrenaline determined inbut not in endothelium-denuded arteries. In some ves-arteries with and without endothelium and in the ab-sels (Toda and Okamura, 1990) as well as in other tis-sence and in the presence of NG-nitro-l-arginine methylsues (Gillespie et al., 1989; Tucker et al., 1990), nitricester or/and indomethacin.oxide might be directly released from perivascularnerve fibers as a transmitter and induces relaxation.

3. DiscussionMany studies concerning nerve-evoked relaxation haveemployed preparations whose adrenergic and choliner-The aim of our study was to examine the possible

modulation by the endothelium of neurogenic-medi- gic receptors were blocked by specific antagonists. This

G. Segarra et al./General Pharmacology 32 (1999) 583–589 587

viously contracted. Thus we were able to evaluate theexistence and importance of nitric oxide and prosta-glandins in the modulation of the adrenergic contrac-tion in vessels with as little compromise as possible ofother mechanisms that might enhance or impair the ac-tion of endothelial nitric oxide and prostaglandins.Under these experimental conditions the results showthat the presence of intact endothelium is a prerequisitefor the release of nitric oxide and vasodilator prosta-glandins to attenuate adrenergic vasoconstriction. Theseresults are consistent with the findings in bronchial ar-teries of rabbits in which two different types of endo-thelium-derived relaxant factors (nitric oxide and vaso-dilator protaglandins) are released during contractionselicited by noradrenaline (Zschauer et al., 1997).

The participation of vasodilator protaglandins in theattenuation of neurogenic responses of pulmonary ar-

Fig. 4. Concentration-response curves for noradrenaline determined teries could be a characteristic of some species. Barnardin artery rings with (solid circles) (n 5 7) and without (open circles) et al. (1993) have shown that the release of nitric oxide(n 5 6) endothelium and in the presence of NG-nitro-l-arginine

or its metabolites produces a decrease in vascular resis-methyl ester (1024 M) (solid squares) (n 5 6), indomethacin (1025 M)tance in the rat lung, whereas some cyclooxygenase(solid triangles) (n 5 6), and NG-nitro-l-arginine methyl ester 1 indo-

methacin (open squares) (n 5 5). Results are expressed as percentage products mediate the same phenomenon in the dogof the contraction developed by 60 mM KCl by each preparation. Val- lung. Vasodilator protaglandins also mediate the en-ues are presented as mean 6 SEM shown by vertical bars (n 5 num- dothelium-dependent relaxation of dog pulmonary ar-ber of animals).

teries induced by bradykinin and calcium ionophore(A23187), but not that to acetylcholine (Gambone etal., 1997).as done to reveal a non-adrenergic, non-cholinergic

Previous studies have shown the interactions be-relaxation (Gillespie et al., 1989; Ignarro et al., 1990;tween nitric oxide and prostacyclin. Prostacyclin stimu-Liu et al., 1992). However, we gave special attention tolates nitric oxide release from endothelial cells and en-preparations without adrenergic and cholinergic block-hances the dilator activity of nitric oxide on vascularade when exploring the actions of neurogenic stimula-smooth muscle (Shimokawa et al., 1988; Maurice et al.,tion. Although adrenergic and cholinergic inhibitors1991). In isolated dog pulmonary arteries these syner-might not directly interfere with the production of nitricgistic interactions occur during endothelium-dependentoxide, they alter the expected constriction which nor-relaxations evoked by bradykinin, but no interactionsmally appears during stimulation of perivascular adren-were observed during responses to acetylcholine (Gam-ergic nerves. In addition, we used preparations under

basal, resting conditions, instead of preparations pre- bone et al. 1997). This suggests that synergistic interac-

Table 1Geometric mean EC50 values and maximal responses to noradrenaline in dog pulmonary arteries

EC50 (M) Maximal responseArterial preparations (confidence interval) (mean 6 SEM)

With endothelium 1.85 3 1026 3643 6 661(1.34 3 1026 – 2.55 3 1026)

With endothelium 1 3.77 3 1027* 5297 6 305*NG-nitro-l-arginine (2.41 3 1027 – 5.88 3 1027)methyl ester

With endothelium 1 2.50 3 1027* 4505 6 319Indomethacin (2.00 3 1027 – 3.11 3 1027)

With endothelium 1 1.65 3 1027* 6846 6 435*Indomethacin 1 (1.05 3 1027 – 2.74 3 1027)NG-nitro-l-argininemethyl ester

Without endothelium 5.44 3 1027* 6571 6 712*(3.92 3 1027 – 7.57 3 1027)

* Significant difference versus untreated arteries with endothelium (p , 0.05).

588 G. Segarra et al./General Pharmacology 32 (1999) 583–589

Aminergic histofluorescence and contractile responses to transmu-tions are specific to certain endothelial cells activators.ral electrical field stimulation and norepinephrine of human mid-In our results the incubation of the segments withdle cerebral arteries obtained promptly after death. Circ Res 65,NG-nitro-l-arginine methyl ester and indomethacin po- 316–321.

tentiates the responses to adrenergic stimulation as Fishman, A.P., 1990. The normal pulmonary circulation. In: Frohman,compared to the segments incubated with NG-nitro-l- A.P. (Ed.), Pulmonary disease and disorders, Volume 1, McGraw-

Hill, New York, NY, pp. 975–997.arginine methyl ester or indomethacin alone but thisFleming, W.W., Westfall, D.P., De la Lande, I.S., Jellet, L.B., 1972.potentiation was not greater than the sum of those due

Long-normal distribution of equieffective doses of norepineph-to NG-nitro-l-arginine methyl ester or indomethacin.rine and acetylcholine in several tissues. J Pharmacol Exp Ther

The augmentation of neurogenic responses by endo- 181, 339–345.thelial removal appears to be associated with adrener- Furchgott, R.F., Zawadzki, J.V., 1980. The obligatory role of endothe-

lial cells in the relaxation of arterial smooth muscle by acetylcho-gic neurotransmitter. The evidence for this being thatline. Nature 288, 373–376.the endothelial mediated inhibition was observed in re-

Gambone, L.M., Murray, P.A., Flavahan, N.A., 1997. Synergistic in-sponse to electrical field stimulation and to noradrena-teraction between endothelium-derived NO and prostacyclin in

line. As postulated in previous reports (Liu et al., 1991; pulmonary artery: potencial role for K1ATP channels. Br J Pharma-

Shinozuka et al., 1992), it is possible that during nerve col 121, 271–279.Gillespie, J.S., Liu, X.R., Martin, W., 1989. The effects of L-argininestimulation other transmitters are released and induce

and NG-mono-metyl-L-arginine on the response of the rat ano-relaxing factors production by endothelial cells, thuscoccygeus muscle to NANC nerve stimulation. Br J Pharmacol 98,leading to partial inhibition of contraction. However,1080–1082.

under the present experimental conditions, noradrena- Gonzalez, C., Estrada, C., 1991. Nitric oxide mediates neurogenic va-line appears to be the main transmitter which triggers sodilation of bovine cerebral arteries. J Cereb Blood Flow Metabthe release of nitric oxide and prostacyclin. 11, 366–370.

Hyman, A.L., Lippton, H.L., Dempesy, C.W., Fontana, C.J., Richard-In conclusion, from these results it can be concludedson, D.E., Rieck, R.W., Kadowitz, P.J., 1989. Autonomic controlthat endothelial cells of dog pulmonary arteries depressof the pulmonary circulation. In: Weir, E.K., Reeves, J.T., (Eds).,the contractile response to electrical field stimulation ofPulmonary vascular physiology and phatophysiology. Marcel

intramural nerves and to noradrenaline by a mechanism Dekker, New York, NY, pp. 291–324.involving the release of both nitric oxide and vasodila- Hynes, M.R., Dang, H., Duckles, S.P., 1988. Contractile responses to

adrenergic nerve stimulation are enhanced with removal of endo-tor prostaglandins.thelium in rat caudal artery. Life Sci 42, 357–365.

Ignarro, L.J., Bush, P.A., Buga, G.M., Wood, K.S., Fukoto, J.M.,Rajfer, J., 1990. Nitric oxide and cyclic GMP formation upon elec-Acknowledgments trical field stimulation cause relaxation of corpus cavernosumsmooth muscle. Biochem Biophys Res Commun 170, 843–850.The present work was supported by the Comision In-

Ignarro, L.J., Byrns, R.E., Buga, G.M., Wood, K.S. 1987. Endothe-terministerial de Ciencia y Tecnologıa, Ministerio de lium-derived relaxing factor from pulmonary artery and vein pos-Sanidad and Generalitat Valenciana. This investigation sesses pharmacological and chemical properties that are identicalconforms with the Guide for the Care and Use of Labo- to those of nitric oxide radical. Circ Res 61, 866–879.

Liu, S.F., Crawley, D.E., Evans, T.W., Barnes, P.J., 1991. Endogenousratory Animals published by the U.S. National Insti-nitric oxide modulates adrenergic neural vasoconstriction intutes of Health (NIH Publication No. 85-23, revisedguinea-pig pulmonary artery. Br J Pharmacol 104, 565–569.1985) and all experimental procedures were performed

Liu, S.F., Crawley, D.E., Evans, T.W., Barnes, P.J., 1992. Endothe-in accordance with institutional guidelines for animal lium-dependent noadrenergic, noncholinergic neural relaxation inwelfare. guinea pig pulmonary artery. J Pharmacol Exp Ther 260, 541–548.

MacLean, M.R., McCulloch, K.M., MacMillan, J.B., McGrath, J.C.,1993. Influences of the endothelium and hypoxia on neurogenictransmission in the isolated pulmonary artery of the rabbit. Br JReferencesPharmacol 108, 150–154.

Martınez, C., Vila, J.M., Aldasoro, M., Medina, P., Chuan, P., Lluch,Aldasoro, M., Martınez, C., Vila, J.M., Flor, B., Lluch, S., 1993. Endo-S., 1994. The human deferential artery: endothelium-mediatedthelium-dependent component in the contractile responses of hu-contraction in response to adrenergic stimulation. Eur J Pharma-man omental arteries to adrenergic stimulation. Eur J Pharmacolcol 261, 73–78.250, 103–107.

Martınez, C., Cases, E., Vila, J.M., Aldasoro, M., Medina, P., Marco,Aldasoro, M., Martınez, C., Vila, J.M., Medina, P., Lluch, S., 1996. In-V., Lluch, S., 1995. Influence of endothelial nitric oxide on neuro-fluence of endothelial nitric oxide on adrenergic contractile re-genic contraction of human pulmonary arteries. Eur Resp J 8,sponses of human cerebral arteries. J Cereb Blood Flow Metabol1328–1332.16, 623–628.

Maurice, D.H., Crankshaw, D., Haslam, R.J., 1991. Synergistic ac-Barnard, J.W., Wilson, P.S., Moore, T.M., Thompson, W.J., Taylor,tions of nitrovasodilators and isoprenaline on rat aortic smoothA.E., 1993. Effect of nitric oxide and cyclooxygenase products onmuscle. Eur J Pharmacol 192, 235–242.vascular resistance in dog and rat lungs. J Appl Physiol 74 (6),

Moncada, S., Gryglewski, R., Bunting, S., Vane, J.R., 1976. An en-2940–2948.zyme isolated from arteries transforms prostaglandin endoperox-Caplan, B.A., Schwartz, C.J., 1973. Increased endothelial cell turn-ides to an unstable substance that inhibits platelet aggregation.over in areas of in vivo Evans blue uptake in the pig aorta. Ath-Nature 263, 663–665.erosclerosis 6, 713–719.

Duckworth, J.W., Wellman, G.C., Walters, C.L., Bevan, J.A., 1989. Moncada, S., Palmer, R.M.J., Higgs, E.A., 1988. The discovery of ni-

G. Segarra et al./General Pharmacology 32 (1999) 583–589 589

tric oxide as the endogenous nitrovasodilator. Hypertension 12, Shinozuka, K., Kobayashi, Y., Shimoura, K., Hattori, K., 1992. Role365–372. of nitric oxide from the endothelium on the neurogenic contractile

Palmer, R.M.J., Ferrige, A.G., Moncada, S., 1987. Nitric oxide release responses of rabbit pulmonary artery. Eur J Pharmacol 222, 113–account for the biological activity of endothelium-derived relaxing 120.factor. Nature 327, 524–526. Tesfamariam, B., Halpern, W., Osol, G., 1985. Effects of perfusion

Rabinovitch, M., Mullen M., Rosenberg, H.C., Maruyama, K., O’Bro- and endothelium on the reactivity of isolated resistance arteries.dovich, H., Olley, P.M., 1988. Angiotensin II prevents hypoxic Blood Vessels 22, 301–305.pulmonary hypertension and vascular changes in rat. Am J Phys- Toda, N., Okamura, T., 1990. Mechanism underlying the response toiol 254, H500–H508. vasodilator nerve stimulation in isolated dog and monkey cerebral

Samuelsson, B., Goldyne, M., Granstrom, E., Hamberg, M., Ham- arteries. Am J Physiol 259, H1511–H1517.marstrom, S., Malmsten, C., 1978. Prostaglandins and thrombox- Tucker, J.F., Brave, S.R., Charalambous, L., Hobbs, A.J., Gibson, A.,anes. Annu Rev Biochem 7, 997–1029. 1990. L-NG-nitro arginine inhibits non-adrenergic, non-choliner-

Shaul, P.W., Kinane, B., Farrar, M.A., Buja, L.M., Magness, R.R., gic relaxations of guinea-pig isolated tracheal smooth muscle. Br1991. Prostacyclin production and mediation of adenylate cyclase J Pharmacol 100, 663–664.activity in the pulmonary artery. J Clin Invest 88:447–455. Zschauer, A.O.A., Sielczak, M.W., Smith, D.A.S., Wanner, A., 1997.

Shimokawa, H., Flavahan, N.A., Lorenz, R.R., Vanhoutte, P.M., Norepinephrine-induced contraction of isolated rabbit bronchial1988. Prostacyclin releases endothelium-derived relaxing factor

artery: role of a1- and a2-adrenoceptor activation. J Appl Physioland potentiates its action in coronary arteries of the pig. Br J Phar-

82(6), 1918–1925.macol 95, 1197–1203.