The hypothesis that endothelium-derived relaxing factor (EDRF) modulates hypoxic pulmonary vasoconstriction (HPV) was tested in isolated, blood-perfused rat lungs ventilated with gas mixtures of 21% O2-5% CO2-74% N2 (normoxia) or of 3% O2-5% CO2-92% N2 (hypoxia); 30 microM NG-monomethyl-L-arginine (L-NMMA), an inhibitor of EDRF production, caused a reduction in the endothelium-dependent relaxant response to acetylcholine (ACh) from 62 +/- 7, 88 +/- 4, and 100 +/- 4% to 26 +/- 8, 49 +/- 12, and 75 +/- 7% at ACh concentrations of 1, 10, and 100 microM, respectively (p less than 0.05 at all concentrations), indicating that L-NMMA acts via the inhibition of EDRF production. L-NMMA induced a concentration-related augmentation in HPV of 20 +/- 5, 32 +/- 8, and 34 +/- 8% at concentrations of 30, 300, and 1,000 microM (p less than 0.05, compared with a vehicle control group at all concentrations). The pressor response to a dose of angiotensin II (A-II), which produced the same increase in pulmonary artery pressure as that induced by hypoxia, was also significantly augmented (2 +/- 0.6%), but to a lesser extent. The augmentation of HPV by 30 microM L-NMMA was completely reversed by 1 mM L-arginine (a precursor of EDRF), but not by D-arginine (an isomer of L-arginine). One and 6 mM L-arginine, but not 6 mM D-arginine caused a significant inhibition of HPV by 20 +/- 2 and 47 +/- 12% (p less than 0.05, compared with the vehicle control group) and a small but not significant reduction in A-II-mediated contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the effects of exposing rats to hypoxia (10% fractional inspired O2 concentration) for 2 and 7 days on endothelium-dependent and -independent vasodilation and also on the ability of guanosine 3',5'-cyclic monophosphate (cGMP) to activate cGMP-dependent protein kinase (G-kinase) in rat conduit pulmonary arteries (PA). The ability of acetylcholine (ACh) and sodium nitroprusside (SNP) to both relax PA rings and elevate tissue cGMP levels was significantly attenuated in PA from hypoxic animals. The ability of atrial natriuretic peptide to relax and generate cGMP in PA rings was unchanged by hypoxia. Relaxation and elevation of cGMP levels induced by SNP in aortic rings was unaltered by hypoxia. Similarly, hypoxia did not alter the concentration-dependent activation by exogenous cGMP of G-kinase. We conclude that chronic exposure of rats to hypoxia results in a selective impairment of soluble guanylyl cyclase in rat PA, leading to an attenuation of ACh- and SNP-induced cGMP accumulation and relaxation.

1. The inhibitory role of endothelium-derived relaxing factor was studied in both rat and human pulmonary arteries in vitro by inhibiting its synthesis with the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA). 2. In rat pulmonary arteries, L-NMMA pretreatment (10-300 microM) dose-dependently inhibited acetylcholine-induced relaxation (which is endothelium-dependent). NG-monomethyl-D-arginine (D-NMMA, 100 microM) was without effect. L-Arginine, but not D-arginine, dose-dependently reversed this inhibition. L-NMMA had no effect on relaxation induced by sodium nitroprusside. 3. In human small pulmonary arteries L-NMMA (100 microM) pretreatment similarly inhibited the acetylcholine-induced relaxation but had no effect on the sodium nitroprusside-induced relaxation. 4. In both rat and human pulmonary arteries, L-NMMA, but not D-NMMA, always caused contraction of preconstricted tissues whereas it had no effect on baseline tone. In the rat this contraction was completely prevented by prior treatment with L-arginine. 5. L-NMMA (100 microM) pretreatment mimicked the effect of endothelium removal on phenylephrine-induced vasoconstriction, both resulting in an increase in tension development at each concentration of phenylephrine. This enhancement was greatest at low concentrations of phenylephrine but was still present even at the highest concentrations. Pretreatment with L-NMMA (100 microM) also significantly increased the responses to single doses of phenylephrine. 6. These results suggest that endothelium-derived relaxing factor from endothelial cells both mediates the relaxation response to acetylcholine and also acts as a physiological brake against vasoconstriction in pulmonary vessels.

1 Nonadrenergic, noncholinergic (NANC) nerves mediate vasodilatation in guinea-pig pulmonary artery (PA) by both endothelium-dependent and endothelium-independent mechanisms. The transmitter(s) involved in the endothelium-independent pathway have not yet been identified. We have therefore investigated the possibility that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) may mediate this neural vasodilator response in guinea-pig branch PA rings denuded of endothelium. 2 Electric field stimulation (EFS, 50 V, 0.2 ms) induced a frequency-dependent (1-24 Hz), tetrodotoxin-sensitive relaxation of the U44069-precontracted PA rings in the presence of adrenergic and cholinergic blockade. 3 The NO synthase inhibitors NG-monomethyl L-arginine (L-NMMA, 100 pM) and NG-nitro L-arginine methyl ester (L-NAME, 30 pM), and the guanylyl cyclase inhibitor methylene blue (5 pM) inhibited the EFS (16 Hz)-induced relaxation by 53 ± 5, 74 + 9 and 82 ± 9% respectively (n = 5-7, P<0.01, compared with control rings).4 Excess concentrations of L-, but not D-arginine (300 gM) completely reversed the inhibitory effect of L-NMMA. 5 The EFS-elicited relaxation (4 Hz) was potentiated by 1 gM zaprinast, a type V phosphodiesterase inhibitor which inhibits guanosine 3':5'-cyclic monophosphate (cyclic GMP) degradation, but was unaffected by 0.1 gM zardaverine, a type III/IV phosphodiesterase inhibitor which inhibits cyclic AMP degradation. 6 EFS (50 V, 0.2 ms, 16 Hz) induced a 3 fold increase in tissue cyclic GMP content, an action which was inhibited by L-NMMA (100;LM).7 Pyrogallol (100IM), a superoxide anion generator, also inhibited the EFS-induced relaxation by 53 ± 9%, and this effect was prevented by superoxide dismutase. 8 Chemical sympathetic denervation with 6-hydroxydopamine had no effect on the relaxant response to EFS in the endothelium-denuded PA rings.9 In endothelium-denuded branch PA rings at resting tone, L-NMMA (100 JM) significantly augmented the adrenergic contractile response, an effect which was completely reversed by L-arginine, but not by D-arginine. In the same groups of vessel rings, L-NMMA had no significant effect on the matched contractile response to exogenous noradrenaline. 10 These results suggest that NO may be released from intramural nerve endings other than adrenergic nerves (probably NANC nerves), and this leads to vasodilatation via activation of guanylyl cyclase.

1 Electrical field stimulation (EFS) of guinea-pig isolated pulmonary artery induced a frequencydependent contraction. This was abolished by tetrodotoxin (1,UM) and prevented by phentolamine and prazosin (both 1,UM), indicating a role for ax-adrenoceptors activated by noradrenaline (NA) released from perivascular adrenergic nerves. 2 L-N0-monomethyl arginine (L-NMMA, 0.3-1OOpM) caused a concentration-dependent enhancement of the EFS-induced contraction with a 3.4 + 0.5 fold increase at 100pM (n = 6). The augmenting effect of 30,UM L-NMMA on the contraction to EFS was completely reversed by 100-300PM L-arginine, but not by an identical concentration of D-arginine. 3 The contractile response to exogenous NA was similarly enhanced by 304UM L-NMMA (2.9 + 0.6 fold increase, n = 5).4 The contractile responses to exogenous phenylephrine and prostaglandin F2. which matched the contraction to EFS (4Hz) were equally augmented by 304uM L-NMMA. 5 In vessel rings submaximally contracted with the thromboxane analogue U44069 (2pM), the selective a2-adrenoceptor agonist UK14304 induced concentration-dependent relaxation, which was abolished by removal of endothelium. NA had little relaxant effect on these precontracted vessel rings unless in the presence of prazosin (1 uM). 6 Indomethacin had no significant effect on the contractile response to EFS or NA, indicating that vasodilator cyclo-oxygenase products such as prostacyclin are not involved in modulating these responses.7 Our results suggest that endogenous nitric oxide inhibits the contractile response to adrenergic nerve stimulation in the guinea-pig pulmonary artery by a postjunctional mechanism, but release of prostacyclin does not modulate these responses. Basal release of nitric oxide from endothelial cells may account for this inhibition.