Magnus G. Persson

Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans

The presence of nitric oxide (NO) in the exhaled air of humans and of anaesthetized rabbits and guinea pigs was demonstrated by chemiluminescence, diazotization and mass spectrometry. This NO is endogenously produced in the lung by an NO synthase, since its generation in guinea pigs and rabbits was inhibited by N omega-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, inhibitors of this enzyme. The effect of the inhibitors was reversed by the precursor of NO synthesis, L-arginine. Since NO is produced by normal vascular endothelium for the physiological regulation of blood flow and pressure and also by activated macrophages to contribute to non-specific immunity, our experiments suggest that NO may play both vascular regulatory and host defence roles in pulmonary physiology and pathophysiology.

Endogenous nitric oxide as a modulator of rabbit skeletal muscle microcirculation in vivo

1 Intravital microscopy of rabbit tenuissimus muscle microvasculature was used for in vivo studies of the role of endogenous nitric oxide (NO) in local vascular control. Derivatives of arginine were applied topically in order to modulate the formation of NO from l‐arginine. 2 l‐NG‐monomethylarginine (l‐NMMA) (10–100 μm), but not d‐NG‐monomethylarginine (d‐NMMA), dose‐dependently reduced microvascular diameters. The vasoconstriction induced by l‐NMMA (100 μm) was prevented by pretreatment with l‐arginine (1 mm) but not with d‐arginine (1 mm). Intravenous infusions of l‐arginine (300 mg kg−1) reversed the effect of l‐NMMA (100 μm). l‐Arginine or d‐arginine applied topically at 1 mm per se had no effect on microvascular diameters. 3 Vasodilatation by acetylcholine (0.03‐3 μm) was significantly inhibited by l‐NMMA (100 μm), whereas vasodilatation by adenosine (0.1–100 μm) or sodium nitroprusside (100 nm) was not affected. 4 The hyperaemic response after tenuissimus muscle contractions induced by motor nerve stimulation was unaffected by the presence of l‐NMMA (100 μm). 5 Aggregates of platelets and white blood cells were seen in venules during superfusion with l‐NMMA (100 μm), but not with d‐NMMA (100 μm). 6 Our results suggest that endogenous NO formed from l‐arginine is a modulator of microvascular tone and platelet and white cell‐vessel wall interaction in vivo. Nitric oxide does not, however, appear to play a role in the mediation of functional hyperaemia in this tissue.

Ethanol can inhibit nitric oxide production.

Endogenous nitric oxide (NO) in exhaled air from anaesthetized rabbits was monitored by chemiluminescence. Ethanol (3-30 mmol kg-1) infused i.v. dose dependently reduced the levels of exhaled NO, with an IC50 of 23 +/- 3 mmol kg-1. L-Arginine (1 g kg-1) did not reverse the effect of ethanol. These results demonstrate the inhibition of NO formation by ethanol in vivo.

Endogenous nitric oxide counteracts antigen-induced bronchoconstriction

In anesthetized, artificially ventilated guinea pigs immunized against ovalbumin, challenge with aerosolized ovalbumin (0.1 mg) elicited a substantial and sustained increase of insufflation pressure. The inhibitor of endogenous nitric oxide (NO) synthesis, L-NAME (N omega-nitro-L-arginine methylester, 30 mg kg-1 i.v.), markedly augmented the response, the potentiation of which could be prevented by NO (20 p.p.m.) in the inhaled air. The results indicate an inhibitory effect of endogenous NO on antigen-induced bronchoconstriction.

Detection of nitric oxide in exhaled air during administration of nitroglycerin in vivo.

1 Direct evidence for nitric oxide (NO) formation from nitroglycerin (GTN) was obtained by measurements of NO concentrations in exhaled air in artifically‐ventilated, pentobarbitone‐anaesthetized rabbits. 2 The concentration of endogenously formed NO was 23 ± 5 parts per billion (p.p.b.). Infusions of GTN (1–100 μg kg−1 min−1, i.v.) induced dose‐dependent and biphasic increments in exhaled NO and concomitant reductions in systemic blood pressure. 3 Tolerance to the blood pressure reduction developed in parallel with a decrease in GTN‐induced exhaled NO, a pattern which was unaffected by administration of Nω‐nitro‐l‐arginine methyl ester (l‐NAME, 30 mg kg−1), l‐cysteine (200 mg kg−1), N‐acetylcysteine (200 mg kg−1) or glutathione (200 mg kg−1). 4 Intravenous infusions of adenosine (0.7 mg ml−1, 250 μl kg−1 min−1) and GTN (1 mg ml−1, 250 μl kg−1 min−1) elicited similar decrements in pulmonary vascular resistance. GTN elicited a substantial increase in exhaled NO (50 ± 10 p.p.b.) whereas adenosine evoked a markedly smaller increase (7 ± 1 p.p.b.). l‐NAME (30 mg kg−1, i.v.) abolished NO in exhaled air, and evoked an increase in pulmonary vascular resistance from 116 ± 19 to 147 ± 9 pulmonary vascular resistance units. After l‐NAME the change in pulmonary vascular resistance induced by adenosine or GTN was increased to a similar degree. However, while the increase in exhaled NO induced by nitroglycerin was unaffected, the response to adenosine was abolished. 5 The present data demonstrate that NO is formed from GTN in vivo. Furthermore, thiol availability, or nitric oxide synthase activity are not limiting factors in the conversion of nitroglycerin to NO in vivo. Finally, pulmonary haemodynamic changes per se do not explain the observed increase in NO upon nitroglycerin infusion.

Direct demonstration of no formation in vivo from organic nitrites and nitrates, and correlation to effects on blood pressure and to in vitro effects

Previous studies, utilizing nitric oxide synthase inhibitors and nitric oxide application, indicate that nitric oxide has the capacity to modulate contractile responses in pulmonary vessels. In the present study, in vitro effects of organic nitrates/nitrites were compared with their in vivo ability to generate nitric oxide and their effects on blood pressure. Glyceryl trinitrate, ethyl nitrite, isobutyl nitrate, isobutyl nitrite, isoamyl nitrite and butyl nitrite inhibited contractions in response to nerve stimulation in guinea pig pulmonary artery and vas deferens. Glyceryl trinitrate (also known as nitroglycerin) was the most potent and isobutyl nitrate the least potent substance with this action (IC50 4.5 +/- 0.2 x 10(-10) and 1.1 +/- 0.1 x 10(-5) M, respectively). Contractile responses to noradrenaline were inhibited, whereas noradrenaline release was unaffected by organonitrates/nitrites, indicating a post-junctional inhibitory effect. When infused intravenously to anaesthetized rabbits glyceryl trinitrate, ethyl nitrite and isobutyl nitrate generated dose-dependent increments of nitric oxide in exhaled air and dose-dependent decrements in systemic blood pressure. Significant correlations were obtained between in vivo NO generation and effects on blood pressure, as well as between NO generation in vivo and the in vitro activity of the organic nitrites and organic nitrates. In conclusion, organic nitrites and organic nitrates can modulate adrenergic neuroeffector transmission in guinea pig pulmonary artery and vas deferens, and produce detectable concentrations of nitric oxide in exhaled air in vivo, in the rabbit. The observations give direct in vivo evidence that organic nitrites and nitrates generate NO, and strongly support them exerting their action via NO formation.

Modulation of neuroeffector transmission in the guinea pig pulmonary artery by endogenous nitric oxide.

The influence of endogenous nitric oxide (NO) on neuroeffector transmission in segments of guinea pig pulmonary artery was analyzed by application of NG-monomethyl-L-arginine (L-NMMA). L-NMMA enhanced contractile responses to nerve stimulation and this enhancement was counteracted by L-arginine. The enhancement remained after removal of the endothelium. L-NMMA enhanced contractions to exogenous noradrenaline. After blockade of adrenergic transmission by phentolamine, L-NMMA enhanced contractions induced by nonadrenergic-noncholinergic (NANC) neurotransmission. Stimulation-induced release of [3H]noradrenaline was unchanged by L-NMMA. The results suggest that endogenous NO exerts a postjunctional inhibition on adrenergic neurotransmission in the guinea pig pulmonary artery. A concomitant pre- and/or postjunctional inhibition of NANC transmission is implicated. The neuromodulation by NO does not require an intact endothelium.

Nerve-induced tachykinin-mediated vasodilatation in skeletal muscle is dependent on nitric oxide formation

Nerve-induced vasodilatation was studied by intravital microscopy of the rabbit tenuissimus muscle, pretreated with pancuronium, phentolamine, and guanethidine. Nerve stimulation of the tenuissimus nerve induced a vasodilatation which was frequency and pulse duration-dependent and insensitive to atropine and propanolol but abolished by tetrodotoxin. The nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM), but not its enantiomer, D-NAME, markedly inhibited the vasodilation induced by nerve stimulation or by exogenous substance P or neurokinin A. Vasodilatation due to calcitonin gene-related peptide, prostaglandin E2 or nitroprusside was unaffected. The substance P antagonist, spantide (30 microM), significantly attenuated nerve-induced vasodilatation, in parallel with L-NAME. Our results indicate that nerve-induced vasodilatation in skeletal muscle can be attributed to the release of substance P and/or other tachykinins and that nitric oxide subsequently mediates the response to endogenous tachykinins released from nerves.

Mechanisms of nitric oxide generation from nitroglycerin and endogenous sources during hypoxia in vivo

Nitroglycerin (GTN), often used in conditions of cardiovascular ischaemia, acts through the liberation of nitric oxide (NO) and the local concentration of NO in the tissue is responsible for any biological effect. However, little is known about the way in which the concentration of NO from GTN and other NO‐donors is influenced by low oxygen tension in the target tissues. To evaluate the impact of changes in oxygen tension in the metabolism of NO‐donors we measured exhaled NO in anaesthetized rabbits in vivo and expired NO and perfusate nitrite (NO2−) in buffer‐perfused lungs in situ. The impact of acute hypoxia on NO formation from GTN, isosorbide‐5‐mononitrate (ISMN), dissolved authentic NO, NO2− and NO generated from endogenous NO‐synthase (NOS) was studied in either model. Acute hypoxia drastically increased exhaled NO concentrations from all NO‐donors studied, both in vivo and in the perfused lung. During similar conditions endogenous NO generation from NOS was strongly inhibited. The effects were most pronounced at less than 3% inspired oxygen. The mechanisms for the increased NO‐formation during hypoxia seems to differ between GTN‐ and NO2−‐derived NO. The former phenomenon is likely due to diminished breakdown of NO. In conclusion, hypoxic conditions preserve very high local NO concentrations generated from organic nitrates in vivo and we suggest that this might benefit preferential vasodilation in ischaemic tissue regions. Our findings point out the necessity to consider the influence of oxygen tension when studying the action of NO‐donors.

The promotion of patent airways and inhibition of antigen‐induced bronchial obstruction by endogenous nitric oxide

1 The aim of the present study was to investigate the role of nitric oxide (NO), histamine and leukotrienes in bronchial obstruction. For this, guinea‐pigs immunised against ovalbumin were studied under anaesthesia during challenge with antigen or agonists. 2 Challenge with nebulised antigen (0.1‐1 mg) elicited dose‐dependent increases in insufflation pressure which were abolished by combined administration of histamine and leukotriene antagonists. 3 Challenge with nebulised antigen (0.1‐1 mg) also elicited dose‐dependent increases in the concentration of endogenous nitric oxide in the exhaled air. After an initial peak, exhaled NO concentrations returned to pre‐challenge levels. 4 The increase in insufflation pressure and in exhaled NO caused by ovalbumin challenge was inhibited by combined administration of histamine and leukotriene antagonists. 5 In non‐immunised guinea‐pigs, challenge of the airways with nebulised histamine (10–1000 nmol) or leukotriene C4 (LTC4, 30–300 pmol) elicited dose‐dependent increases in insufflation pressure and in concentrations of endogenous NO in exhaled air. 6 The increase in exhaled NO correlated with the increase in insufflation pressure in response to ovalbumin, histamine and LTC4. An inhibitor of endogenous NO synthesis, Nω‐nitro‐l‐arginine methylester (l‐NAME, 30 mg kg−1 i.v.) abolished NO exhalation, and markedly augmented the airway responses to ovalbumin, histamine, or LTC4. 7 The potentiation by l‐NAME of the increase in insufflation pressure in response to ovalbumin or histamine was prevented by exogenous NO (20 p.p.m.) in the inhaled air. 8 The results indicate that endogenous NO has an inhibitory effect on bronchial obstruction. Increased NO release during allergen challenge is likely to be due to actions of histamine and leukotrienes.