Stephen L. Archer

The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels.

Hypoxia causes constriction in small pulmonary arteries and dilatation in systemic arteries. Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary blood flow is controlled in the fetus and by which local lung perfusion is matched to ventilation in the adult. HPV reduces the flow of desaturated blood through underventilated areas of lung. Even though many vasoactive substances have been examined as possible mediators of HPV, these appear more likely to be modulators than mediators. Hypoxic contraction has been demonstrated in single pulmonary vascular smooth muscle cells (PVSMC). The ability to sense changes in oxygen tension is observed in PVSMC and type 1 cells of the carotid body. In both cells, hypoxia has been shown to inhibit an outward potassium current, thus causing membrane depolarization and calcium entry through the voltage‐dependent calcium channels. In both cells there is evidence to suggest that changes in the redox status of the oxygen‐sensitive potassium channel or channels may control current flow, so that the channel is open when oxidized and closed when reduced. The redox status may be determined by the effects of hypoxia on mitochondrial/peroxisomal function or on the activity of an oxidase similar to NAD(P)H oxidase. More studies are needed to precisely define the individual potassium channels responsive to hypoxia and to confirm the gating mechanism. In systemic arteries hypoxia causes an increased current through ATP‐dependent potassium channels and vasodilatation, whereas in the pulmonary arteries hypoxia inhibits potassium current and causes vasoconstriction.—Weir, E. K., Archer, S. L. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J. 9, 183–189 (1995)

Anorexic Agents Aminorex, Fenfluramine, and Dexfenfluramine Inhibit Potassium Current in Rat Pulmonary Vascular Smooth Muscle and Cause Pulmonary Vasoconstriction

BACKGROUND The appetite suppressant aminorex fumarate is thought to have caused an epidemic of pulmonary hypertension in Europe in the 1960s. More recently, pulmonary hypertension has been described in some patients taking other amphetamine-like, anorexic agents: fenfluramine and its d-isomer, dexfenfluramine. No mechanism has been demonstrated that might account for the association between anorexic drugs and pulmonary hypertension. METHODS AND RESULTS Using the whole-cell, patch-clamp technique, we found that aminorex, fenfluramine, and dexfenfluramine inhibit potassium current in smooth muscle cells taken from the small resistance pulmonary arteries of the rat lung. Dexfenfluramine causes reversible membrane depolarization in these cells. These actions are similar to those of hypoxia, which initiates pulmonary vasoconstriction by inhibiting a potassium current in pulmonary vascular smooth muscle. In the isolated, perfused rat lung, aminorex, fenfluramine, and dexfenfluramine induce a dose-related increase in perfusion pressure. When the production of endogenous NO is inhibited by N-nitro-L-arginine methyl ester, the pressor response to dexfenfluramine is greatly enhanced. CONCLUSIONS These observations indicate that anorexic agents, like hypoxia, can inhibit potassium current, cause membrane depolarization, and stimulate pulmonary vasoconstriction. They suggest one mechanism that could be responsible for initiating pulmonary hypertension in susceptible individuals. It is possible that susceptibility is the result of the reduced production of an endogenous vasodilator, such as NO, but this remains speculative.

The non specificity of specific nitric oxide synthase inhibitors

L-NAME (Nw-Nitro-L-arginine methylester) and L-NMMA (NG- Monomethyl-L-arginine, monoacetate) are used widely as nitric oxide (NO) synthase inhibitors. Because of their functional groups (alcohols, amines and carboxylates), it appeared that they could interact with iron in a variety of systems. Using three in vitro models we observed these two compounds had inhibitory effects on cytochrome C reduction by ferrous iron, by ferrous iron accelerated by an unsaturated fatty acid or by epinephrine. This suggests that L-NAME and L-NMMA could have effects in iron containing systems found intracellularly apart from their inhibition of (NO) synthesis.

Impairment of hypoxic pulmonary vasoconstriction in mice lacking the voltage-gated potassium channel Kv1.5

Hypoxic pulmonary vasoconstriction (HPV) is initiated by the inhibition of several 4‐aminopyridine (4‐AP)‐sensitive, voltage‐gated, K+ channels (Kv). Several O2‐sensitive candidate channels (Kv1.2, Kv1.5, Kv2.1, and Kv3.1b) have been proposed, based on similarities between their characteristics in expression systems and the properties of the O2‐sensitive K+ current (IK) in pulmonary artery smooth muscle cells (PASMCs). We used gene targeting to delete Kv1.5 in mice by creating a SWAP mouse that is functionally a Kv1.5 knockout. We hypothesized that SWAP mice would display impaired HPV. The Kv1.5 α‐subunits present in the endothelium and PASMCs of wild‐type mice were absent in the lungs of SWAP mice, whereas expression of other channels Kv (1.1, 1.2, 2.1, 3.1, 4.3), Kir 3.1, Kir 6.1, and BKCa was unaltered. In isolated lungs and resistance PA rings, HPV was reduced significantly in SWAP versus wild‐type mice. Consistent with this finding, PASMCs from SWAP PAs were slightly depolarized and lacked IKv1.5, a 4‐AP and hypoxia‐sensitive component of IK that activated between ‐50 mV and ‐30 mV. We conclude that a K+ channel containing Kv1.5 α‐subunits is an important effector of HPV in mice.

Enhanced chemiluminescence as a measure of oxygen-derived free radical generation during ischemia and reperfusion.

It has been suggested that oxygen-derived free radicals may contribute to the myocardial injury associated with ischemia and reperfusion. As the presence of enhanced free radical generation is a prerequisite for such damage, several techniques have been used to provide evidence of increased oxygen free radical production during reperfusion; however, all such techniques have substantial limitations. In this study, we used enhanced chemiluminescence to evaluate oxygen free radical generation during ischemia and reperfusion in the isolated Langendorff-perfused rat heart. The chemiluminescent technique, which has high sensitivity and can monitor radical generation continuously, avoids some of the limitations of earlier methods. Chemiluminescence (expressed as counts per second) decreased from 219 +/- 11 at baseline to 142 +/- 9 during ischemia and markedly increased to a peak of 476 +/- 36 during the first 3-5 minutes of reperfusion. This was followed by a slow decline over 11-16 minutes to a steady-state level of 253 +/- 14 (each sequential change in chemiluminescence was highly significant; p less than 0.001). Superoxide dismutase (2,000 units/min) significantly decreased peak reperfusion chemiluminescence to 316 +/- 17 (p less than 0.01). Hearts subjected to a second period of ischemia and reperfusion had a higher peak chemiluminescence (626 +/- 62), which also was significantly attenuated by 1,000 units/min superoxide dismutase (398 +/- 16; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

NG-monomethyl-l-arginine causes nitric oxide synthesis in isolated arterial rings: Trouble in paradise☆

Arginine analogs are commonly used as inhibitors of the synthesis of endothelium-derived relaxing factor, nitric oxide. However, their effect on nitric oxide levels is rarely measured. Using a chemiluminescence assay for nitric oxide, we found that NG-monomethyl-L-arginine enhanced, rather than reduced, nitric oxide synthesis in pulmonary arterial and aortic rings. NG-monomethyl-L-arginine inhibited relaxation to the endothelium-dependent vasodilator A23187 in aortic but not pulmonary arterial rings. In contrast, N omega-nitro-L-arginine did not stimulate nitric oxide synthesis and it inhibited relaxation to A23187 in all rings. We conclude that NG-monomethyl-L-arginine is a partial agonist for nitric oxide synthesis.

Nebulized nitric oxide/nucleophile adduct reduces chronic pulmonary hypertension

OBJECTIVE Inhaled nitric oxide (NO) is a selective pulmonary vasodilator, but its use has been restricted almost exclusively to the intensive care setting due to the complexity of its delivery. NO/nucleophile adducts, such as diethylenetriamine/NO (DETA/NO), spontaneously release NO in aqueous solutions. We hypothesized that a nebulized DETA/NO (half-time of NO release > 20 h) would stay in the lower airways and continuously supply sufficient NO to achieve sustained vasodilation in chronic pulmonary hypertension. METHODS Chronic pulmonary hypertension was induced in rats by a monocrotaline injection. Nineteen days later, nebulizations of DETA/NO were given on 4 consecutive days (5 and 50 mu mol; 10 min/day). One day after the last nebulization, pulmonary and systemic arterial pressure and cardiac output were measured after thoracotomy. The lungs were isolated and perfused to study the pressure-flow relationship. The effect of DETA/NO nebulization on acute vasoconstrictor reactivity was studied in additional isolated lungs. RESULTS Total pulmonary, but not systemic, vascular resistance was significantly reduced by both DETA/NO doses, suggesting that DETA/NO, like NO, causes preferential dilation of the pulmonary circulation. The pulmonary perfusion pressure-flow curves were shifted downwards by DETA/NO treatment, indicating improved resistive properties of the pulmonary vasculature. DETA/NO nebulization into isolated lungs increased exhaled NO levels and progressively reduced vasoconstrictor responses to angiotensin II and acute hypoxia. These effects were not reversed by perfusate exchange. In intact rats, carotid artery pressure and plasma NO2- + NO3- levels did not change during and after DETA/NO nebulization. CONCLUSION DETA/NO nebulization offers a possibility of once a day, ambulatory delivery of NO and is a potential treatment for chronic pulmonary hypertension, although further studies are needed to establish safety and selectivity.

Coronary Nitric Oxide Production in Response to Exercise and Endothelium-Dependent Agonists

BACKGROUND Endothelium-derived nitric oxide (NO) contributes to epicardial coronary artery vasodilation during exercise. However, blockade of NO production does not impair the increase in coronary blood flow (CBF) during exercise, suggesting that NO is not obligatory for exercise-induced coronary resistance vessel dilation. In contrast, the increases in CBF produced by endothelium-dependent agonists are decreased after NO blockade. Consequently, this study was performed to determine whether the increase in coronary NO production in response to agonists is greater than that which occurs during exercise. METHODS AND RESULTS We measured the oxidation products of NO (nitrate+nitrite=NO(x)) in aortic and coronary sinus plasma using chemiluminescence to assess NO(x) production across the coronary circulation in chronically instrumented dogs during a 3-stage treadmill exercise protocol and in response to intracoronary administration of the endothelium-dependent agonists acetylcholine (37.5 microg/min) and bradykinin (3.0 microg/min). No coronary NO(x) production could be detected at rest or during the first 2 stages of exercise; only at the highest level of exercise was a small increase in coronary NO(x) production measured. In contrast, coronary production of NO(x) was significantly increased in response to endothelium-dependent agonists. CONCLUSIONS Coronary NO production in response to endothelium-dependent agonists is greater than in response to the increase in shear stress associated with exercise. These findings support previous studies suggesting that NO is not required for the coronary vasodilation that occurs in the normal heart during exercise.

Utility of a nitric oxide electrode for monitoring the administration of nitric oxide in biologic systems

Amperometric techniques for the detection of nitric oxide (NO) are commercially available, but their sensitivity and specificity are not well described. We evaluated the sensitivity and specificity of a Clark-style, platinum NO electrode. The electrode has a lower limit of detection for NO of <25 pmol/ml in vitro and is linear over the range from 25 pmol/ml to 4 nmol/ml. The electrode is specific for NO so long as the protective membrane that covers the electrode is intact. Any defect in this membrane results in the detection of other redox agents such as hydrogen peroxide. Because of its ease of handling, specificity, and sensitivity, the NO electrode is a useful tool for quantification of administered NO in vitro and in various biologic systems.

GTP (γS) and GDP (βS) as electron donors: New wine in old bottles

Abstract G proteins are membrane-bound regulatory proteins which modulate the activity of ion channels and other effector systems. The GTP and GDP analogs GTP (γS) and GDP (βS) have been used to study the role of G proteins in numerous physiologic systems. The prolonged effects of these analogs have been thought to be due to the fact that they are nonhydrolyzable. However, in this paper we show that the GTP (γS) and GDP (βS) analogs are potent reducing agents at physiologic pH. This observation suggests that previous data obtained using these compounds may need to be reinterpreted.