Daniel P. Nelson

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.

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)

Redox control of oxygen sensing in the rabbit ductus arteriosus

How the ductus arteriosus (DA) closes at birth remains unclear. Inhibition of O2‐sensitive K+ channels may initiate the closure but the sensor mechanism is unknown. We hypothesized that changes in endogenous H2O2 could act as this sensor. Using chemiluminescence measurements with luminol (50 μm) or lucigenin (5 μm) we showed significantly higher levels of reactive O2 species in normoxic, compared to hypoxic DA. This increase in chemiluminescence was completely reversed by catalase (1200 U ml−1). Prolonged normoxia caused a significant decrease in K+ current density and depolarization of membrane potential in single fetal DA smooth muscle cells. Removal of endogenous H2O2 with intracellular catalase (200 U ml−1) increased normoxic whole‐cell K+ currents (IK) and hyperpolarized membrane potential while intracellular H2O2 (100 nm) and extracellular t‐butyl H2O2 (100 μm) decreased IK and depolarized membrane potential. More rapid metabolism of O2−· with superoxide dismutase (100 U ml−1) had no significant effect on normoxic K+ currents. N‐Mercaptopropionylglycine (NMPG), duroquinone and dithiothreitol all dilated normoxic‐constricted DA rings, while the oxidizing agent 5,5′‐dithiobis‐(2‐nitrobenzoic acid) constricted hypoxia‐dilated rings. NMPG also increased IK. We conclude that increased H2O2 levels, associated with a cytosolic redox shift at birth, signal K+ channel inhibition and DA constriction.

Role of Store-Operated Calcium Channels and Calcium Sensitization in Normoxic Contraction of the Ductus Arteriosus

Background— At birth, the increase in oxygen causes contraction of the ductus arteriosus, thus diverting blood flow to the lungs. Although this contraction is modulated by substances such as endothelin and dilator prostaglandins, normoxic contraction is an intrinsic property of ductus smooth muscle. Normoxic inhibition of potassium channels causes membrane depolarization and calcium entry through L-type calcium channels. However, the studies reported here show that after inhibition of this pathway there is still substantial normoxic contraction, indicating the involvement of additional mechanisms. Methods and Results— Using ductus ring experiments, calcium imaging, reverse-transcription polymerase chain reaction, Western blot, and cellular electrophysiology, we find that this depolarization-independent contraction is caused by release of calcium from the IP3-sensitive store in the sarcoplasmic reticulum, by subsequent calcium entry through store-operated channels, and by increased calcium sensitization of actin-myosin filaments, involving Rho-kinase. Conclusions— Much of the normoxic contraction of the ductus arteriosus at birth is related to calcium entry through store-operated channels, encoded by the transient receptor potential superfamily of genes, and to increased calcium sensitization. A clearer understanding of the mechanisms involved in normoxic contraction of the ductus will permit the development of better therapy to close the patent ductus arteriosus, which constitutes ≈10% of all congenital heart disease and is especially common in premature infants.

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.

Pergolide Is an Inhibitor of Voltage-Gated Potassium Channels, Including Kv1.5, and Causes Pulmonary Vasoconstriction

Background—Pergolide produces clinical benefit in Parkinson disease by stimulating dopamine D1 and D2 receptors. An increased incidence of carcinoid-like heart valve disease (CLHVD) has been noted in pergolide users, reminiscent of that induced by certain anorexigens used for weight reduction. Anorexigens that modulate serotonin release and reuptake, such as dexfenfluramine, were withdrawn from sale because of CLHVD. Interestingly, the anorexigens also caused pulmonary arterial hypertension (PAH). Anorexigens were shown to enhance hypoxic pulmonary vasoconstriction, in part by inhibiting voltage-gated K+ channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). Although PAH has not been associated with pergolide use, we hypothesized that pergolide might have similar effects on hypoxic pulmonary vasoconstriction and Kv channels. Methods and Results—Pergolide enhanced hypoxic pulmonary vasoconstriction in the isolated perfused rat lung compared with control lungs (mean pulmonary artery pressure 32±3 versus 21±2 mm Hg; P<0.01). Pergolide also caused vasoconstriction in rat pulmonary artery rings. Pergolide inhibited PASMC potassium current density, resulting in membrane depolarization (from −51±2 to −44±1 mV) and increased cytosolic calcium in both rat and human PASMCs. Pergolide directly inhibited heterologously expressed Kv1.5 and KCa channels. Conclusions—Pergolide causes Kv channel inhibition and, despite being from a different class of drugs, has pulmonary vascular effects reminiscent of dexfenfluramine. Coupled with their shared proclivity to induce CLHVD, these findings suggest that clinical monitoring for pergolide-induced PAH should be considered.

Effects of fluoxetine, phentermine, and venlafaxine on pulmonary arterial pressure and electrophysiology

The anorexic agents dexfenfluramine and fenfluramine plus phentermine have been associated with outbreaks of pulmonary hypertension. The fenfluramines release serotonin and reduce serotonin reuptake in neurons. They also inhibit potassium current ( I K), causing membrane potential depolarization in pulmonary arterial smooth muscle cells. The recent withdrawal of the fenfluramines has led to the use of fluoxetine and phentermine as an alternative anorexic combination. Because fluoxetine and venlafaxine reduce serotonin reuptake, we compared the effects of these agents with those of phentermine and dexfenfluramine on pulmonary arterial pressure, I K, and membrane potential. Fluoxetine, venlafaxine, and phentermine caused minimal increases in pulmonary arterial pressure at concentrations < 100 μM but did cause a dose-dependent inhibition of I K. The order of potency for inhibition of I K at +50 mV was fluoxetine > dexfenfluramine = venlafaxine > phentermine. Despite the inhibitory effect on I K at more positive membrane potentials, fluoxetine, venlafaxine, and phentermine, in contrast to dexfenfluramine, had minimal effects on the cell resting membrane potential (all at a concentration of 100 μM). However, application of 100 μM fluoxetine to cells that had been depolarized to -30 mV by current injection elicited a further depolarization of >18 mV. These results suggest that fluoxetine, venlafaxine, and phentermine do not inhibit I K at the resting membrane potential. Consequently, they may present less risk of inducing pulmonary hypertension than the fenfluramines, at least by mechanisms involving membrane depolarization.The anorexic agents dexfenfluramine and fenfluramine plus phentermine have been associated with outbreaks of pulmonary hypertension. The fenfluramines release serotonin and reduce serotonin reuptake in neurons. They also inhibit potassium current (IK), causing membrane potential depolarization in pulmonary arterial smooth muscle cells. The recent withdrawal of the fenfluramines has led to the use of fluoxetine and phentermine as an alternative anorexic combination. Because fluoxetine and venlafaxine reduce serotonin reuptake, we compared the effects of these agents with those of phentermine and dexfenfluramine on pulmonary arterial pressure, IK, and membrane potential. Fluoxetine, venlafaxine, and phentermine caused minimal increases in pulmonary arterial pressure at concentrations < 100 microM but did cause a dose-dependent inhibition of IK. The order of potency for inhibition of IK at +50 mV was fluoxetine > dexfenfluramine = venlafaxine > phentermine. Despite the inhibitory effect on IK at more positive membrane potentials, fluoxetine, venlafaxine, and phentermine, in contrast to dexfenfluramine, had minimal effects on the cell resting membrane potential (all at a concentration of 100 microM). However, application of 100 microM fluoxetine to cells that had been depolarized to -30 mV by current injection elicited a further depolarization of >18 mV. These results suggest that fluoxetine, venlafaxine, and phentermine do not inhibit IK at the resting membrane potential. Consequently, they may present less risk of inducing pulmonary hypertension than the fenfluramines, at least by mechanisms involving membrane depolarization.

Low potassium dextran lung preservation solution reduces reactive oxygen species production

BACKGROUND Low potassium dextran lung preservation solution has reduced primary graft failure in animal and human studies. Though the mechanism of reducing primary graft failure is unknown, low potassium dextran differs most significantly from solutions such as Euro-Collins (EC) and University of Wisconsin in its potassium concentration. The aim of this study was to investigate the impact that potassium concentration in lung preservation solutions had on pulmonary arterial smooth muscle cell depolarization and production of reactive oxygen species. METHODS Using isolated pulmonary artery smooth muscle cells from Sprague-Dawley rats, the patch-clamp technique was used to measure resting cellular membrane potential and whole cell potassium current. Measurements were recorded at base line and after exposure to low potassium dextran, EC, and University of Wisconsin solutions. Pulmonary arteries from rats were isolated from the main pulmonary artery to the fourth segmental branch. Arteries were placed into vials containing low potassium dextran, EC, low potassium EC, Celsior, and University of Wisconsin solutions with reactive oxygen species measured by lucigenin-enhanced chemiluminescence. RESULTS Pulmonary artery smooth muscle cell membrane potentials had a significant depolarization when placed in the University of Wisconsin or EC solutions, with changes probably related to inhibition of voltage-gated potassium channels. Low potassium dextran solution did not alter the membrane potential. Production of reactive oxygen species as measured by chemiluminescence was significantly higher when pulmonary arteries were exposed to University of Wisconsin or EC solutions (51,289 +/- 5,615 and 35,702 +/- 4353 counts/0.1 minute, respectively) compared with low potassium dextran, Celsior, and low potassium EC (12,537 +/- 3623, 13,717 +/- 3,844 and 15,187 +/- 3,792 counts/0.1 minute, respectively). CONCLUSIONS Preservation solutions with high potassium concentration are clearly able to depolarize the pulmonary artery smooth muscle cells and increase pulmonary artery reactive oxygen species production. Low potassium preservations solutions may limit reactive oxygen species production and thus reduce the incidence of primary graft failure in lung transplantation.

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.

Triple-bonded unsaturated fatty acids are redox active compounds.

Unsaturated fatty acids with triple bonds are used as inhibitors of unsaturated fatty acid metabolism or cytochrome P450 reactions because they are believed to be chemically inert. In this paper we use in vitro cytochrome C reduction to show that two commonly used triple-bonded unsaturated fatty acids are in fact potent electron transfer agents and could affect the multiple cellular systems that are redox-modulated.