Helen L. Reeve

Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes.

Hypoxia initiates pulmonary vasoconstriction (HPV) by inhibiting one or more voltage-gated potassium channels (Kv) in the pulmonary artery smooth muscle cells (PASMCs) of resistance arteries. The resulting membrane depolarization increases opening of voltage-gated calcium channels, raising cytosolic Ca2+ and initiating HPV. There are presently nine families of Kv channels known and pharmacological inhibitors lack the specificity to distinguish those involved in control of resting membrane potential (Em) or HPV. However, the Kv channels involved in Em and HPV have characteristic electrophysiological and pharmacological properties which suggest their molecular identity. They are slowly inactivating, delayed rectifier currents, inhibited by 4-aminopyridine (4-AP) but insensitive to charybdotoxin. Candidate Kv channels with these traits (Kv1.5 and Kv2.1) were studied. Antibodies were used to immunolocalize and functionally characterize the contribution of Kv1. 5 and Kv2.1 to PASMC electrophysiology and vascular tone. Immunoblotting confirmed the presence of Kv1.1, 1.2, 1.3, 1.5, 1.6, and 2.1, but not Kv1.4, in PASMCs. Intracellular administration of anti-Kv2.1 inhibited whole cell K+ current (IK) and depolarized Em. Anti-Kv2.1 also elevated resting tension and diminished 4-AP-induced vasoconstriction in membrane-permeabilized pulmonary artery rings. Anti-Kv1.5 inhibited IK and selectively reduced the rise in [Ca2+]i and constriction caused by hypoxia and 4-AP. However, anti-Kv1.5 neither caused depolarization nor elevated basal pulmonary artery tone. This study demonstrates that antibodies can be used to dissect the whole cell K+ currents in mammalian cells. We conclude that Kv2. 1 is an important determinant of resting Em in PASMCs from resistance arteries. Both Kv2.1 and Kv1.5 contribute to the initiation of HPV.

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.

Oxygen-induced constriction of rabbit ductus arteriosus occurs via inhibition of a 4-aminopyridine-, voltage-sensitive potassium channel.

The ductus arteriosus is a vital fetal structure allowing blood ejected from the right ventricle to bypass the pulmonary circulation in utero. Closure of the ductus arteriosus at birth, essential for postnatal adaptation, is initiated by an increase in oxygen (O2) tension. We recently demonstrated the presence of O2-sensitive potassium channels in the fetal and adult pulmonary circulation which regulate vascular tone in response to changes in O2 tension. In this study, we assessed the cellular mechanisms underlying O2-induced constriction of the ductus arteriosus in late-gestation fetal rabbits. We report that O2 reversibly inhibits a 58-pS voltage- and 4-aminopyridine-sensitive potassium channel, causing membrane depolarization, an increase in intracellular calcium through L-type voltage-gated calcium channels, and constriction of the ductus arteriosus. We conclude that the effector mechanism for O2 sensing in the ductus arteriosus involves the coordinated action of delayed rectifier potassium channels and voltage-gated calcium channels.

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.

Redox Signaling in Oxygen Sensing by Vessels

In response to the increase in oxygen tension at birth, the resistance pulmonary arteries dilate, while the ductus arteriosus constricts. Although modulated by the endothelium, these opposite responses are intrinsic to the vascular smooth muscle. While still controversial, it seems likely that during normoxia the production of reactive oxygen species (ROS) increases and the smooth muscle cell cytoplasm is more oxidized in both pulmonary arteries and ductus, compared to hypoxia. However, the effect of changes in the endogenous redox status or the addition of a redox agent, oxidizing or reducing, is exactly opposite in the two vessels. A reducing agent, dithiothreitol, like hypoxia, in the pulmonary artery will inhibit potassium current, cause depolarization, increase cytosolic calcium and lead to contraction. Responses to dithiothreitol in the ductus are opposite and removal of endogenous H(2)O(2) by intracellular catalase in the ductus increases potassium current. Oxygen sensing in both vessels is probably mediated by redox effects on both calcium influx and calcium release from the sarcoplasmic reticulum (SR).

Dexfenfluramine increases pulmonary artery smooth muscle intracellular Ca2+, independent of membrane potential

The anorexic agent dexfenfluramine causes the development of primary pulmonary hypertension in susceptible patients by an unknown mechanism that may include changes in K+-channel activity and intracellular Ca2+ concentration ([Ca2+]i). We investigated the dose-dependent effects of dexfenfluramine on [Ca2+]i, K+ current, and membrane potential in freshly dispersed rat pulmonary artery smooth muscle cells. Dexfenfluramine caused a dose-dependent (1-1,000 μM) increase in [Ca2+]i, even at concentrations lower than those necessary to inhibit K+ currents (10 μM) and cause membrane depolarization (100 μM). The [Ca2+]iresponse to 1 and 10 μM dexfenfluramine was completely abolished by pretreatment of the cells with 0.1 μM thapsigargin, whereas the response to 100 μM dexfenfluramine was reduced. CoCl2 (1 mM), removal of extracellular Ca2+, and pretreatment with caffeine (1 mM) reduced but did not abolish the response to 100 μM dexfenfluramine. We conclude that dexfenfluramine increases [Ca2+]iin rat pulmonary artery smooth muscle cells by both release of Ca2+ from the sarcoplasmic reticulum and influx of extracellular Ca2+.

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.

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.

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.

Vascular K+ Channel Expression and Function at Birth and in the Neonate

The fetal pulmonary circulation is a unique vascular bed which undergoes dramatic changes during the transition from fetus to neonate. Since the developing fetus has no need of its lungs for oxygen exchange, the pulmonary vasculature remains constricted and is characterized by high pulmonary vascular resistance (PVR) and minimal blood flow. Blood enters the right side of the heart and is directly shunted away into the systemic circulation via the ductus arteriosus (DA), a dilated blood vessel connecting the main pulmonary artery (PA) and the descending aorta. At birth, as the lungs become the primary site of oxygen exchange, two important changes occur in the pulmonary circulation. First, within 24 hours of birth, the pulmonary arterial bed dilates, resulting in an 8 to 10-fold increase in blood flow to the lungs and a decrease in pulmonary pressure to half systemic levels (Cassin et al., 1964; Emmanouilides et al., 1964; Dawes et al., 1953). Second, the DA constricts to remove the right-to-left shunt pathway (Heymann and Rudolph, 1975). The closure of the DA occurs in two stages, with an initial O2-dependent functional closure (characterized by constriction of the ductus) followed by intimal and medial necrosis and fibrosis (the anatomic phase). The time course over which these two phases occur is strongly species-dependent; in the normal human neonate, functional closure is generally complete within 48 hours of birth, with full anatomic closure occurring by 2–3 weeks of age (Eldridge and Hultgre, 1955). Although much of the decrease in PVR that occurs at birth is due to the mechanical process of lung ventilation (Dawes et al., 1953), there is also an O2-dependent factor in the fall in PVR.