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E K Weir

Publications and source records attributed to E K Weir.

At least 19 recordsLinked to original sources
PubMed2005

Effect of normoxia and hypoxia on K(+) current and resting membrane potential of fetal rabbit pulmonary artery smooth muscle.

At birth, the increase in O(2) tension (pO(2)) is an important cause of the decrease in pulmonary vascular resistance. In adult animals there are impressive interspecies differences in the level of hypoxia required to elicit a pulmonary vasoconstrictor response and in the amplitude of the response. Hypoxic inhibition of some potassium (K(+)) channels in the membrane of pulmonary arterial smooth muscle cells (PASMCs) helps to initiate hypoxic pulmonary vasoconstriction. To determine the effect of the change in pO(2) on fetal rabbit PASMCs and to investigate possible species-dependent differences, we measured the current-voltage relationship and the resting membrane potential, in PASMCs from fetal resistance arteries using the amphotericin-perforated patch-clamp technique under hypoxic and normoxic conditions. Under hypoxic conditions, the K(+) current in PASMCs was small, and could be inhibited by 4-aminopyridine, iberiotoxin and glibenclamide, reflecting contributions by Kv, K(Ca) and K(ATP) channels. The average resting membrane potential was -44.3+/-1.3 mV (n=29) and could be depolarized by 4-AP (5 mM) and ITX (100 nM) but not by glibenclamide (10 microM). Changing from hypoxia, that mimicked fetal life, to normoxia dramatically increased the K(Ca) and consequently hyperpolarized (-9.3+/-1.7 mV; n=8) fetal rabbit PASMCs. Under normoxic conditions K(+) current was reduced by 4-AP with a significant change in resting membrane potential (11.1+/-1.7 mV; n=8). We conclude that resting membrane potential in fetal rabbit PASMCs under both hypoxic and normoxic conditions depends on both Kv and K(Ca) channels, in contrast to fetal lamb or porcine PASMCs. Potential species differences in the K(+) channels that control resting membrane potential must be taken into consideration in the interpretation of studies of neonatal pulmonary vascular reactivity to changes in O(2) tension.

Animals↗
PubMed2003

The effect of the anorectic agent, d-fenfluramine, and its primary metabolite, d-norfenfluramine, on intact human platelet serotonin uptake and efflux.

Dexfenfluramine, a drug formerly prescribed for treatment of obesity, caused heart valve damage and pulmonary hypertension in some people. The cause of the toxicity has not been defined, but 5-HT has been implicated. The objective of this study was to evaluate the effect of the anorectic agent, d-fenfluramine, and its major metabolite, d-norfenfluramine, on intact human platelet serotonin (5-HT) transport in vitro. The effects of d-fenfluramine and d-norfenfluramine on platelet uptake and efflux of 3H-5-HT were measured in buffer at pH 6.7, to optimize serotonin transporter (SERT) function, and at pH 7.4. Uptake of 3H-5-HT at pH 6.7 and 7.4 was inhibited by both agents at micro m concentrations (IC50, d-fenfluramine approximately 3 microM; d-norfenfluramine approximately 10 microM). However, no efflux of 3H-5-HT from labeled platelets at either pH 6.7 or 7.4 occurred at similar concentrations of d-fenfluramine or d-norfenfluramine. With inhibition of platelet dense granule 3H-5-HT uptake by reserpine, efflux of 3H-5-HT was observed at pH 6, but not at pH 7.4. Fluoxetine, a SERT inhibitor, was a more potent inhibitor of uptake (IC50 0.05 microM) than d-fenfluramine, but the anorectic agent, phentermine, had no effect. Therefore, d-fenfluramine and d-norfenfluramine inhibit human platelet uptake of 5-HT in vitro at tissue concentrations attainable in vivo, but they do not stimulate 5-HT efflux due to dense granule sequestration. Inhibition of platelet 5-HT uptake may play a role in the cardiopulmonary toxicity of d-fenfluramine, but other factors probably contribute, since similar toxicity has not been observed with fluoxetine.

Appetite Depressants↗
PubMed2001

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 [mu]M) or lucigenin (5 [mu]M) 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 [mu]M) 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.

Animals↗
PubMed2001

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.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed2001

The pathobiology of pulmonary hypertension. Smooth muscle cells and ion channels.

Chronic hypoxic pulmonary arterial hypertension, APAH, and PPAH are characterized by vasoconstriction and vascular remodeling and are associated with decreased Kv currents in PA smooth muscle cells. Although Kv2.1 is less well studied, it seems that Kv1.5 is particularly important in the pulmonary circulation in animals and humans because it has been implicated in physiologic phenomena (HPV) and all of the aforementioned pulmonary hypertensive disorders. This occurrence is perhaps because of the fact that it controls Em in the PA smooth muscle cells and it has a short turnover half time. It is also certain that the pathogenesis of PPAH is multifactorial and not a result of a single abnormality. The recently discovered "PPAH gene" in chromosome 2q in patients with familial PPAH (6%-12% of patients) does not seem to encode for any Kv channels. Kv1.5 abnormalities, however, are likely to be a strong predisposing factor that, in association with others such as endothelial dysfunction, [figure: see text] anorexigen use, or viral infections, will initiate a process that eventually leads to PPAH. The selective Kv1.5 down-regulation leaves wide open the door to replacement gene therapy in pulmonary hypertension research.

Animals↗
PubMed2001

Anorectic drugs and pulmonary hypertension from the bedside to the bench.

Anorectic drugs have been used for more than 30 years as an aid in weight reduction for obese persons. The use of aminorex, an amphetamine analog that increases norepinephrine levels in the central nervous system, led to an epidemic of primary pulmonary hypertension (PPH) in Europe in the late 1960s and early 1970s. The use of fenfluramine and later dexfenfluramine [drugs that inhibit 5-hydroxytryptamine (5-HT) release and reuptake and increases 5-HT and thus 5-HT secretion in the brain] was associated with a second epidemic of PPH. All of these drugs have been voluntarily withdrawn from the market. The pathogenesis of PPH in patients treated with these agents is uncertain, but recent evidence suggests that potassium channel abnormalities and vasoactive and proliferative properties of 5-HT may play a role. There is increasing experimental evidence suggesting that aminorex, fenfluramine and dexfenfluramine inhibit 4-aminopyridine-sensitive currents in potassium channels resulting in vasoconstriction in pulmonary resistance vessels and perhaps smooth muscle cell proliferation. 5-HT causes pulmonary artery vasoconstriction and smooth muscle cell proliferation. Its levels are known to be high in those with fenfluramine-induced PPH. However, a firm cause-and-effect relationship has not yet been established. One potentially beneficial effect of the epidemics of anorectic-related PPH is that it may have provided important insights into the causes of PPH unrelated to anorectic agents.

Aminorex↗
PubMed2001

Potassium channels regulate tone in rat pulmonary veins.

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K(+) channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (K(V)) and inward rectifier (K(ir)) K(+) channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba(2+), and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several K(V), K(ir), and large-conductance Ca(2+)-sensitive K(+) channels are present in PVs. Immunohistochemistry showed that K(ir) channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K(+) channels are present and functionally important in rat PVs. PVCMs form sphincters rich in K(ir) channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.

4-Aminopyridine↗
PubMed2001

Alterations in a redox oxygen sensing mechanism in chronic hypoxia.

The mechanism of acute hypoxic pulmonary vasoconstriction (HPV) may involve the inhibition of several voltage-gated K+ channels in pulmonary artery smooth muscle cells. Changes in PO2 can either be sensed directly by the channel(s) or be transmitted to the channel via a redox-based effector mechanism. In control lungs, hypoxia and rotenone acutely decrease production of activated oxygen species, inhibit K+ channels, and cause constriction. Two-day and 3-wk chronic hypoxia (CH) resulted in a decrease in basal activated oxygen species levels, an increase in reduced glutathione, and loss of HPV and rotenone-induced constriction. In contrast, 4-aminopyridine- and KCl-mediated constrictions were preserved. After 3-wk CH, pulmonary arterial smooth muscle cell membrane potential was depolarized, K+ channel density was reduced, and acute hypoxic inhibition of whole cell K+ current was lost. In addition, Kv1.5 and Kv2.1 channel protein was decreased. These data suggest that chronic reduction of the cytosol occurs before changes in K+ channel expression. HPV may be attenuated in CH because of an impaired redox sensor.

Animals↗
PubMed2000

Molecular identification of O2 sensors and O2-sensitive potassium channels in the pulmonary circulation.

Small, muscular pulmonary arteries (PAs) constrict within seconds of the onset of alveolar hypoxia, diverting blood flow to better-ventilated lobes, thereby matching ventilation to perfusion and optimizing systemic PO2. This hypoxic pulmonary vasoconstriction (HPV) is enhanced by endothelial derived vasoconstrictors, such as endothelin, and inhibited by endothelial derived nitric oxide. However, the essence of the response is intrinsic to PA smooth muscle cells in resistance arteries (PASMCs). HPV is initiated by inhibition of the Kv channels in PASMCs which set the membrane potential (EM). It is currently uncertain whether this reflects an initial inhibitory effect of hypoxia on the K+ channels or an initial release of intracellular Ca2+, which then inhibits K+ channels. In either scenario, the resulting depolarization activates L-type, voltage gated Ca2+ channels, which raises cytosolic calcium levels [Ca2+]i and causes vasoconstriction. Nine families of Kv channels are recognized from cloning studies (Kv1-Kv9), each with subtypes (i.e. Kv1.1-1.6). The contribution of an individual Kv channel to the whole-cell current (IK) is difficult to determine pharmacologically because Kv channel inhibitors are nonspecific. Furthermore, the PASMC's IK is an ensemble, reflecting activity of several channels. The K+ channels which set EM, and inhibition of which initiates HPV, conduct an outward current which is slowly inactivating, and which is blocked by the Kv inhibitor 4-aminopyridine (4-AP) but not by inhibitors of Ca(2+)- or ATP-sensitive K+ channels. Using anti-Kv antibodies to immunolocalize and inhibit Kv channels, we showed that the PASMC contains numerous types of Kv channels from the Kv1 and Kv2 families., Furthermore Kv1.5 and Kv2.1 may be important in determining the EM and play a role as effectors of HPV in PASMCs. While the Kv channels in PASMCs are the "effectors" of HPV, it is uncertain whether they are intrinsically O2-sensitive or are under the control of an "O2 sensor". Certain Kv channels are rich in cysteine, and respond to the local redox environment, tending to open when oxidized and close when reduced. Candidate sensors vary the PASMC redox potential in proportion to O2. These include Nicotinamide Adenine Dinucleotide Phosphate Oxidase, (NADPH oxidase) and the cytosolic ratio of reduced/oxidized redox couples (i.e. glutathione GSH/GSSG), as controlled by electron flux in the mitochondrial electron transport chain (ETC). Using a mouse that lacks the gp91phox component of NADPH oxidase, we have recently shown that loss of the gp91phox-containing NADPH oxidase as a source of activated oxygen species does not impair HPV. However, inhibition of complex 1 of the mitochondrial electron transport chain mimics hypoxia in that it inhibits IK, reduces the production of activated O2 species and causes vasoconstriction. We hypothesize that a redox O2 sensor, perhaps in the mitochondrion, senses O2 through changes in the accumulation of freely diffusible electron donors. Changes in the ratio of reduced/oxidized redox couples, such as NADH/NAD+ and glutathione (GSH/GSSG) can reduce or oxidize the K+ channels, resulting in alterations of PA tone.

Animals↗
PubMed1999

O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase.

The rapid response to hypoxia in the pulmonary artery (PA), carotid body, and ductus arteriosus is partially mediated by O2-responsive K+ channels. K+ channels in PA smooth muscle cells (SMCs) are inhibited by hypoxia, causing membrane depolarization, increased cytosolic calcium, and hypoxic pulmonary vasoconstriction. We hypothesize that the K+ channels are not themselves "O2 sensors" but rather respond to the reduced redox state created by hypoxic inhibition of candidate O2 sensors (NADPH oxidase or the mitochondrial electron transport chain). Both pathways shuttle electrons from donors, down a redox gradient, to O2. Hypoxia inhibits these pathways, decreasing radical production and causing cytosolic accumulation of unused, reduced, freely diffusible electron donors. PASMC K+ channels are redox responsive, opening when oxidized and closing when reduced. Inhibitors of NADPH oxidase (diphenyleneiodonium) and mitochondrial complex 1 (rotenone) both inhibit PASMC whole-cell K+ current but lack the specificity to identify the O2-sensor pathway. We used mice lacking the gp91 subunit of NADPH oxidase [chronic granulomatous disease (CGD) mice] to assess the hypothesis that NADPH oxidase is a PA O2-sensor. In wild-type lungs, gp91 phox and p22 phox subunits are present (relative expression: macrophages > airways and veins > PASMCs). Deletion of gp91 phox did not alter p22 phox expression but severely inhibited activated O2 species production. Nonetheless, hypoxia caused identical inhibition of whole-cell K+ current (in PASMCs) and hypoxic pulmonary vasoconstriction (in isolated lungs) from CGD vs. wild-type mice. Rotenone vasoconstriction was preserved in CGD mice, consistent with a role for the mitochondrial electron transport chain in O2 sensing. NADPH oxidase, though a major source of lung radical production, is not the pulmonary vascular O2 sensor in mice.

Animals↗
PubMed1999

GTP (gammaS) and GDP (betaS) as electron donors: new wine in old bottles.

G proteins are membrane-bound regulatory proteins which modulate the activity of ion channels and other effector systems. The GTP and GDP analogs GTP (gammaS) and GDP (betaS) 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 (gammaS) and GDP (betaS) analogs are potent reducing agents at physiologic pH. This observation suggests that previous data obtained using these compounds may need to be reinterpreted.

Cytochrome c Group↗
PubMed1999

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 (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.

Animals↗
PubMed1999

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 microM) increase in [Ca2+]i, even at concentrations lower than those necessary to inhibit K+ currents (10 microM) and cause membrane depolarization (100 microM). The [Ca2+]i response to 1 and 10 microM dexfenfluramine was completely abolished by pretreatment of the cells with 0.1 microM thapsigargin, whereas the response to 100 microM 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 microM dexfenfluramine. We conclude that dexfenfluramine increases [Ca2+]i in rat pulmonary artery smooth muscle cells by both release of Ca2+ from the sarcoplasmic reticulum and influx of extracellular Ca2+.

Animals↗
PubMed1999

Dexfenfluramine elevates systemic blood pressure by inhibiting potassium currents in vascular smooth muscle cells.

Appetite suppressants, such as dexfenfluramine (dex), are associated with primary pulmonary hypertension, valvular heart disease, and systemic vascular complications, such as coronary, cerebral, or mesenteric ischemia. These drugs suppress appetite by enhancing release and inhibiting reuptake of serotonin in the central nervous system. The effects of dex on the systemic circulation have not been studied. K(+) channels regulate vascular tone in most vascular beds. We hypothesized that dex is a systemic vasoconstrictor acting primarily by inhibiting K(+) channels, independent of effects on serotonin. The effects of clinically relevant concentrations of dex (10(-6) to 10(-4) M) on outward K(+) current and membrane potential were studied with whole-cell patch clamping in freshly isolated smooth muscle cells from rat renal, carotid, and basilar arteries. Tone was measured in tissue baths. Blood pressure, cardiac output, and left ventricular end diastolic pressure were assessed in open- and closed-chest anesthetized rats. At 10(-4) M, dex inhibits outward K(+) current (50%) and increases membrane potential (by >35 mV), an effect comparable with 4-aminopyridine (5 mM). Furthermore, dex constricts rings and acutely elevates systemic pressure (+17 +/- 3 mm Hg) and systemic vascular resistance in the presence of ketanserin. Dex vasoconstriction is dose-dependent (threshold dose 10(-6) M; 156 microg/ml) and enhanced in L-NAME-fed rats. We conclude that dex causes acute systemic vasoconstriction, at least in part by inhibition of voltage-gated K(+) channels, independent of effects on serotonin. To our knowledge, this is the first time that a commonly prescribed drug with voltage-gated K(+) channel-blocking properties is shown to have significant hemodynamic effects in vivo.

4-Aminopyridine↗
PubMed1998

A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent.

The mechanism responsible for the abrupt decrease in resistance of the pulmonary circulation at birth may include changes in the activity of O2-sensitive K+ channels. We characterized the electrophysiological properties of fetal and adult ovine pulmonary arterial (PA) smooth muscle cells (SMCs) using conventional and amphotericin B-perforated patch-clamp techniques. Whole cell K+ currents of fetal PASMCs in hypoxia were small and characteristic of spontaneously transient outward currents. The average resting membrane potential (RMP) was -36 +/- 3 mV and could be depolarized by charybdotoxin (100 nM) or tetraethylammonium chloride (5 mM; both blockers of Ca2+-dependent K+ channels) but not by 4-aminopyridine (4-AP; 1 mM; blocker of voltage-gated K+ channels) or glibenclamide (10 microM; blocker of ATP-dependent K+ channels). In hypoxia, chelation of intracellular Ca2+ by 5 mM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid further reduced the amplitude of the whole cell K+ current and prevented spontaneously transient outward current activity. Under these conditions, the remaining current was partially inhibited by 1 mM 4-AP. K+ currents of fetal PASMCs maintained in normoxia were not significantly reduced by acute hypoxia. In normoxic adult PASMCs, whole cell K+ currents were large and RMP was -49 +/- 3 mV. These 4-AP-sensitive K+ currents were partially inhibited by exposure to acute hypoxia. We conclude that the K+ channel regulating RMP in the ovine pulmonary circulation changes after birth from a Ca2+-dependent K+ channel to a voltage-dependent K+ channel. The maturational-dependent differences in the mechanism of the response to acute hypoxia may be due to this difference in K+ channels.

Aging↗
PubMed1998

A role for potassium channels in smooth muscle cells and platelets in the etiology of primary pulmonary hypertension.

Plasma serotonin levels are markedly elevated in patients with primary pulmonary hypertension (PPH) and platelet levels of serotonin are low. Furthermore, plasma serotonin levels remain elevated after bilateral lung transplantation, in the absence of any pulmonary hypertension. Dexfenfluramine can cause the anorexigen-induced form of PPH that is clinically and histologically indistinguishable from PPH. We find that dexfenfluramine releases serotonin from platelets and inhibits its reuptake. These observations suggest that serotonin might be involved in, or be a marker for, the mechanism responsible for both forms of PPH. Dexfenfluramine causes inhibition of voltage-sensitive potassium (Kv) channels, membrane depolarization, and calcium entry in pulmonary artery smooth muscle cells and vasoconstriction in isolated perfused rat lungs. We have recently found that dexfenfluramine also inhibits Kv channels in megakaryocytes, the stem cell for platelets. In smooth muscle cells, taken from the pulmonary arteries of PPH patients, Kv channels appear to be dysfunctional. The underlying defect in PPH is likely to be an abnormality of one or more Kv channels in both pulmonary artery smooth muscle cells and platelets. Relatively few patients exposed to dexfenfluramine develop PPH. The factors responsible for susceptibility might be a difference in expression of potassium channels and/or a decrease in the endogenous production of nitric oxide.

Animals↗