(Circulation. 1996;94:2216-2220.)
© 1996 American Heart Association, Inc.
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the Department of Medicine, Veterans Affairs Medical Center and University of Minnesota, Minneapolis, Minn.
Correspondence to Dr E. Kenneth Weir, Department of Medicine, VA Medical Center (111C), One Veterans Dr, Minneapolis, MN 55417.
| Abstract |
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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.
Key Words: hypertension, pulmonary obesity muscle, smooth vasoconstriction
| Introduction |
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The mechanism by which aminorex or fenfluramine might cause pulmonary hypertension is unknown but might resemble that responsible for hypoxic pulmonary vasoconstriction, in which K+ channel inhibition leads to membrane depolarization and Ca2+ entry through the voltage-dependent Ca2+ channels.13 Vasoconstriction is important in both hypoxic pulmonary hypertension13 and primary pulmonary hypertension.14 We hypothesized that inhibition of an IK, which initiates hypoxic pulmonary vasoconstriction in pulmonary vascular smooth muscle,13 15 might play a role in the pulmonary hypertension sometimes associated with aminorex and fenfluramine ingestion.
| Methods |
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after the application of Sylgard coating and fire polishing.
Cell Electrophysiology
Currents were evoked from a holding potential of -70 mV using test pulses of 200-msec duration at a rate of 0.033 to 0.1 Hz. Series resistance was generally compensated by 90% to 95%. Currents were filtered at 1 kHz and sampled at 4 kHz. All data were recorded and analyzed using pClamp 6.02 software (Axon Instruments). Experiments using the perforated-patch configuration were performed at low light intensity. Results are presented as mean±SEM.
In the aminorex (n=5) and fenfluramine (n=5) dose-response experiments, whole-cell IK were elicited by 20-mV step depolarizations (500-msec pulse duration) from a holding potential of -70 mV. The cells were characterized by recording I-V curves in the presence of 4-AP (2 mmol/L) and TEA (10 mmol/L). In the whole-cell technique, the cytoplasm of the cell is dialyzed by the larger volume of the solution in the electrode. In the perforated-patch technique, the patch is permeable to monovalent ions, but cytosolic factors are not lost. This might be considered more physiological. Consequently, in the dexfenfluramine experiments (n=23), IK was recorded using the amphotericin-perforated patch-clamp technique17 by +10-mV step depolarizations from -70 to +40 mV. Membrane depolarization caused by dexfenfluramine was recorded using current-clamp mode. Cells were monitored in current-clamp mode at their resting potential, and controls were recorded for >1 minute before application of the drug to ensure membrane potential stability. All patch-clamp experiments were performed at 32°C. In four additional cells, the inhibition of IK induced by 4 mmol/L 4-AP was compared with that induced by 4 mmol/L 4-AP plus 100 µmol/L dexfenfluramine (amphotericin-perforated patch technique).
Isolated, Perfused Lungs
For pressure studies, lungs that had been surgically removed from adult Sprague-Dawley rats were perfused at a constant flow rate with a physiological salt solution containing albumin (4%) and meclofenamate (1.7x10-5 mol/L) as previously described.18 Initially, two cycles of Ang II followed by hypoxia were performed to assess the reactivity of the lungs. For each cycle, a bolus injection of 0.15 µg of Ang II was made into the pulmonary artery line, followed after 8 minutes by a 6-minute hypoxic challenge (FIO2 2.5%). In one series of lungs (n=6), increasing bolus injections of aminorex were made at 18-minute intervals during normoxia, and the resulting changes in pressure were measured. In the second series of lungs (n=6), increasing bolus injections of fenfluramine were made at the same time intervals. In the third series of lungs (n=5), increasing bolus injections of dexfenfluramine were made at 5-minute intervals. (Doses are shown in the figure legends.) In the fourth series of lungs (n=5), L-NAME (5x10-5 mol/L) was added to the reservoir, and the same succession of increasing bolus doses of dexfenfluramine was made as in the third series. The experimental protocols were approved by the institutional animal studies committee.
Statistical Analysis
Data are expressed as mean±SEM. The effects of drugs on IK and pulmonary arterial pressure were compared using a repeated measures ANOVA (StatView II, Version 4.0, Abacus Concepts). A value of P<.05 was considered significant.
| Results |
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In the isolated, perfused rat lung, aminorex, fenfluramine, and dexfenfluramine caused a modest dose-dependent increase in pulmonary arterial pressure (Figs 3 and 4![]()
). However, in the presence of the NO synthase inhibitor L-NAME, dexfenfluramine caused a much greater pressor response (Fig 4
). There was a correlation between the severity of the maximal pressor response to dexfenfluramine, in the absence of L-NAME, and the pressor response to the preceding hypoxic challenge (P<.05) but not between the pressor response to dexfenfluramine and that to Ang II.
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| Discussion |
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1.5% of those exposed to contaminated rapeseed oil had pulmonary hypertension on follow-up 4 years later.19 It is not known whether those who develop pulmonary hypertension metabolize aminorex more slowly and consequently have higher tissue levels or whether their pulmonary arteries are more susceptible in some way, such as a lower endogenous production of NO. In more recent years, the anorexic agents fenfluramine and dexfenfluramine have been widely used for weight reduction12 20 and have also been associated with sporadic cases of pulmonary hypertension.3 4 5 6 7 8 9 10 11 Despite many studies, the mechanism by which these drugs might initiate pulmonary hypertension is unknown.
The patch-clamp experiments show that aminorex, fenfluramine. and dexfenfluramine cause a dose-dependent inhibition of whole-cell IK in smooth muscle cells freshly dispersed from resistance arteries of the rat lung. Dexfenfluramine produces membrane depolarization in these cells, indicating that the inhibition of IK has a physiological effect (Fig 2
). Thus, like hypoxia,13 these drugs may initiate pulmonary vasoconstriction by permitting Ca2+ entry through voltage-gated Ca2+ channels. The K+ channel or channels blocked by dexfenfluramine appear to be sensitive to low-dose 4-AP, suggesting that they include a delayed rectifier. It is important to note that fenfluramine is as effective in reducing whole-cell IK as 4-AP, a classic K+ channel blocker (Fig 1
). It is also possible that dexfenfluramine, like 4-AP,21 could have actions in addition to K+ channel blockade, such as inhibition of the Ca2+-ATPase in the sarcoplasmic reticulum.
The sustained plasma concentration of fenfluramine that "correlates with the best rate of weight loss" is said to be 1 µmol/L.22 This is slightly less than the 10 and 100 µmol/L concentrations of dexfenfluramine that acutely inhibited IK but comparable to the 100 nmol/L and 1 µmol/L concentrations that increased pulmonary artery pressure in the presence of L-NAME. The somewhat higher concentration of dexfenfluramine found necessary to inhibit IK, compared with the plasma concentration in patients, may reflect the difference between the effects of chronic drug administration in a susceptible patient and acute exposure of a single cell taken from an unselected rat. In the chronic hypoxia model of pulmonary hypertension, NO activity is upregulated.23 24 25 26 In this study, the acute inhibition of NO synthesis by L-NAME markedly enhanced the vasoconstriction caused by dexfenfluramine. It is possible that the patients who develop pulmonary hypertension while taking an anorexic agent have diminished NO activity. The actual reason for susceptibility remains to be established.
Chronic administration of aminorex to small numbers of monkeys,27 calves,28 and rats29 has not been found to cause pulmonary hypertension. In dogs, three chronic studies failed to show pulmonary hypertension,29 30 31 but another study, in which aminorex was fed for 2 years, resulted in an increase in pulmonary arterial pressure and resistance.32 The authors postulated that precapillary vasoconstriction induced by aminorex might lead to more fixed pulmonary hypertension. Plasma membrane depolarization and consequent Ca2+ influx can stimulate DNA synthesis and cell proliferation in osteoblasts,33 34 so it is possible that this mechanism might also be involved in the intimal proliferation observed in the pulmonary hypertension caused by anorexic agents.
Acute administration of fenfluramine has been observed to increase pulmonary artery pressure in the dog.29 In pigs rendered more susceptible to pulmonary hypertension by prior left pulmonary artery ligation, ingestion of fenfluramine for 3 months increased pulmonary vascular resistance.35 In the present study, aminorex, fenfluramine, and dexfenfluramine produced a dose-related increase in pressure in the isolated, perfused rat lung. The fact that these agents mimic hypoxia by inhibiting IK in smooth muscle cells, and causing pulmonary vasoconstriction, suggests the possibility of a similar mechanism. This analogy is supported by the observation that dexfenfluramine restores acute hypoxic pulmonary vasoconstriction in dogs that otherwise do not respond to hypoxia, while leaving unchanged the hypoxic vasoconstriction in responders.36
Dexfenfluramine inhibits the cellular uptake of 5-HT.37 5-HT causes pulmonary vasoconstriction through its action on 5-HT2 receptors, which can be blocked by ketanserin. It might be considered that dexfenfluramine could inhibit IK through a mechanism involving 5-HT. However, because single cells are studied in the patch-clamp experiments, this would mean that the smooth muscle cell would also have to be the source of the 5-HT. In addition, in the experiment cited above, in which dexfenfluramine restored hypoxic pulmonary vasoconstriction in the dog, ketanserin did not affect the vasoconstriction.36 These observations make it unlikely that 5-HT is involved.
In the future, it would seem wise to consider that drugs that block K+ channels, such as these anorexic agents, might be able to cause pulmonary hypertension in susceptible individuals. The illegal drug Ecstasy (3,4-methylenedioxymethamphetamine) is known to block neuronal K+ channels38 and has been associated with pulmonary hypertension.39 The anorexic agents may give us insight into the origins of pulmonary hypertension. Because aminorex and fenfluramine cause a histological picture that is indistinguishable from that of primary pulmonary hypertension, information on the etiologic mechanisms of drug-induced pulmonary hypertension may provide clues to the causes of primary pulmonary hypertension.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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| Footnotes |
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Received January 17, 1996; revision received May 31, 1996; accepted June 7, 1996.
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