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

Helen L. Reeve1,2, Stephen L. Archer3, Marjorie Soper1, and E. Kenneth Weir1,4

Departments of 1 Medicine and 2 Physiology, University of Minnesota, Minneapolis 55455; 4 Department of Medicine, Veterans Affairs Medical Center, Minneapolis, Minnesota 55417; and 3 Departments of Medicine and Physiology, University of Alberta, Edmonton, Canada T6G 267


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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+]i response 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+]i in rat pulmonary artery smooth muscle cells by both release of Ca2+ from the sarcoplasmic reticulum and influx of extracellular Ca2+.

intracellular calcium; potassium channels; sarcoplasmic reticulum; anorexics


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE USE OF THE ANOREXICS dexfenfluramine or fenfluramine for >3 mo increases the risk ratio of developing primary pulmonary hypertension by a factor of 23 (1). The pathogenesis of primary pulmonary hypertension is poorly understood, and, consequently, the possibilities for treatment are limited (13, 15). It has previously been shown that aminorex, fenfluramine, and dexfenfluramine inhibit K+ currents in rat pulmonary artery (PA) smooth muscle cells (SMCs) and that dexfenfluramine causes reversible membrane depolarization in these cells (12, 16). Although an increase in vascular smooth muscle intracellular Ca2+ concentration ([Ca2+]i) would be expected as a result of membrane depolarization and as a prerequisite for vasoconstriction, changes in [Ca2+]i in response to dexfenfluramine have not been determined. In this study, we measured the changes in [Ca2+]i elicited by dexfenfluramine in rat PASMCs and evaluated the relative contribution of extracellular Ca2+ influx versus release of Ca2+ from intracellular stores. K+ currents and membrane potentials were also measured with the amphotericin-perforated whole cell patch-clamp technique (11).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of rat PASMCs. All animal studies were conducted in accordance with institutional guidelines. Fresh rat PASMCs were dissociated in the same manner for both Ca2+ and patch-clamp studies. Male Sprague-Dawley rats (250-350 g) were anesthetized with pentobarbital sodium (50 mg/kg), and their heart and lungs were removed en bloc. Resistance PAs (third to fifth division) were dissected, cleaned of connective tissue, cut open, and placed in "Ca2+-free" Hanks' solution composed of (in mM) 145 NaCl, 4.2 KCl, 1.0 MgCl2, 1.2 KH2PO4, 10 HEPES, and 0.1 EGTA (pH 7.4 with NaOH) for 10 min at 4°C. The arteries were then transferred to Hanks' solution without EGTA ("low Ca2+") containing 1 mg/ml of papain, 0.8 mg/ml of albumin, and 0.75 mg/ml of dithiothreitol and kept at 4°C for 20 min. Subsequently, the arteries were incubated at 36°C for 12 min. After digestion, the cells were washed in low-Ca2+ Hanks' solution to remove residual enzymes and maintained at 4°C. The arteries were triturated before the experiments to produce a suspension of single cells.

Measurement of intracellular Ca2+. [Ca2+]i was measured by dual-excitation imaging with fura 2 (4). Freshly dispersed cells were transferred to imaging dishes (Molecular Probes, Eugene OR) and incubated in low-Ca2+ Hanks' solution with the cell-permeable acetoxymethyl ester form of fura 2 (0.1 µM) and Pluronic F-127 (0.8 µM) for 15 min at room temperature. The plates were then washed with HEPES buffer containing 1.5 mM Ca2+ (see solution composition in Electrophysiology) and incubated at room temperature for a further 20 min. The plates were then washed again and placed on a heated microscope stage (33°C). The drugs were added directly to the cells as a bolus by microinjection. All drugs were given in 10-µl volumes to remove potential volume-induced artifacts. Ten-microliter injections of saline had no effect on [Ca2+]i. Changes in [Ca2+]i were recorded in individual cells with a MetaFluor (Universal Imaging, West Chester, PA)-driven 340/380 filter imaging system and cooled charge-coupled device camera (Photometrics, Tucson, AZ). This system allows discrete areas within single cells to be imaged so that relaxed SMCs can be identified while other cell types (endothelial cells and fibroblasts) can be excluded. Background fluorescence was recorded from each dish of cells and subtracted before calculation of the 340- to 380-nm ratio. Measurements were made every 5 s. [Ca2+]i was calculated according to the method of Grynkiewicz et al. (4). A dissociation constant of 325 nM was calculated from the in vitro calibration. Maximal and minimal ratio values were determined at the end of each experiment by first treating the cells with 1 µM ionomycin (maximal ratio) and then chelating all free Ca2+ with 2 mM EGTA (minimal ratio). For studies with Ca2+-free medium, the extracellular solution had no added Ca2+ and was supplemented with the Ca2+ chelator EGTA (10 mM) and 1.5 mM MgCl2 to replace the Ca2+. Cells were exposed to the Ca2+-free medium for 1 min before the addition of dexfenfluramine.

Electrophysiology. Triturated cells were divided into aliquots on the stage of an inverted microscope (Nikon Diaphot 200) for amphotericin-perforated patch-clamp studies (11). The perforated patch-clamp technique was used so that cellular conditions mimicked those for the imaging studies as closely as possible (e.g., because EGTA cannot enter the cell with this technique, there was no chelation of intracellular Ca2+). Briefly, the cells were bathed in an extracellular solution composed of (in mM) 145 NaCl, 5.4 KCl, 1.0 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH), and the drugs were added to the perfusate by 10-µl microinjections as for Ca2+ imaging (see Measurement of intracellular Ca2+). The electrodes had resistances of 2-3 MOmega when fire polished and filled with a solution of (in mM) 140 KCl, 1.0 MgCl2, 5 HEPES, and 0.1 EGTA and 120 µg/ml of amphotericin B (pH 7.2 with KOH). Capacitance was corrected, and perforation was monitored by changes in membrane potential and access resistance. Cells were discarded if the access resistance did not decrease to <15 MOmega to minimize voltage errors. The average access resistance was 13.3 ± 0.4 MOmega (n = 15 cells). Series resistance was minimized by electronic compensation (usually 80%). For voltage-clamp studies, the cells were held at a membrane potential of -70 mV and stepped to more depolarized potentials in steps of +20 mV. The dose-dependent effects of dexfenfluramine (1, 10, 100, and 1,000 µM) on K+ currents were recorded. For current-clamp studies, the cells were held at their resting membrane potential (zero-current potential) and exposed to dexfenfluramine (1, 10, or 100 µM) after a 1-min control recording to determine stability. All data were recorded and analyzed with pClamp 6.04 software (Axon Instruments, Foster City, CA).

Drugs. All drugs were purchased from Sigma (St. Louis, MO) except fura 2-AM {1-[2-(5-carboxyloxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester; Molecular Probes} and caffeine (RBI, Natick, MA). The drugs were dissolved in HEPES buffer except fura 2, Pluronic F-127, and ionomycin, which were dissolved in DMSO. Saline and DMSO vehicles had no effect on baseline levels of Ca2+.

Statistics. The results are expressed as means ± SE. Intracellular Ca2+ levels were compared with an unpaired two-tailed Student's t-test. Intergroup differences were assessed with a factorial analysis of variance, with post hoc analysis with Fisher's least significant difference test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dexfenfluramine and intracellular Ca2+ in rat PASMCs. Dexfenfluramine caused a dose-dependent elevation in [Ca2+]i in rat PASMCs, with an initial increase in Ca2+ at 1 µM (P < 0.05; n = 46 cells; Fig. 1). Thirty-seven of the forty-six cells tested in this way (80.4%) responded to 1 µM dexfenfluramine. The source of the rise in [Ca2+]i was determined by multiple recordings of the dexfenfluramine-mediated responses after blockade of the influx of Ca2+ or release of the intracellular pools of Ca2+. At 100 µM (average rise in Ca2+ = 1,234 ± 122 nM; n = 25 cells), pretreatment of the cells for 1 min with 1 mM CoCl2 to block the influx of extracellular Ca2+ reduced but did not abolish the elevation in [Ca2+]i in response to 100 µM dexfenfluramine (Fig. 2A). In the absence of extracellular Ca2+, i.e., with the cells bathed in Ca2+-free perfusate (see METHODS), there was a similar reduction in the response to 100 µM dexfenfluramine (Fig. 2A). The component of the rise in [Ca2+]i that was not prevented by removal or blockade of the influx of extracellular Ca2+ was investigated after pretreatment of the cells with thapsigargin (to deplete all intracellular Ca2+ stores) or caffeine (to deplete the ryanodine-sensitive stores). Thapsigargin (0.1 µM) caused a slow, sustained rise in [Ca2+]i consistent with a leak of Ca2+ from the intracellular stores and then a block of reuptake (n = 11 cells) (14). The increase in [Ca2+]i caused by 1 and 10 µM dexfenfluramine was completely inhibited by pretreatment of the cells with 0.1 µM thapsigargin (Fig. 3A), whereas the response to 100 µM dexfenfluramine was significantly reduced from the control value (n = 11 cells; Figs. 2B and 3A) but was not abolished. Caffeine (1 mM) caused a classic, transient increase in [Ca2+]i, after which the response to 100 µM dexfenfluramine was also significantly reduced to 454 ± 57 nM (P < 0.05; Figs. 2B and 3B).


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Fig. 1.   Changes in intracellular Ca2+ concentration (Delta [Ca2+]i) after sequential applications of 1, 10, and 100 µM dexfenfluramine. [dexfenfluramine], Dexfenfluramine concentration. Values are means ± SE; n = 37 cells. * P < 0.05 compared with baseline.



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Fig. 2.   Delta [Ca2+]i to 100 µM dexfenfluramine (DEX) alone and after pretreatment of cells with Ca2+-free solution and CoCl2 (A) or caffeine, thapsigargin, and ketanserin (B). Values are means ± SE; nos. in parentheses, no. of cells. * P < 0.05 compared with DEX response.



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Fig. 3.   A: representative example of actual changes in 340- to 380-nm (340/380) ratio to DEX after pretreatment with thapsigargin. B: representative example of actual changes in 340- to 380-nm ratio to DEX after pretreatment with caffeine. Arrows, drug application.

The anorexic actions of dexfenfluramine are thought to be related to its ability to release serotonin (5-HT) from neurons. To determine any role of serotonin in the Ca2+ response, the cells were pretreated with 1 µM ketanserin (5-HT2-receptor blocker) before 100 µM dexfenfluramine to determine whether dexfenfluramine had any interaction with 5-HT2 receptors to raise [Ca2+]i. This concentration of ketanserin completely blocks serotonin-induced constriction of the isolated, perfused rat lung (Reeve and Weir, unpublished observations). The addition of 1 µM ketanserin to the cells had no effect on baseline Ca2+ and did not affect the subsequent response to 100 µM dexfenfluramine (1,012 ± 48 nM; not significant; n = 17 cells; Fig. 2B).

Dexfenfluramine, K+ currents, and membrane potential in rat PASMCs. With the use of the amphotericin- perforated patch clamp, outward currents were recorded from single PASMCs. The average current density was 265 ± 33 pA/pF (at +50 mV; n = 15 cells), and the outward currents could be almost completely inhibited by 1 mM 4-aminopyridine. Figure 4A shows a dose-response curve of the inhibitory effect of dexfenfluramine. In contrast to the rise in [Ca2+]i observed with 1 µM dexfenfluramine, the currents were only inhibited at concentrations of 10 µM and above as previously described (12, 16). The average resting membrane potential recorded from the PASMCs was -46.1 ± 2 mV (n = 6 cells). The membrane potential was recorded for 1 min to establish stability, and then doses of 1, 10, and 100 µM dexfenfluramine were applied to the cell for 2 min each. Each dose was washed out before the next one was applied. There was no effect of dexfenfluramine on membrane potential until 100 µM (Fig. 4B).


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Fig. 4.   A: percentage of K+ current (IK) inhibited at -10 mV by increasing doses of DEX in rat pulmonary artery smooth muscle (n = 3 cells/dose). * Significant change in IK inhibition, P < 0.05. B: membrane potential (Em) recorded during control (n = 6 cells) or after 2-min exposure to 1 (n = 3 cells), 10 (n = 5 cells), or 100 (n = 6 cells) µM DEX. Values are means ± SE. * Significant change in Em, P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism of pulmonary hypertension associated with dexfenfluramine intake remains to be elucidated. The present study shows that dexfenfluramine increases [Ca2+]i in rat PASMCs. A similar dose-dependent increase was observed in cultured human SMCs (Soper and Archer, unpublished observations), but because the cells used were an immortalized cell line taken from conduit human PAs, the full study was undertaken with freshly dispersed rat PASMCs. Increased [Ca2+]i is an important signal that activates the contractile apparatus but can also stimulate cellular proliferation (7, 8). Both vasoconstriction and mesenchymal cell proliferation are important in the pathogenesis of pulmonary hypertension (15).

The sustained plasma concentration of fenfluramine that "correlates with the best rate of weight loss" is said to be 1 µM (2). We show an increase in [Ca2+]i at 1 µM dexfenfluramine, whereas there is little effect on K+ current and no effect on membrane potential at this concentration. This change at 1 µM dexfenfluramine could be completely prevented by prior release of intracellular stores by 0.1 µM thapsigargin (14, 18), suggesting that release occurs before influx of extracellular Ca2+ via sarcolemmal depolarization. Furthermore, blockade of the influx of extracellular Ca2+ by CoCl2 or removal of extracellular Ca2+ reduced (by ~58%) but did not abolish the increase in [Ca2+]i caused by 100 µM dexfenfluramine, suggesting that entry of extracellular Ca2+ is a source of dexfenfluramine-induced increase in [Ca2+]i at a high concentration but that release of intracellular Ca2+ also contributes. It should be noted that thapsigargin has been reported to inhibit L-type Ca2+ channels at high concentrations (5 µM) (14). The concentration of thapsigargin used (0.1 µM) was chosen to avoid this potentially misleading effect. Caffeine depletes the sarcoplasmic reticulum of Ca2+ by binding to the ryanodine receptor (10). One millimolar caffeine also reduced the response to 100 µM dexfenfluramine, suggesting a role of the ryanodine-sensitive intracellular stores. Serotonin-induced rises in intracellular Ca2+ in cultured PASMCs have been suggested to be primarily due to release of inositol 1,4,5-trisphosphate stores (17). These data presented here suggest that dexfenfluramine, at least in isolated PASMCs, increases intracellular Ca2+ through a mechanism different from that of serotonin. A previous study (9) in canine PASMCs has suggested that the inhibition of K+ current that occurs in hypoxic pulmonary vasoconstriction may be secondary to a rise in [Ca2+]i. An elegant study by the same group (5) indicated roles for caffeine and ryanodine-sensitive stores in hypoxic pulmonary vasoconstriction. Although it seems likely that dexfenfluramine releases intracellular Ca2+ before its inhibition of K+ current, it remains to be determined whether this rise in Ca2+ is directly responsible for the inhibition of K+-channel activity.

It has recently been reported that patients with both primary pulmonary hypertension (6) and anorexic-induced pulmonary hypertension (3) may have impaired endothelial function as indicated by reduced endogenous nitric oxide levels. In light of these data, it could be speculated that a small rise in [Ca2+]i may occur at plasma levels of dexfenfluramine that occur clinically but that significant pulmonary constriction only occurs in the absence of normal, compensatory vasodilator mechanisms.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Valerie Porter for technical advice.


    FOOTNOTES

H. L. Reeve was supported by National Heart, Lung, and Blood Institute Grant R29-HL-59182-01 and was the 1997 Giles F. Filley Awardee. E. K. Weir was supported by Merit Review funding from the Department of Veterans Affairs, and S. L. Archer was supported by the Medical Research Council of Canada and the Alberta Heart and Stroke Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. L. Reeve, Research 151, VA Medical Center, Minneapolis, MN 55417 (E-mail: reeve007{at}tc.umn.edu).

Received 1 April 1999; accepted in final form 22 June 1999.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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