RAPID COMMUNICATION
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
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ABSTRACT |
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
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INTRODUCTION |
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).
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METHODS |
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 M
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 M
to minimize voltage errors.
The average access resistance was 13.3 ± 0.4 M
(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.
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RESULTS |
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
( [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.
[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.
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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.
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DISCUSSION |
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.
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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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REFERENCES |
1.
Abenhaim, L.,
Y. Moride,
F. Brenot,
S. Rich,
J. Benichou,
X. Kurz,
T. Higenbottam,
C. Oakley,
E. Wouters,
M. Aubier,
G. Simonneau,
and
B. Bgaud.
Appetite-suppressant drugs and the risk of primary pulmonary hypertension.
N. Engl. J. Med.
335:
609-616,
1996[Abstract/Free Full Text].
2.
Anonymous.
Dexfenfluramine.
Lancet
337:
1315-1316,
1991[Medline].
3.
Archer, S. L.,
K. Djaballah,
M. Humbert,
E. K. Weir,
M. Fartoukh,
J. Dall'ava-Santucci,
J. C. Mercier,
G. Simonneau,
and
A. Tuan Dinh-Xuan.
Nitric oxide deficiency in fenfluramine- and dexfenfluramine-induced pulmonary hypertension.
Am. J. Respir. Crit. Care Med.
158:
1061-1067,
1998[Abstract/Free Full Text].
4.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
5.
Jahr, R. I.,
H. Toland,
C. H. Gelband,
X. X. Wang,
and
J. R. Hume.
Prominent role of intracellular Ca2+ release in hypoxic vasoconstriction of canine pulmonary artery.
Br. J. Pharmacol.
122:
21-30,
1997[Abstract].
6.
Kaneko, F. T.,
A. C. Arroliga,
R. A. Dweik,
S. A. Comhair,
D. Laskowski,
R. Oppedisano,
M. J. Thomassen,
and
S. C. Erzurum.
Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension.
Am. J. Respir. Crit. Care Med.
158:
917-923,
1998[Abstract/Free Full Text].
7.
Loza, J.,
L. Carpio,
G. Lawless,
N. Marzec,
and
R. Dziak.
Role of extracellular calcium influx in EGF-induced osteoblastic cell proliferation.
Bone
16, Suppl.:
341S-347S,
1995[Medline].
8.
Loza, J.,
N. Marzec,
S. Simasko,
and
R. Dziak.
Role of epidermal growth factor-induced membrane depolarization and resulting calcium influx in osteoblastic cell proliferation.
Cell Calcium
17:
301-306,
1995[Medline].
9.
Post, J. M.,
C. H. Gelband,
and
J. R. Hume.
[Ca2+]i inhibition of K+ channels in canine pulmonary artery: novel mechanism for hypoxic-induced membrane depolarization.
Circ. Res.
77:
131-139,
1995[Abstract/Free Full Text].
10.
Pozzan, T.,
R. Rizzuto,
P. Volpe,
and
J. Meldolesi.
Molecular and cellular physiology of intracellular calcium stores.
Physiol. Rev.
74:
595-636,
1994[Free Full Text].
11.
Rae, J.,
K. Cooper,
G. Gates,
and
M. Watsky.
Low access resistance perforated patch recordings using amphotericin B.
J. Neurosci. Methods
37:
15-26,
1991[Medline].
12.
Reeve, H. L.,
D. P. Nelson,
S. L. Archer,
and
E. K. Weir.
Effects of fluoxetine, phentermine, and venlafaxine on pulmonary arterial pressure and electrophysiology.
Am. J. Physiol.
276 (Lung Cell. Mol. Physiol. 20):
L213-L219,
1999[Abstract/Free Full Text].
13.
Rubin, L. J.
Primary pulmonary hypertension.
N. Engl. J. Med.
336:
111-117,
1997[Free Full Text].
14.
Treiman, M.,
C. Caspersen,
and
S. B. Christensen.
A tool coming of age: thapsigargin as an inhibitor of sarcoendoplasmic reticulum Ca2+ATPases.
Trends Pharmacol. Sci.
19:
131-135,
1998[Medline].
15.
Voelkel, N. R.,
R. M. Tuder,
and
E. K. Weir.
Pathophysiology of primary pulmonary hypertension: from physiology to molecular mechansims.
In: Primary Pulmonary Hypertension, edited by L. J. Rubin,
and S. Rich. New York: Dekker, 1997, vol. 99, p. 83-129. (Lung Biol. Health Dis. Ser.)
16.
Weir, E. K.,
H. L. Reeve,
J. M. C. Huang,
E. Michelakis,
D. P. Nelson,
V. Hampl,
and
S. L. Archer.
Anorexic agents aminorex, fenfluramine, and dexfenfluramine inhibit potassium current in rat pulmonary vascular smooth muscle and cause pulmonary vasoconstriction.
Circulation
94:
2216-2220,
1996[Abstract/Free Full Text].
17.
Yuan, X.-J.,
R. T. Bright,
A. M. Aldinger,
and
L. J. Rubin.
Nitric oxide inhibits serotonin-induced calcium release in pulmonary artery smooth muscle cells.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L44-L50,
1997[Abstract].
18.
Yuan, Y.-T.,
O.-L. Wang,
and
R. A. Whorton.
Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1258-C1265,
1992[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 277(3):L662-L666
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