1 Department of Molecular
Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden; 2 Department of
Pulmonary and Critical Care Medicine, Catecholamines promote lung edema clearance via
alveolar epithelium; protein kinases; actin cytoskeleton; sodium
transport; early endosomes; late endosomes
PULMONARY EDEMA FORMATION is due to increased
hydrostatic pressure in the pulmonary circulation or increased
pulmonary capillary permeability (43). Regardless of its pathogenesis,
once edema is established, the clearance of pulmonary fluid is
effected by active epithelial
Na+ transport out of the alveoli
(18). Vectorial movement of Na+ in
alveolar epithelial cells depends on the concerted activity of ion
transport proteins localized within structurally and functionally distinct plasma membrane domains:
Na+-K+-ATPase,
confined to the basolateral side, and various ion transporters located
at the apical domain of the cell (34). The electrochemical gradient
generated by the activity of
Na+-K+-ATPase
is responsible for the vectorial transport of
Na+ from the air space, with water
following isosmotically (39, 40, 42, 43).
The alveolar epithelium comprises two distinct cell populations: type I
cells, which are thin and elongated and have not been studied in detail
due to technical difficulties in isolation, and type II cells, which
have been extensively studied. Alveolar epithelial type II
(ATII) cells are cuboidal, and their major distinguishing
characteristic is their capacity for synthesis and secretion of
pulmonary surfactant. In addition to serving as surfactant storage
sites, when ATII cells are isolated and cultured on plastic in
serum-containing medium, they express the Na+-K+-ATPase
Catecholamines increase lung fluid clearance via
The purpose of this study was to determine whether
Na+-K+-ATPase
in ATII cells undergoes acute changes in its catalytic activity in
response to short-term Materials. Phallacidin, Iso, forskolin
(FSK), brefeldin A, Tris-ATP, and the 20-residue cAMP-dependent protein
kinase inhibitor (IP20) were
obtained from Sigma (St. Louis, MO).
[ Isolation and culture of alveolar epithelial
cells. ATII cells were isolated from specific
pathogen-free male Sprague-Dawley rats (200-225 g) as previously
described (15, 28, 33). Briefly, the lungs were perfused via the
pulmonary artery, lavaged, and digested with elastase (30 U/ml). ATII
cells were purified by differential adherence to IgG-pretreated dishes,
and cell viability was assessed by trypan blue exclusion (>95%).
Cells were suspended in Dulbecco's modified Eagle's medium (DMEM;
Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum
with 2 mM L-glutamine, 40 µg/ml of gentamicin, 100 U/ml of penicillin, and 100 µg/ml of
streptomycin. For studies of
[ Determination of
Na+-K+-ATPase
activity in intact cells.
Na+-K+-ATPase
activity was determined as described before (4). Briefly, after the
cell suspensions were preincubated with the desired agonists at room
temperature, they were placed on ice, and aliquots (~10 µg of
protein) were transferred to the Na+-K+-ATPase
assay medium (final volume 100 µl) containing (in mM) 50 NaCl, 5 KCl,
10 MgCl2, 1 EGTA, 50 Tris · HCl, and 7 Na2ATP and
[
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-adrenergic-mediated stimulation of active
Na+ transport across the alveolar
epithelium. Because alveolar epithelial type II cell
Na+-K+-ATPase
contributes to vectorial Na+ flux,
the present study was designed to investigate whether
Na+-K+-ATPase
undergoes acute changes in its catalytic activity in response to
-adrenergic-receptor stimulation.
Na+-K+-ATPase
activity increased threefold in cells incubated with 1 µM
isoproterenol for 15 min, which also resulted in a fourfold increase in
the cellular levels of cAMP. Forskolin (10 µM) also stimulated
Na+-K+-ATPase
activity as well as ouabain binding. The increase in
Na+-K+-ATPase
activity was abolished when cells were coincubated with a
cAMP-dependent protein kinase inhibitor. This stimulation, however, was
not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase
-subunit; rather, it was the result of an increased number of
-subunits recruited from the late endosomes into the plasma membrane. The recruitment of
-subunits to the plasma membrane was
prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but
was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases
Na+-K+-ATPase
activity in alveolar type II epithelial cells by recruiting
-subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1- and
1-subunit isoforms for up to 4 days in culture and maintain active
Na+ transport (28, 33). Monolayers
of ATII cells generate short-circuit currents and
Na+ fluxes that are ouabain
inhibitable (23). Increased edema clearance is associated with higher
Na+-K+-ATPase
activity (31) in alveolar epithelial cells, further supporting the role
of this cell population in Na+ and
water transport accross the alveolar epithelium.
-adrenergic-mediated stimulation of active
Na+ transport across lung
epithelial cells (1). The cellular mechanisms involved are not yet
fully elucidated, although they probably include changes in apical
Na+ channels (29, 44). In
addition, it has been recently proposed that alveolar cell
Na+-K+-ATPase
plays an important role in lung edema clearance by enhancing active
Na+ transport (31). Several
intracellular signaling messengers such as cAMP and protein kinase C,
which modulate
Na+-K+-ATPase
activity in other epithelia (5, 6), have been shown to affect fluid
transport across alveolar epithelial cells (36, 41).
-adrenergic-receptor stimulation by isoproterenol (Iso) and, if so, to explore possible mechanisms involved
in this effect.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP and
[3H]ouabain were from
Amersham (Arlington Heights, IL).
[32P]orthophosphate
was purchased from NEN Life Science Products. Rhodamine-phalloidin was
from Molecular Probes (Eugene, OR). Elastase was from Worthington
Biochemical (Freehold, NJ). The
Na+-K+-ATPase
-antibody was a generous gift from Dr. M. Caplan (Yale University,
New Haven, CT), and the Rab 5 antibody was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA).
-32P]ATP
hydrolysis in intact alveolar epithelial cells and preparation of
membranes for Western blot analysis, 10 ml of the cell suspension (106 cells/ml) were added to
100-mm dishes (Becton Dickinson). For studies evaluating the
cytoskeleton structure, ATII cells were plated onto glass coverslips at
2 × 105 cells/coverslip.
Cells were incubated in a humidified atmosphere of 5%
CO2-95% air at 37°C.
Identification of ATII cells was based on the presence of lamellar
inclusions. Lamellar bodies were stained with Papanicolaou stain (22),
tannic acid (27), and alkaline phosphatase (17).
-32P]ATP (specific
activity 3,000 Ci/mmol) in tracer amounts (3.3 nCi/µl). Cells were
transiently exposed to a thermic shock (10 min at
20°C) to
render the membranes permeable to ATP. The samples were then incubated
at 37°C for 15 min, and the reaction was terminated by the addition
of 700 µl of a TCA-charcoal (5:10% wt/vol) suspension and rapid
cooling to 4°C. After the charcoal phase containing the
unhydrolyzed nucleotide was separated (12,000 g for 5 min), the
liberated 32P was counted in an
aliquot (200 µl) from the supernatant.
Na+-K+-ATPase
activity was calculated as the difference between the test samples
(total ATPase activity) and the samples assayed in the same medium but
devoid of Na+ and
K+ and in the presence of 4 mM
ouabain (ouabain-insensitive ATPase activity).
4 M
[3H]ouabain (25 Ci/mmol), pH 7.2. Nonspecific binding was determined with an identical
incubation medium containing a 100-fold excess of unlabeled ouabain.
The binding reaction was allowed to proceed for 30 min at 37°C.
Unbound [3H]ouabain
was removed by rapidly washing the cells three times with ice-cold
sample buffer.
Phosphorylation and immunoprecipitation of the
Na+-K+-ATPase
-subunit in intact ATII cells.
Chibalin et al. (12) recently quantitated phosphorylation
of the
-subunit in a renal cell line (OK), and we have utilized identical procedures in these experiments. Briefly, ATII cells (2.0-2.5 mg protein/dish) were labeled during 2.5 h at 37°C in a buffer containing (in mM) 120 NaCl, 5 KCl, 4 NaHCO3, 1 CaCl2, 1 MgSO4, 0.2 NaH2PO4,
0.15 Na2HPO4,
5 glucose, 10 lactate, 1 pyruvate, and 20 HEPES, 1% bovine serum
albumin, pH 7.45, and 100 µCi/ml of
[32P]orthophosphate.
The cells were labeled and incubated with different agonists in culture
dishes to preserve the polarized distribution of different ion
transport proteins. The incubation with Iso was performed at room
temperature according to protocols similar to those described for
Na+-K+-ATPase
activity. The incubation was terminated by removing the medium, adding
immunoprecipitation buffer [100 mM NaCl, 50 mM Tris · HCl, 2 mM EGTA, 10 mM NaF, 30 mM
Na4O7P2,
1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml of leupeptin, 4 µg/ml of aprotonin, and 1% Triton X-100, pH 7.45], and placing the samples on ice. The cells were disrupted by gentle homogenization. An aliquot was removed for SDS-PAGE analysis of the total pattern of phosphorylation.
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RESULTS |
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ATII cells after 2 days in culture were resuspended and incubated at
room temperature with 1 µM Iso for 15 min in the presence and absence
of IP20 (Fig.
1A).
Na+-K+-ATPase
activity in nonstimulated ATII cells was 97 ± 7 nmol
Pi · mg
protein1 · min
1
(n = 5). Iso stimulated
Na+-K+-ATPase
activity, and this effect was abolished in the presence of 10 nM
IP20, which by itself did not
significantly change
Na+-K+-ATPase
activity. Consistent with these results, incubation of ATII cells with
Iso was associated with a dose-dependent increase in the cellular
levels of cAMP (Fig. 1B).
|
To determine whether an increase in cellular cAMP (by direct activation
of adenylyl cyclase) affects
Na+-K+-ATPase
activity, ATII cells were also incubated with 10 µM FSK for 15 min at
room temperature. FSK significantly increased
Na+-K+-ATPase
activity as measured by
[-32P]ATP
hydrolysis (Fig.
2A)
as well as by
[3H]ouabain binding
(Fig. 2B).
|
Because
Na+-K+-ATPase
activity is stimulated by cAMP in several tissues (2, 14, 26),
including the lung epithelia (41), it has been suggested that this
effect could be mediated by direct phosphorylation of the -subunit.
We therefore examined in ATII cells metabolically labeled with
32P whether incubation with Iso
resulted in increased phosphorylation of the catalytic
-subunit.
Immunoprecipitation of the
-subunit from cells treated with Iso
revealed no increase in its state of phosphorylation compared with that
from vehicle-treated cells (Fig. 3).
Alternatively, the FSK-induced increase in
[3H]ouabain binding
within 15 min might suggest that in ATII cells cAMP stimulates
Na+-K+-ATPase
activity by increasing the number of
Na+-K+
pump units in the plasma membrane.
|
Stabilizing the cortical cytoskeleton with the fungal toxin phallacidin has proven to be an important tool in studying the relevance of actin dynamics in several physiological processes (38). The next series of experiments was therefore performed in ATII cells pretreated with phallacidin (1 µM) overnight. This treatment did not affect the cell morphology or the distribution of actin polymers within the cell cytoplasm (Fig. 4). However, monolayers pretreated with phallacidin did not exhibit the increments in Na+-K+-ATPase activity and [3H]ouabain binding (Fig. 5, A and B, respectively) observed in response to FSK alone, even though the ability of FSK to increase the cellular levels of cAMP was not affected (Fig. 5C).
|
|
We next evaluated the effect of Iso and FSK on
Na+-K+-ATPase
-subunit abundance in the BLM using Western blot analysis. BLMs were
prepared from monolayers incubated with 1 µM Iso or 10 µM FSK for
15 min at room temperature. Iso and FSK increased the
-subunit
abundance to a similar extent, and this effect was completely blocked
by pretreatment with phallacidin (Fig.
6).
|
Further support for traffic of
Na+-K+-ATPase
molecules as a mechanism for increased activity was provided by studies
performed in the presence of the fungal toxin brefeldin A. Preincubation of ATII cells with brefeldin A (5 µg/ml at room
temperature for 20 min) inhibited
Na+-K+-ATPase
activity (vehicle: 61.2 ± 10 nmol
Pi · mg
protein1 · min
1,
n = 10; brefeldin A: 40.7 ± 5 nmol
Pi · mg
protein
1 · min
1,
n = 5;
P < 0.05). Under this condition, Iso
(15 min at room temperature) failed to stimulate enzyme activity (Iso + brefeldin A: 39.5 ± 4 nmol
Pi · mg
protein
1 · min
1;
n = 4 experiments). The effects of Iso
and brefeldin A on
Na+-K+-ATPase
-subunit abundance in BLMs were also evaluated in ATII monolayers
(Fig. 7). Iso increased
Na+-K+-ATPase
-subunit abundance, and this effect was blocked by brefeldin A
treatment.
|
The stimulatory effect of Iso on
Na+-K+-ATPase
-subunit abundance in the BLM appeared to be independent of
Na+ permeability because it was
not altered by coincubation with 10 µM amiloride (Fig.
8). In addition, the
stimulatory effect of Iso (210 ± 14% of control value;
P < 0.05;
n = 5 experiments) on
Na+-K+-ATPase
activity was also not abolished by coincubation with amiloride (238 ± 38% of control value; n = 4 experiments), and amiloride alone did not affect
Na+-K+-ATPase
activity (95.7 ± 25% of control value;
n = 4 experiments).
|
The increased -subunit abundance is not likely to represent
increased de novo synthesis of
Na+-K+-ATPase
molecules because the Iso effect occurs within 15 min. Thus we
hypothesized that the increased number of
Na+-K+-ATPase
molecules within the BLM may be caused by recruitment of existing units
from intracellular compartments. We therefore prepared early and late
endosomes from ATII cells, the purity and identity of which were
confirmed by enrichment of Rab 5- and mannose 6-receptor
immunoreactivity, respectively (11). Both populations of endosomes
contained
Na+-K+-ATPase
-subunits (Fig. 9). In
late but not in early endosomes prepared from Iso-treated ATII cells,
there was a significant decrease in
-subunit abundance, with a
concomitant increase in their presence in BLMs from the same cell
population (Fig. 9).
|
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DISCUSSION |
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In this study, we demonstrate that Iso increases
Na+-K+-ATPase
activity of ATII cells independently of
Na+ permeability and that this
effect is mediated by increasing the number of
Na+-K+-ATPase
molecules within the BLM. The stimulatory action of Iso is associated
with increased levels of cAMP but does not involve phosphorylation of
the
Na+-K+-ATPase
-subunit.
Although regulation of ATII cell
Na+-K+-ATPase
activity by -adrenergic agonists has been previously suggested (41),
the intrinsic mechanisms of this regulation as well as the
intracellular signaling systems involved have not yet been completely elucidated.
Here we report that short-term (15-min) incubation with 1 µM Iso
stimulated
Na+-K+-ATPase
activity in ATII cells (measured as hydrolytic activity in cell
suspensions or ouabain binding in cell monolayers) and that this effect
was associated with a significant increase in the cellular levels of
cAMP (Fig. 1B). Furthermore, the
stimulatory effect of Iso was blocked by a cAMP-dependent protein
kinase inhibitor. However, Suzuki et al. (41) could not demonstrate a
direct correlation between the increase in
Na+-K+-ATPase
activity and the rise in cellular cAMP levels. In that report, a cAMP
analog increased
Na+-K+-ATPase
activity in alveolar cells cultured for 2 and 5 days, whereas the
-adrenergic agonist terbutaline, although able to increase cAMP
levels at both intervals, failed to stimulate
Na+-K+-ATPase
activity in cells cultured for 2 days. Our experiments were performed
in resuspended ATII cells (hydrolytic activity) or cell monolayers
(ouabain binding) after 2 days in culture, and we indeed found a
correlation between the changes in
Na+-K+-ATPase
activity and the increase in cAMP (see Figs. 1 and 2). At present, we
do not have an explanation for the differences between these two
studies. Furthermore, the effect of Iso was blocked by a cAMP-dependent
protein kinase inhibitor.
Short-term regulation of
Na+-K+-ATPase
activity by hormones and the signaling systems involved have been
extensively studied (5, 6). Accumulating evidence indicates that most
receptor signals converge in the activation of protein kinases.
Phosphorylation of the purified
Na+-K+-ATPase
-subunit preparations by both protein kinase C and cAMP-dependent protein kinase resulted in a significant change in its catalytic activity (3, 13, 25). In addition, studies in intact cells also
demostrated that regulation of
Na+-K+-ATPase
activity may be accomplished by phosphorylation of its catalytic
subunit. Specifically, activation of cAMP kinase in intact cells has
been associated with increased
Na+-K+-ATPase
activity (10). In our studies, Iso stimulation of
Na+-K+-ATPase
activity, although associated with increased cellular levels of cAMP
and presumably activation of a cAMP-dependent protein kinase, was not
due to increased phosphorylation of the
Na+-K+-ATPase
catalytic subunit (Fig. 3). The lack of
-subunit phosphorylation suggests that cAMP and cAMP-dependent kinase may be necessary at other
stages of
Na+-K+
pump regulation, possibly during the unit's traffic to the BLM.
An alternative mechanism by which Iso could stimulate
Na+-K+-ATPase
activity is by increasing the number of functioning units in the plasma
membrane. This effect could be brought about by alterations in the rate
of protein synthesis, consequently leading to changes in enzymatic
activity. However, this process (which usually occurs over a period of
hours) cannot explain the observed Iso effect, which occurred within 15 min. Because stimulation of
Na+-K+-ATPase
activity was associated with an increase in the number of units at the
cell surface (Fig. 2B, ouabain
binding, and Fig. 6, -subunit abundance), we reasoned that pump
units present in one or more intracellular compartments may have been
recruited for insertion in the plasma membrane. Contrary to Suzuki et
al. (41), we did find an increase in
-subunit abundance at the BLM.
Although speculative, a possible explanation why Suzuki et al. did not
observe such an increase is because the high-speed centrifugation
utilized in their protocols to obtain the membrane preparations could
have caused the inclusion of endosomes and other organelles in the
isolated material, thus making it difficult to distinguish between the
proportion of molecules that are incorporated in the basolateral
membranes and those from the intracellular compartments.
We have demonstrated in renal epithelial cells the presence of defined
intracellular compartments in which
Na+-K+-ATPase
is located (11). On incubation with dopamine, the resultant decrease in
Na+-K+-ATPase
activity was associated with a stepwise transfer of
Na+-K+-ATPase
- and
-subunits from the plasma membrane into early and late
endosomes via a clathrin vesicle-dependent mechanism (11). In the
present study, we have also identified
Na+-K+-ATPase
subunits within these compartments in ATII cells, although the transfer
seems to occur in the opposite direction, i.e., from late endosomes to
the plasma membrane. In cells incubated with Iso for 15 min, there is
an increased incorporation of
Na+-K+-ATPase
-subunits in the plasma membranes, associated with a decrease in
their abundance in the late endosomal compartment without a significant
change in early endosomes (see Figs. 6 and 9). It is possible that the
-subunits present in the early endosomes are there as the
consequence of a constitutive or a regulated endocytic pathway as
demonstrated in renal cells (11), whereas in late endosomes, they may
represent a pool that can recycle to the plasma membrane on rapid
demand. This pool could be replenished by newly synthesized
Na+-K+-ATPase
subunits or by those from the endocytic pathway that have not proceeded
to degradation in lysosomes.
The reason(s) why the percent increase of the -subunit in the BLM
(~80%) does not correspond to its decrease in the late endosomes
(~30%) is not entirely clear. An important factor determining the
relative changes in
Na+-K+-ATPase
in the two compartments is the relative size of these compartments
accessible to analysis. A likely explanation, although speculative at this point, could therefore be that different recoveries are obtained during the isolation of endosomes and BLMs (because, after
the flotation gradient fractionation used to separate endosomes, the
fraction containing BLMs was further enriched with Percoll gradient
centrifugation), making these determinations (percent changes)
semiquantitative. However, when fractions were prepared by the same
method (in kidney cells), we found a decrease in
-subunit abundance
in the fraction containing BLMs comparable to the increase in the
fraction containing endosomes (11). Another possibility to be
considered is that of "serial compartmentalization" of
-subunit traffic between the late endosomes and the plasma membrane:
-subunits will not reach the plasma membrane solely from the late
endosomes but also from intermediate compartments yet unidentified or
not accessible to isolation. Also, from our studies, it cannot be excluded that there may be additional mechanisms responsible for the
effect under discussion.
Additional evidence suggesting that Iso stimulation of
Na+-K+-ATPase
activity is accomplished by favoring the movement of pump molecules
from intracellular pools to the plasma membrane is provided by the
finding that this process was inhibited by stabilizing the actin
cytoskleton with phallacidin. The mechanisms underlying the regulatory
role of actin cytoskeleton dynamics in these transport events are still
unclear, but one possibility would be by permitting the motion of the
late endosomes containing
Na+-K+-ATPase
toward the BLM and their fusion with it. Also, incubation with the
fungal toxin brefeldin A, which prevents anterograde transport,
abolished the increased -subunit abundance in BLMs induced by Iso.
Therefore, this study also raises the possibility that Iso might
regulate the actin cytoskeleton to favor the traffic of
Na+-K+
pump molecules to their cellular destination.
Vectorial transport of Na+ in the
alveolar epithelium is critical for modulation of alveolar fluid
clearance (31, 32, 37). Administration of -adrenergic agonists has
been shown to increase active Na+
transport and lung edema clearance (35), but the mechanisms whereby
-adrenergic stimulation results in increased lung edema clearance
are only partially understood. It is postulated that activation of
apical Na+ channels (29, 44) and
stimulation of
Na+-K+-ATPase
activity are the cellular mechanisms involved (42). This coordinated
transport would entail changes in vectorial movement of
Na+ without affecting its
intracellular concentration as reported for other tissues (7), thus
preventing further changes in cellular homeostasis.
The concept that Iso would independently affect
Na+ permeability and active
transport is supported by the present results, which suggest that the
action of Iso on
Na+-K+-ATPase
activity is not mediated by, or dependent on, an increase in
intracellular Na+. Amiloride, at
concentrations that should have blocked the majority of
Na+ entry pathways, did not
prevent Iso-dependent stimulation of Na+-K+-ATPase
activity or the increase in -subunit abundance in the BLM (Fig. 8).
This observation and that of a previous report on ATII cells (41)
differ from the situation in renal proximal tubule cells where
Iso-dependent regulation of
Na+-K+-ATPase
activity appears to be the result of increased
Na+ permeability (39).
In conclusion, this study demonstrates that -adrenergic agonists
(Iso) regulate
Na+-K+-ATPase
activity in transporting epithelia (such as ATII cells) by increasing
the number of
Na+-K+-ATPase
units in the BLM. This regulation is rapid and probably does not
involve synthesis of new molecules. Rather, the new
Na+-K+-ATPase
units incorporated in the BLM are apparently recruited from defined
intracellular compartments, e.g., the late endosomes. Once in the BLM,
these newly inserted transport proteins contribute to the overall
increase in cellular
Na+-K+-ATPase
activity and consequently to vectorial
Na+ flux across the alveolar epithelium.
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ACKNOWLEDGEMENTS |
---|
We thank Chris Waters for help with this work.
![]() |
FOOTNOTES |
---|
This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-48129 (to J. I. Sznajder); a National Research Science Award from the National Institutes of Health (to K. M. Ridge); an American Heart Association grant (to J. I. Sznajder); and a Swedish Medical Research Council grant (to A. M. Bertorello)
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: A. M. Bertorello, Dept. of Molecular Medicine L6B:02, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Received 22 April 1998; accepted in final form 2 September 1998.
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