1 Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90089; and 2 Institute of Physiology, University of Aarhus, 8000 Aarhus C, Denmark
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ABSTRACT |
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Fourteen-day adrenal steroid treatment increases
[3H]ouabain binding sites 22-48% in muscle biopsies
from patients treated with adrenal steroids for chronic obstructive
lung disease and in rats treated with dexamethasone (Dex). Ouabain
binding measures plasma membrane sodium pumps
(Na+-K+-ATPase) with isoform-dependent
affinity. In this study we have established the specific pattern of Dex
regulation of sodium pump isoform protein and mRNA levels in muscle.
Rats were infused with Dex (0.1 mg/kg per day) or vehicle for 14 days.
Abundance of sodium pump catalytic 1- and
2-subunits and glycoprotein
1- and
2-subunits was determined by immunoblot in soleus,
extensor digitorum longus, whole gastrocnemius, and diaphragm and was
normalized to the mean vehicle control value. Dex increased
2 and
1 protein in all muscle types by
53-78% and ~50%, respectively. Dex increased
1 protein only in diaphragm (65 ± 7%). At the mRNA level in whole hindlimb muscle, Dex increased
2 (6.4 ± 0.5-fold)
and
1 (1.54 ± 0.15-fold) and decreased
2 (to 0.36 ± 0.6 of control). In summary,
2
1 is the Dex-responsive pump in all
skeletal muscles, and changes in
2 and
1
mRNA levels can drive the 50% change in
2
1-subunits, which can account for the
reported increase in [3H]ouabain binding.
Na+-K+-ATPase isoforms; dexamethasone; soleus; extensor digitorum longus; gastrocnemius; diaphragm
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INTRODUCTION |
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RAVN AND DORUP (34) discovered that patients treated in hospital with adrenal steroids and diuretics for chronic obstructive lung disease (COLD) had reduced concentrations of skeletal muscle potassium and elevated muscle sodium pump number, as estimated by [3H]ouabain binding. This was unexpected because a previous study (12) on patients treated with diuretics (and not steroids) showed that the accompanying K+ depletion was associated with decreased concentration of sodium pumps in muscle. In a parallel study in rats, Dorup and Clausen (11) demonstrated that 7-day treatment with dexamethasone, a potent synthetic corticosteroid, increases [3H]ouabain binding to rat skeletal muscle biopsies by 22-42%. This finding indicated that the increased muscle sodium pump number in the COLD patients was due to the glucocorticoid treatment, which could apparently override any decrease in sodium pump number due to diuretic-induced potassium depletion.
Glucocorticoids have been reported to have effects on sodium pumps in a number of tissues. These steroids return sodium pump levels to control levels in kidney cells from adrenalectomized rats (5, 35) and increase sodium pump number in heart cells (15, 21) and cultured aortic smooth muscle cells (40).
Glucocorticoids are routinely administered for a variety of medical
conditions (45), and the accompanying effect on muscle sodium pump expression could precipitate potentially dangerous complications (11). For instance, the results summarized
above suggest that when a COLD patient is treated with systemic
glucocorticoids to reduce smooth muscle inflammation, there will be an
increase in skeletal muscle sodium pump number; when an acute attack of airway constriction in these same patients is treated with a
2-adrenergic agonist, there will be an acute activation
of the pool of sodium pumps already enlarged by glucocorticoid
treatment (6-8). The activation of K+
transport into the cell could lower extracellular K+ to the
point where it could reduce cardiac excitability/contractility to
life-threatening levels (11).
The isoform dependence of glucocorticoid regulation of sodium pumps in
muscle has not been previously studied. The sodium pump exists as an
-catalytic,
-glycoprotein heteromer, and muscle expresses two
isoforms of each subunit in a muscle fiber type-specific manner
(18, 42). Slow oxidative soleus expresses
1-,
2- and
1-isoforms (as
1
1 and
2
1
heteromers), while at the opposite phenotypic extreme, fast glycolytic
white gastrocnemius expresses
1-,
2-, and
2-isoforms (presumably as
1
2 and
2
2)
(42). Extensor digitorum longus (EDL), a fast
metabolically mixed glycolytic and oxidative-glycolytic muscle,
expresses all four isoforms.
3- and
3-isoforms are not expressed at significant levels in adult skeletal muscle (28, 32, 39, 41).
2
abundance is about the same, per muscle protein, in all muscle types
examined, while
1 abundance is twice as high in
oxidative soleus and diaphragm as in fast glycolytic white
gastrocnemius (41). We have calculated that the ratio of
1 to
2 is around 1:1, on the basis of
relative changes in
1,
2, and
1 in soleus, and
2 in white
gastrocnemius, during K+ deprivation (41).
[3H]ouabain binding can be used as a measure of the
concentration of sodium pumps because the cardiac glycoside binds to
the extracellular face of -catalytic subunits of plasma
membrane-localized sodium pumps. However, there are two caveats to this
use of ouabain in rat muscle biopsies: 1) the
[3H]ouabain will not bind to pumps located in endosomes
(17); and 2) in rat, the
1-isoform has far less affinity for ouabain [dissociation constant (Kd) = 5 × 10
5 M] than the
2-isoform
(Kd = 10
7-10
9 M). Because of the low
affinity for
1-isoform, it has been proposed that the
[3H]ouabain binding assay only accurately detects the
higher affinity
2-isoform in muscle (11).
On the basis of these observations, we aimed to determine whether the
previously reported increase in [3H]ouabain binding to
skeletal muscle driven by dexamethasone treatment could be accounted
for by an increase in the 1 and/or
2
protein abundance and, if so, whether the changes were driven by
changes in the mRNA levels of the subunits. We report that
2 and
1 protein and mRNA levels increase
after glucocorticoid treatment in all muscles examined, enough to
account for the observed increase in [3H]ouabain binding.
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METHODS |
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Animals and diets.
Two series were conducted, one for protein and another for mRNA
analysis, both as previously described (11). In short,
female Wistar rats, ~10 wk of age (~200 g), were maintained on
standard chow at constant temperature (21°C) under a 12:12-h
artificial light cycle with unlimited access to water. Dexamethasone
(0.1 mg/kg per day) or vehicle (polyethylene glycol 400) was
continuously infused for 14 days via dorsally implanted osmotic
minipumps (model 2002; Alzet, Palo Alto, CA). For protein analysis,
day 14 rats (n = 5 treated and 5 controls)
were anesthetized and decapitated, and the soleus, EDL, whole
gastrocnemius, and diaphragm were removed, frozen in liquid nitrogen,
and stored at 80°C pending analyses. For RNA analysis
(n = 5-6 in each group), whole hindlimb muscle was
removed and either extracted directly or frozen in liquid nitrogen
pending extraction.
Na+-K+-ATPase subunit protein analysis. Skeletal muscle was homogenized with a Polytron homogenizer 1:20 (wt/vol) in 5% sorbitol, 25 mM histidine imidazole (pH 7.4), 0.5 mM Na2EDTA, and the proteolytic enzyme inhibitors phenylmethylsulfonyl fluoride (PMSF; 0.5 mM), leupeptin (1 µl/ml), and 4-aminobenzamidine dichloride (pABAD; 1 mM), as previously described (42). Protein concentration was determined after TCA precipitation of 10-µl aliquots of the homogenate in triplicate, resuspension of the pellet in NaOH, and assay by the Lowry method (26).
A constant amount of homogenate protein (20 µg forNa+-K+-ATPase activity.
Whole hindlimb muscle was homogenized by using a procedure specific for
the measurement of Na+-K+-ATPase activity in
muscle (1), distinct from that used for the immunoblot
assay. Specifically, hindlimb muscle was dissected, weighed, and minced
with scissors in ice-cold homogenization medium (250 mM mannitol, 30 mM
L-histidine, 5 mM Tris-EDTA, and 0.1% sodium deoxycholate,
brought to pH 6.8 with NaOH at room temperature). The minced suspension
was homogenized with Ultra Turrax T25 (IKA-Labortechnik) at 22,000 rpm
for 15 s twice before it was transferred to a glass homogenizer
tube and further homogenized for six full strokes with a motor-driven
Teflon pestle (setting 5; Dyna-Mix, Fisher Scientific). The homogenate
was centrifuged at 3,000 g for 20 min (at 4°C) and frozen
at 80°C overnight. The next day, the Na+-K+-ATPase activity in the supernatant was
measured in triplicate as the ouabain-sensitive ATPase activity:
homogenate was preincubated with or without 2 mM ouabain for 30 min,
and the reaction was started with the addition of ATP.
Na+-K+-ATPase activity was calculated as the
difference in inorganic phosphate (Pi) generated over 15 min in the absence (total activity) and presence (Mg-ATPase activity)
of 2 mM ouabain and expressed as micromoles of Pi liberated
per milligram of protein per hour. In addition, the samples were
reassayed for establishment of reproducibility on another day.
Na+-K+-ATPase subunit mRNA analysis.
At day 14 of dexamethasone or vehicle treatment, whole
hindlimbs were removed, and either RNA was isolated immediately or muscles were flash frozen in liquid nitrogen and stored at 80°C until RNA isolation; no effects of freezing on degradation of RNA were
evident when muscles from the same animal were compared. Total RNA was
isolated from rat skeletal muscle and probed as previously detailed
(41). In short, RNA was isolated with Tri Reagent
(Molecular Research Center, Cincinnati, OH) according to the
manufacturer's protocol, concentrations were determined by optical
density OD260, and purity was estimated by
OD260/OD280. Total RNA was assayed by Northern
analysis on NitroPure nitrocellulose transfer membrane (Micron
Separations, Westborough, MA). Immobilized RNA was probed with either
isoform-specific restriction endonuclease fragments (~300 bp) of
1,
2, and
1 or the rat
2 cDNA clone (provided by P. Martin-Vasallo) labeled for
similar specific activity with [32P]dCTP probes with the
use of a multiprimer DNA-labeling technique, as described previously
(3, 41). Blots were washed three times in 2× SSC (0.3 M
NaCl-0.03 M sodium citrate, pH 7.0) with 0.05% SDS at room temperature
for 10 min each and then twice for 20 min each in 0.1× SSC with 0.1%
SDS at 55°C.
Quantitation. Film exposure time was adjusted to a range wherein the samples run at one-half the amount of protein on each gel had one-half the autoradiographic density relative to the same samples run at twice that amount of protein. Autoradiograms were quantitated with the Bio-Rad GS670 Imaging Densitometer and dedicated software. All data are expressed as means ± SE. Significance was assessed by using the two-tailed Student's t-test, and differences were considered significant at P < 0.05.
Materials. Chemicals were reagent grade, spectroquality, or electrophoresis purity reagents. SDS-PAGE reagents were from Bio-Rad (Hercules, CA). Leupeptin, PMSF, pABAD, and SDS-PAGE molecular weight standards were from Sigma (St. Louis, MO). PNGase F was from Genzyme (Cambridge, MA). 125I-labeled protein A and [32P]dCTP were from ICN (Costa Mesa, CA).
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RESULTS |
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Because skeletal muscle is a heterogeneous tissue consisting of different muscle fiber types and because there is a muscle fiber-specific pattern of sodium pump isoform expression, the effects of 14-day dexamethasone treatment (0.1 mg/kg per day) were examined in a panel of distinct skeletal muscles including soleus, a predominantly slow oxidative (type I) muscle; EDL, a classically fast muscle that is metabolically both glycolytic (IIB) and oxidative-glycolytic (IIA); gastrocnemius, a mixed muscle type; and diaphragm, also a highly mixed muscle type (2, 30, 37).
Na+-K+-ATPase isoform protein and activity
regulation by dexamethasone.
To determine whether dexamethasone regulated the total pool size of the
1-,
2-,
1-, or
2-subunits expressed in skeletal muscle, we evaluated
subunit pool size by immunoblotting a constant amount of total
homogenate protein. Samples for quantitation of
1- or
2-subunit were deglycosylated before analysis, as
described in METHODS, because glycosylation appears to
interfere with antibody binding to both
-subunits (38).
Typical autoradiograms of skeletal muscle homogenate samples, probed
with isoform-specific antibodies against
1,
2,
1, and
2, are shown in
Fig. 1.
1 and
2 were detected at ~110 kDa. Deglycosylated core
1 and
2 proteins were detected at
32-35 kDa. In Fig. 1, the relative autoradiographic densities
reflect the subunit expression levels when control vs. dexamethasone-treated samples are compared within a specific muscle for
a specific isoform because these samples were run on the same blot, in
which autoradiographic exposure time was adjusted to assure the
linearity of signal density with the amount of sample loaded (as
described in METHODS). In comparison, the relative densities of signals from different muscles or isoforms cannot be
compared because these were obtained from different films with different exposure times and because each antibody has a unique affinity for its subunit epitope.
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Na+-K+-ATPase isoform mRNA regulation by
dexamethasone.
RNA was extracted from whole hindlimb, a mixture of different fiber
types, because RNA isolated from distinct muscles may be degraded
during the dissection process, while hindlimb muscle can be very
quickly removed and extracted or frozen (41). As summarized in Fig. 3A,
1 mRNA was detected as a single band at 3.7 kb,
2 was detected as two bands at 5.3 and 3.4 kb,
1 was detected primarily as two main bands at 2.7 and
2.3 kb (which were scanned for quantitation), and
2 was
detected as a single band at ~2.8 kb. Kidney RNA was loaded as a
control sample that contains high levels of
1 and
1 but little or no
2 or
2
(Fig. 3A, first lane), and brain RNA was added as a control
sample, also enriched in
1 and
1 but
containing
2 and
2 as well (Fig. 3A, last lane). To verify the linearity of the
autoradiographic signal with the amount of RNA loaded, we loaded
one-half the amount for two of the samples (Fig. 3, 1/2C and
1/2D). As summarized in Fig. 3B, 14-day dexamethasone
treatment did not significantly increase
1 mRNA but
increased
2 mRNA more than sixfold in whole hindlimb. Dexamethasone also increased Na+-K+-ATPase
1 mRNA levels 1.54 ± 0.15-fold, in line with the
change in
2 and
1 protein levels and
ouabain binding, but also decreased
2 levels in hindlimb
to 0.36 ± 0.6 of levels for vehicle-treated controls. Thus, on
the whole, the changes in
2 and
1 mRNA
levels in hindlimb can account for the changes in these subunit protein levels and ouabain binding measured in individual muscles.
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DISCUSSION |
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In the previous study by Dorup and Clausen (11), the
infusion of graded doses of dexamethasone (0.02-0.1 mg/kg per day) increased the binding of the cardiac glycoside
[3H]ouabain to skeletal muscle biopsies by 22-48%.
However, the molecular mechanism responsible for this change in ouabain
binding was not determined. Ouabain is not likely to bind to all the
pumps in a muscle biopsy preparation because it will have access only to active sodium pumps in the sarcolemma (25) and not to
the endosomal pools of 2-type pumps identified in
oxidative muscle fibers (29). These sequestered pumps are
known to shuttle between sarcolemma and endosomal pools in response to
insulin (23, 29), so they are physiologically relevant. In
addition, the fairly ubiquitous
1 has such a low
affinity for ouabain that it is termed "ouabain resistant" and may
not significantly bind [3H]ouabain under these assay
conditions (25). In addition, there are likely to be
significant pools of
1 in muscle: we recently calculated
that the ratio of
1 to
2 protein levels
was near 1 by using regression analysis of the relative changes in
1- and
2-isoforms vs.
1-isoform in soleus or vs.
2-isoform in white gastrocnemius in response to potassium deprivation
(41). These observations prompted us to determine the
effect of dexamethasone treatment on specific
-catalytic and
-glycoprotein pool sizes in skeletal muscle.
After 14 days of dexamethasone infusion, the increases in
[3H]ouabain binding (40-48%) and in the
ouabain-sensitive 2-isoform abundance (53%) were
similar in soleus and EDL (Fig. 2). This finding suggests that there
are similar changes in the pools of
2-type pumps present
at the plasma membrane and in endosomes. In contrast, in gastrocnemius
and diaphragm, the increase in
2 protein abundance
exceeded the increase in [3H]ouabain binding by 39% and
53%, respectively. One interpretation is that in these muscles,
dexamethasone stimulates a larger increase in the ouabain-inaccessible
internal pools of
2 than in the ouabain-accessible sarcolemmal pool. Another possibility is that there are pools of
unassembled inactive
2 in these muscles. In fact, in
diaphragm, the change in expression of
1 (1.39 ± 0.07) protein after dexamethasone treatment is less than the increase
in changes in
1 or
2 levels (1.65 ± 0.07 and 1.78 ± 0.09, respectively), suggesting the possibility that
1 (not the
-subunits) is rate limiting for the
production of heterodimers capable of moving to the cell surface and
binding [3H]ouabain and suggesting the corollary that
there may be spare unassembled
1- and
2-subunits that would not bind ouabain.
Dexamethasone stimulates an increase in 2 protein
independent of fiber type. Conversely,
1 abundance is
increased significantly only in diaphragm (to 1.65 of control). There
was also an increase in the mean
1 expression in
gastrocnemius, but the variability of the response was large. We
verified that these muscle specific changes were not loading artifacts
by reprobing the blots with calsequestrin and glucose transporter 4 (19), neither of which showed an analogous increase after
glucocorticoid treatment (not shown).
The glucocorticoid-mediated increase in relative abundance of sodium
pump subunits, whether direct or indirect, is a result of either
increased subunit synthesis or decreased subunit degradation. The
analysis of the mRNA levels in whole hindlimb indicates that the change
in 2 mRNA, >6-fold, can more than account for the change in the
2 protein levels observed in the
individual muscles and that the change in
1 mRNA, ~1.5
fold, is equivalent to the change in
1 protein levels.
Thus we conclude that the changes in
2 and
1 protein levels are mediated by increased synthesis driven by increases in their mRNA levels.
The relative changes in 2 and
1 mRNA vs.
protein levels demonstrate that the large induction of
2
is not translated into a similarly enlarged stable pool of
2 and suggest that the changes in
1
synthesis may limit the magnitude of increase in functional
2
1 protein heteromers. In other words,
these comparisons suggest that formation of
2
1 heteromers stabilizes
2
during or after translation and that unassembled
2-subunits are degraded. We have previously described
similar discoordinate changes in sodium pump subunit protein vs. mRNA
levels in heart and muscle in response to thyroid hormone (T3)
treatment (3, 16). In rat muscle, T3 increased
2 and
2 mRNA five- and fourfold but
increased protein levels only three- and twofold, respectively,
suggesting an inefficiency in the translation and assembly of the
heteromers or an accompanying T3-driven increase in
degradation rate (3). In the rat heart, 8- to 16-day
thyroid hormone treatment increased
1 mRNA and protein levels 2- to 3-fold but increased
2 mRNA and protein
levels 6- and 15-fold and
1 mRNA and protein levels 15- and 3-fold, respectively. The very discoordinate changes in
2 mRNA vs. protein levels are not easily explained but
suggest that
1 levels are rate limiting in the heart and
that the 15-fold increases in
1 mRNA stabilize nascent
2 proteins by
assembly. This effect would
decrease the degradation rate of
2 during T3 treatment
and thus amplify the 6-fold increase in
2 mRNA into a
15-fold increase in
2 protein levels during T3 treatment
(16). Similarly, if
1 mRNA is rate limiting
for the synthesis of sodium pumps in muscle, the dexamethasone-driven increase could also explain the tendency for
1 protein
increase in gastrocnemius and diaphragm in the absence of a change in
1 mRNA by increasing
1
1
heteromer formation and decreasing
1 degradation rate.
Additional support for this theory comes from experiments in rat
alveolar epithelial cells, where dexamethasone increases only
1 mRNA expression but increases both
1
and
1 protein levels relative to controls
(4), suggesting increased stabilization of
1
1 during or after translation by
assembly of nascent
1 with an increased pool of newly
synthesized
1.
The significant dexamethasone-driven decrease in 2 mRNA
was unexpected given the unchanged levels of
2 in
gastrocnemius and the tendency for
2 increase in EDL,
the two muscles in which
2 was detected at the protein
level. By employing the reasoning developed to explain the
discoordinate changes in
2 and
1, we can
postulate that
2 mRNA is not rate limiting for the
formation of sodium pump heteromers and that
2 mRNA may
be in such excess in the control state that decreases in
2 mRNA levels by 50% by dexamethasone may not impact
the formation of stable
1
2 or
2
2 heteromers in these muscles. While
both EDL and gastrocnemius are represented in the hindlimb sample used
for preparation of the RNA, it will be important to measure the
2 mRNA levels and protein levels in the same specific
muscles to further test this idea that
2 mRNA is not
rate limiting for the formation of sodium pumps in muscle.
Dexamethasone regulation of sodium pump subunit mRNA levels appears to
be quite tissue or cell dependent. Orlowski and Lingrel (31) determined in primary cultures of myocardiocytes that
dexamethasone treatment induced 2 mRNA levels but did
not influence
1,
3, or
1
mRNA levels (
2 not tested) (31). While the
increase in
2 (but not
1) mRNA agrees
with the finding in this study, the lack of an effect on
1 mRNA is in contrast to most other studies, including
the current study. The increase in
1 is predicted
because there are glucocorticoid receptors in muscle, and the
Na+-K+-ATPase
1 gene promoter
contains a glucocorticoid-responsive element that is specifically
activated by glucocorticoid receptors (9). In addition,
while this study in skeletal muscle as well as the others discussed
(4, 31) found no direct dexamethasone regulation of
1, there are a number of reports in the literature that
do support specific glucocorticoid activation of
Na+-K+-ATPase
1 expression,
especially in epithelia. Wang et al. (44) provided
evidence that glucocorticoids regulate
1 transcription in infant rat kidney. Lee et al. (24) also observed a
glucocorticoid-driven increase in
1 and
1
mRNA and protein in cultured adult proximal tubule and, furthermore,
provided evidence that the response is indirect, requiring ongoing
protein synthesis.
When examining regulators of sodium pump expression, it is always important to ask whether the effects are mediated indirectly by changes in cell [Na+] or [K+]. In the previous, related study (11), 7-day dexamethasone infusion did not significantly change plasma [Na+] or [K+] and had no, or minute, effects on tissue cations in this same panel of muscles. Furthermore, no direct in vitro effects of dexamethasone on 22Na influx were detected, indicating that glucocorticoid-driven increases in sodium pump isoform mRNAs and proteins in skeletal muscle are not driven by primary changes in cell cations. These results agree with those of earlier studies in rat heart (21) and outer medullary kidney tubules (36), in which investigators demonstrated that glucocorticoid-mediated increases in sodium pump activity were not mediated by changes in intracellular Na+ or K+ levels.
Our previous studies of ionic and hormonal regulation of sodium pump
isoform expression in muscle have established that 2 is
highly regulated while
1 expression is not, that
hypokalemia and hypothyroid status depresses
2 but not
1 abundance, and that thyroid hormone increases
2 but not
1 abundance (3, 41,
42). Similarly, glucocorticoid treatment increases
2 expression in all muscles tested but increases
1 only in diaphragm. These findings provide the
rationale for future investigations on how multiple stimuli affect
muscle sodium pump expression. For example, chronic obstructive lung
disease patients are often given diuretic therapy that is known to
provoke hypokalemia, which depresses muscle
2
expression, along with glucocorticoids, which are shown here to raise
muscle
2 expression. Because muscle sodium pump
expression is a critical regulator of plasma potassium and an important
determinant of muscle endurance, the net influence of these multiple
therapies may have important health consequences that should be understood.
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ACKNOWLEDGEMENTS |
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This work was supported by National Science Foundation Grant IBN 9S13958, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34316, and an American Heart Association-Western Affiliate Grant-in-Aid (to A. A. McDonough) as well as grants from the Nordic Insulin Foundation, The NOVO Industry Research Foundation, the Alfred Benzons Foundation, and the Danish Biomembrane Research Center (to I. Dorup).
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. A. McDonough, Dept. of Physiology and Biophysics, Univ. of Southern California School of Medicine, 1333 San Pablo St., Los Angeles, CA 90089-9142.
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. Section 1734 solely to indicate this fact.
Received 16 September 1999; accepted in final form 18 September 2000.
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