Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163
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ABSTRACT |
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Skeletal muscle
Na+-K+-2Cl cotransporter (NKCC)
activity provides a potential mechanism for regulated K+
uptake.
-Adrenergic receptor (
-AR) activation stimulates
skeletal muscle NKCC activity in a MAPK pathway-dependent manner. We
examined potential G protein-coupled pathways for
-AR-stimulated
NKCC activity. Inhibition of Gs-coupled PKA blocked
isoproterenol-stimulated NKCC activity in both the slow-twitch soleus
muscle and the fast-twitch plantaris muscle. However, the
PKA-activating agents cholera toxin, forskolin, and 8-bromo-cAMP
(8-BrcAMP) were not sufficient to activate NKCC in the plantaris and
partially stimulated NKCC activity in the soleus.
Isoproterenol-stimulated NKCC activity in the soleus was abolished by
pretreatment with pertussis toxin (PTX), indicating a
Gi-coupled mechanism. PTX did not affect the
8-BrcAMP-stimulated NKCC activity. PTX treatment also precluded the
isoproterenol-mediated ERK1/2 MAPK phosphorylation in the soleus,
consistent with NKCC's MAPK dependency. Inhibition of
isoproterenol-stimulated ERK activity by PTX treatment was associated
with an increase in Akt activation and phosphorylation of Raf-1 on the
inhibitory residue Ser259. These results demonstrate a
novel, muscle phenotype-dependent mechanism for
-AR-mediated NKCC
activation that involves both Gs and Gi
protein-coupled mechanisms.
potassium; mitogen-activated protein kinases; protein kinase A; pertussis toxin; Raf-1
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INTRODUCTION |
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SKELETAL MUSCLE
IS WELL RECOGNIZED for its contribution to the regulation
of plasma K+ concentration via insulin-,
catecholamine-, and contraction-regulated Na+-K+ ATPase activity (4, 8, 20,
27). Even during moderate exercise, however, total calculated
K+ efflux can exceed the capacity of the sodium pump for
K+ reuptake in rat muscle (9). In addition,
the complex kinetics of increased K+ reuptake by exercising
muscle cannot be solely explained by simple stimulation of the sodium
pump by the ion substrates (14). There is accumulating
evidence for additional inward K+ transport mechanisms in
skeletal muscle (12, 21, 31). We have reported that one of
these mechanisms, bumetanide-sensitive Na+-K+-2Cl cotransporter (NKCC)
activity, contributes as much as one-third of the total inward
K+ flux in skeletal muscle in response to catecholamines
and contractile activity (12, 30, 31). Thus skeletal
muscle NKCC activity may contribute significantly to the regulation of
plasma K+ concentration.
We are just beginning to understand the intracellular signaling
pathways that regulate NKCC activity in skeletal muscle. Understanding these mechanisms provides a broader perspective on the manner in which
muscle responds to its environment. The isoproterenol-stimulated cotransporter activity is dependent on mitogen-activated protein kinase
kinase (MAPKK1/2, or MEK1/2) activity that stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2) MAPK (12, 31).
The -adrenergic agonist isoproterenol acts through receptors that couple to heterotrimeric GTP-binding proteins (G proteins, G
) to elicit its effects (5, 32). The activation of the
ERK1/2 cascade has been established for G proteins of diverse classes, including stimulatory (Gs) and inhibitory (Gi)
G proteins (22, 28). In skeletal muscle, isoproterenol
stimulates
2-adrenergic receptors (
2-AR).
Depending on cell type, it is thought that the
2-AR can
stimulate ERK MAPK by both Gs and Gi proteins
(11, 18, 26). In the case of Gs, isoproterenol
is reported to activate the ERK pathway through the cascade of adenylyl
cyclase, protein kinase A (PKA), Raf, and MEK1/2 (10, 26).
There is also evidence for a
2-AR-stimulated, pertussis
toxin (PTX)-sensitive Gi pathway (including also
Go and Gt family members) for stimulation of
MAPKK1/2 and ERK1/2 MAPK through a Src, phosphatidylinositol 3-kinase
(PI 3-K) 110
/Ras/Raf-1 complex (11, 28). In
addition to the linear pathways initiated either by Gs or
Gi proteins, sequential activation of both G
protein-coupled pathways has also been reported. Daaka et al.
(11) describe a mechanism for a PKA-dependent switch of
2-AR signaling from Gs to Gi to
stimulate MAPK in HEK-293 cells. It is relevant, therefore, to wonder
which mechanisms are involved in isoproterenol-stimulated, MAPK
pathway-dependent NKCC activation in skeletal muscle. In particular, we
were curious as to whether the mechanism exhibited phenotypic
differences between slow-twitch and fast-twitch muscle.
In this study, we investigated whether inhibition of key components of
putative 2-AR-stimulated pathways affected
isoproterenol-stimulated, MAPK pathway-dependent NKCC activity in
skeletal muscle. Our results indicate that muscle fiber
phenotype-dependent signaling involves both Gs and
Gi proteins.
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METHODS |
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Animal care and muscle preparation. Female Sprague-Dawley rats (100-150 g) were used for all experiments. The rats were housed in light- and temperature-controlled quarters where they received food and water ad libitum. Animals were randomly assigned to experimental groups, and all animals were handled identically. The rats were anesthetized with pentobarbital sodium (45 mg/kg ip) for tissue removal. Ankle extensor muscles, soleus (~87% type I and 13% type II), and plantaris (~9% type I and 91% type II) (3) were used for the experiments. Soleus and plantaris muscles were removed by careful dissection of the proximal tendons at the muscle origin and severing of the distal tendon. The muscles were placed in oxygenated Krebs-Ringer buffer at 25°C in preparation for further treatment (see below). The Animal Care and Use Committee of the University of Tennessee, Health Science Center, approved all procedures.
Materials. 86RbCl was from New England Nuclear (Boston, MA). Isoproterenol, bumetanide, and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were purchased from Sigma-Aldrich (St. Louis, MO). PTX and cholera toxin were obtained from List Biological Laboratories (Campbell, CA). Forskolin, 8-bromo-cAMP (8-BrcAMP), and H-89 were from CalBiochem (La Jolla, CA). GW5074 was from Tocris Cookson (Ellisville, MO). Phosphospecific antibody to ERK, anti-ERK-2, and anti-Raf-1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit phosphospecific Akt Ser473, anti-Akt, and anti-Raf-1 Ser259 antibodies and HRP-conjugated anti-rabbit IgG antibody assay kits for MAPK and Akt activity were from New England Biolabs (Beverly, MA). Anti-Raf-1 Ser338 antibody was from Upstate Biotechnology (Lake Placid, NY). Enhanced chemiluminescence (ECL) kit was from Amersham Life Sciences (Piscataway, NJ).
Muscle incubation and 86Rb influx constant
calculation.
Each soleus and plantaris muscle was attached with 4-0 surgical silk
suture to glass wands for rapid transfer among bathing solutions. The
muscles were preincubated for 15 min at 30°C in preincubation medium
[oxygenated Krebs-Ringer containing bumetanide (105 M)
or vehicle (DMSO) for the contralateral muscle]. For stimulation, muscles were taken directly to incubation medium (oxygenated
Krebs-Ringer containing 1 µCi/ml 86Rb and either
bumetanide or vehicle) at 30°C, which contained 30 µM
isoproterenol, 0.5 mM 8-BrcAMP, 20 µM forskolin, or 5 µg/ml cholera
toxin. Incubation was for 10 min. Muscles were immediately washed with
ice-cold 0.9% saline solution. After being washed, the muscles were
blotted, weighed, and homogenized in 2 ml of 0.3 M trichloroacetic acid
(TCA). 86Rb uptake by the muscle was measured by Cerenkov
counting (Beckman LS 5000 TA). 86Rb transport was expressed
as a rate constant, as described previously (31).
NKCC activity. The bumetanide-sensitive 86Rb uptake was used as an index of NKCC activity, as described previously (31). The bumetanide-sensitive portion of 86Rb uptake was calculated by subtracting the bumetanide treatment transport rate constant for the muscle of one hindlimb from the vehicle treatment rate constant of the contralateral muscle. Thus the bumetanide and vehicle treatments were statistically paired.
PKA assay.
Skeletal muscle was excised from anesthetized rats and incubated for 25 min in oxygenated Krebs-Ringers solution. After incubation, the tissues
were treated according to the protocol described by Keely et al.
(17). Briefly, after incubation, tissues were immediately frozen with Wollenberger tongs that had been cooled with liquid nitrogen. The muscles were pulverized in liquid nitrogen by using a
percussion mortar, which had also been cooled with liquid nitrogen. The
powder was stored at 80°C until use. The powdered tissue was
suspended on ice in three times its weight of 10 mM potassium phosphate
buffer containing 10 mM EDTA and 0.5 mM 1-methyl-3-isobutylxanthine, at
pH 6.8, and homogenized with a Teflon pestle. The homogenate was
immediately centrifuged at 12,000 g for 20 min at 4°C. The supernatant was diluted fivefold with the homogenization buffer. PKA
activity was detected by using the PepTag Assay for nonradioactive detection of cAMP-dependent protein kinase activity (Promega). Briefly,
5 µl of reaction buffer (100 mM Tris · HCl, 50 mM
MgCl2, and 5 mM ATP, pH 7.4), 5 µl of PepTag A1 peptide
(0.4 µg/µl kemptide), 5 µl of activator solution (5 µM cAMP,
100 µM forskolin, or 2.5 mM 8-BrcAMP), and 5 µl of deionized water
were mixed on ice. The tubes were removed from the ice and incubated at
30°C for 1 min. Sample (5 µl) was added to the tube and incubated
at 30°C for 30 min. The reaction was stopped by placing the tube in a
boiling water bath for 10 min. Eighty percent glycerol (1 µl) was
added to each sample, followed by electrophoresis on an 0.8% agarose gel (50 mM Tris · HCl, pH 8.0) for analysis. To block the
activation of PKA, 5 µl of H-89 (10 µM) were substituted for the
deionized water in the reaction mixture.
Immunoblotting. Whole muscle was preincubated and incubated as described for the 86Rb assay. Incubation medium included no 86Rb. After incubation with isoproterenol (as before), the muscles were placed in ice-cold lysis buffer (10 mM Tris · HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 30 mM Na4P2O7, 50 mM NaF, 100 µM Na3VO4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml antipain, and 1 µg/ml pepstatin A), homogenized with a Teflon pestle, and centrifuged at 4°C for 5 min at 5,000 g. The protein concentration of the supernatant was measured by the bicinchoninic acid assay (Pierce, Rockford, IL). Equal amounts of protein were mixed with SDS denaturing buffer, warmed to 95°C for 5 min, and electrophoresed on a 10% SDS-PAGE gel. The gels were electroblotted onto polyvinylidene difluoride membrane with the semidry blotter from Buchler Instruments (Fairfield, NJ). The membrane was incubated overnight at 4°C in the Western blocking buffer (1.5 mM NaH2PO4, 8 mM Na2HPO4, 0.15 M NaCl, and 0.5% Tween 100, pH 7.4) supplemented with 3% BSA. The membranes were then incubated at room temperature for 1.5 h in blocking buffer containing 1% BSA and the specific antibody (1:1,000). Phosphospecific antibodies to ERK1/2 dually phosphorylated on Thr202 and Tyr204 to Akt phosphorylated on Ser473 were used to detect the catalytically activated forms of the kinases. Phosphospecific Raf-1 antibodies recognized Raf-1 phosphorylated only at Ser259 or Ser338. The membranes were probed with HRP-conjugated anti-rabbit or anti-mouse IgG for 1 h at room temperature, and immunoreactive proteins were detected by using ECL reagents. After determination of ERK1/2, Raf-1, or Akt phosphorylation, membranes were stripped for 30 min at 50°C, washed extensively, and used to determine total ERK1/2, Raf-1, or Akt protein expression with an appropriate antibody (1:1,000). Antibody signals were quantitated by video densitometry of film images of the blots. The extent of ERK or Akt phosphorylation was determined by comparing the amount of phosphorylated protein with the total ERK, Raf-1, or Akt expression, respectively.
Kinase assays for MAPK/ERK and Akt activity. The activity of MAPK and Akt was determined by using kinase assay kits from New England Biolabs. Homogenates of the muscles were prepared as described earlier. Supernatants of muscle lysates were mixed with immobilized phosphospecific p44/42 MAPK or Akt monoclonal antibody and incubated with gentle rocking overnight at 4°C. The beads were washed three times with ice-cold lysis and kinase buffers. The kinase assays were performed by incubating the suspended pellet in kinase buffer containing 200 µm ATP and 2 µg of Elk-1 or GSK-3 fusion protein for MAPK and Akt assays, respectively, for 30 min at 30°C. The samples were analyzed by 12% SDS-PAGE. Phospho-Elk-1-Ser383 or phospho-glycogen synthase kinase-3-Ser21/9 was detected with phosphospecific antibody (1:1,000 dilution) by using Western blot analysis.
Data analysis and statistics. Comparisons within and among treatments were made by analysis of variance. Differences between treatments were considered significant at P < 0.05. Data are reported as means ± SE.
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RESULTS |
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Involvement of Gs-stimulated PKA in regulation of
NKCC activity.
As we have previously reported (12, 31), isoproterenol
activation of soleus and plantaris muscle
-AR stimulated
bumetanide-sensitive 86Rb uptake (Fig.
1A); bumetanide is a specific
inhibitor of NKCC activity. It is well known that activation of PKA
through a G
s-coupled mechanism is an important element
for signal transduction by the
2-AR, the predominant
adrenergic receptor expressed in skeletal muscle (16).
Preincubation of the muscles with 2 µM H-89, an inhibitor of PKA
(7), abolished the isoproterenol-stimulated NKCC activity
in both the soleus and plantaris muscles (Fig. 1A). To
determine whether PKA activation was sufficient to stimulate NKCC
activity, we used three chemically and mechanistically different agents
to activate PKA: cholera toxin, forskolin, and 8-BrcAMP. These agents
significantly stimulated bumetanide-sensitive 86Rb uptake
only in the predominantly slow-twitch soleus muscle (Fig.
1B). The increased NKCC activity caused by direct activation of PKA was sensitive to H-89 (not shown). However, the NKCC activity stimulated by the PKA activators was less than that stimulated by
isoproterenol. To verify that these agents did indeed stimulate PKA
activity in muscle, we performed an in vitro PKA assay using skeletal
muscle lysates. Consistently, 8-BrcAMP, forskolin, and cAMP (as a
control) stimulated PKA activity in whole muscle homogenates of both
the soleus and plantaris muscles, and H-89 abolished this activity
(Fig. 2). These results indicate that PKA
activation is necessary for
-AR-stimulated NKCC activity but appears
to be only partially sufficient in the predominantly slow-twitch soleus
muscle.
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PTX sensitivity of stimulated NKCC activity.
2-AR agonists can activate both Gs- and
Gi-coupled mechanisms, and Gs-stimulated PKA
activity is required for the Gi activation in some cell
types (11, 26, 32). These reports also emphasize that
involvement of a PTX-sensitive Gi protein is a key step in the activation of the ERK1/2 MAPK cascade. Because
isoproterenol-stimulated NKCC activity is MAPK dependent (12,
31), it was of interest to examine the effect of PTX treatment
on isoproterenol-stimulated NKCC activity. Thirty hours after injection
of PTX into rats (48 µg/kg body wt ip), isoproterenol-stimulated,
NKCC-mediated 86Rb uptake was abrogated in the soleus
muscle but not in the plantaris muscle (Fig.
3). Total isoproterenol-stimulated
86Rb uptake decreased by the value of the
bumetanide-sensitive portion (not shown). The portion of the NKCC
activity that could be stimulated by 8-BrcAMP in the soleus muscle was
not affected by the PTX treatment (Fig. 3). PTX treatment affected
neither total nor bumetanide-sensitive 86Rb uptake in the
unstimulated soleus and plantaris muscles (not shown).
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PTX regulation of isoproterenol-mediated ERK1/2 activation.
We have recently reported that inhibition of muscle MAPKK1/2 (MEK1/2)
abolishes isoproterenol-stimulated NKCC activity and ERK1/2
phosphorylation (31). To investigate whether the PTX treatment that abolished isoproterenol-stimulated NKCC activity in
soleus muscle (Fig. 3) affected -AR-dependent ERK signaling pathways, we injected animals with PTX and assessed the ability of
isoproterenol to activate ERK1/2. Isoproterenol stimulation of normal
muscle resulted in a significant increase in ERK phosphorylation, with
a greater extent observed in slow-twitch muscle (P < 0.05) (Fig. 4A). Consistent
with the ERK phosphorylation, ERK1/2 activity was also stimulated, as
determined by in vitro kinase assay by using Elk-1 as the substrate
(Fig. 4B). PTX treatment prevented isoproterenol-stimulated
ERK phosphorylation and activity in the soleus muscle but not in the
plantaris muscle (Fig. 4). PTX treatment did not change the basal level
of ERK phosphorylation, ERK activity, or total ERK protein expression
in either soleus or plantaris muscles (not shown).
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DISCUSSION |
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-AR activation stimulates NKCC-mediated K+
transport in smooth (1) and striated muscles (2, 12,
31). In addition, it has been shown that stimulation of NKCC
activity is MAPK dependent in striated muscle (2, 31). The
present study demonstrates that
-AR stimulation can activate fiber
phenotype-specific signaling cascades that involve both Gs-
and Gi-coupled processes. Furthermore, the data indicate
that the Gi-coupled signals may suppress an Akt pathway
that inhibits Raf-1 activation upstream of ERK1/2 MAPK. We discuss
below the dual G protein-coupled signaling mechanisms that appear to be
involved in the regulation of skeletal muscle NKCC activity.
It is well established that one consequence of -AR activation is the
dissociation of the G
s subunit from the heterotrimeric G
protein complex, with subsequent activation of adenylyl cyclase and
production of cAMP. cAMP can activate PKA by causing dissociation of
the inhibitory subunit from the catalytic subunit. Our data show that
H-89, an inhibitor of PKA, abolished isoproterenol-stimulated NKCC
activity (Fig. 1A). Whether PKA stimulation was also
sufficient for NKCC stimulation was addressed by stimulation of PKA
activity independent of the
-AR G protein-coupled mechanism. NKCC
activity was stimulated only in the predominantly slow-twitch soleus
muscle (Fig. 1B). Phenotype differences in
-AR-stimulated
PKA pathway components have been noted previously. In rat skeletal
muscle,
-AR density significantly correlates with muscle oxidative
capacity (16, 29). Moreover, isoproterenol-stimulated
adenylyl cyclase activity is considerably greater in slow-twitch
oxidative muscle (29). In agreement, Hoover et
al. (15) showed that the slow-twitch soleus
muscle expresses twice the PKA catalytic activity as fast-twitch muscle. Furthermore, isoproterenol-mediated ERK1/2 MAPK activation was
significantly greater in the soleus muscle than in the plantaris muscle
(Fig. 4), which may reflect phenotype-related differences in ERK
expression (12). Thus clear phenotypic differences are evident in the expression and activity of the
-AR, PKA, and ERK MAPK, and likely many other phenotypic differences in signal pathways exist. As an example, despite the necessity for PKA activation for NKCC
activity in both the soleus and plantaris muscles, PKA activation
through receptor-independent pathways was partially sufficient for NKCC
activation only in the slow-twitch soleus muscle (Figs. 1 and 3).
Cholera toxin (which stimulates G
s activity), forskolin
(which upregulates adenylyl cyclase), and 8-BrcAMP were unable to
completely mimic the isoproterenol-stimulated NKCC activity (Fig.
1B). Although the evidence that PKA-stimulating agents
cannot mimic the effects of isoproterenol on ERK MAPK is not new
(10), we raise several possibilities that could explain
the incomplete NKCC activation by the PKA stimulators. It is entirely
possible that
-AR activation stimulates covalent modification of
PKA, in addition to the allosteric activation of PKA by cAMP.
Indeed, phosphatidylinositol 3,4,5-trisphosphate
(PIP3)- dependent phosphoinositide-dependent protein kinase-1 (PDK-1) activity has been implicated in the
phosphorylation of Thr197 in the activation loop of the PKA
catalytic subunit (6). Our recent results provide support
for this possibility. Inhibition of PI 3-K, which produces
PIP3, decreases both isoproterenol-stimulated ERK1/2
phosphorylation and NKCC-mediated transport by half in the soleus
muscle (12). Another possibility is simply a physically incomplete activation of PKA by the PKA activators, although it is
curious that all three mechanistically and chemically dissimilar compounds produced strikingly similar results (Fig. 1B). Yet
another possibility, and one which the data here support, is that
additional pathways participate in regulation of ERK-dependent NKCC
activity in the soleus muscle, as discussed below.
PTX treatment abolished isoproterenol-mediated ERK phosphorylation and
NKCC activation in the soleus muscle but not in the plantaris muscle.
PTX causes ADP ribosylation of the heterotrimeric Gi/o/t
proteins, thereby preventing receptor-mediated dissociation of
G subunits. Both G
i and G
are known to
activate Raf-1 and, subsequently, the ERK cascade (11, 13,
28). The data for the soleus muscle consistently show the
necessity of active Gi proteins to stimulate the Raf-1/ERK
MAPK pathway, in addition to a Gs mechanism. Whereas
isoproterenol can activate Raf-1 through a PTX-insensitive
phosphorylation on Ser338, active G
i/o/t
subunits are required to prevent hyperphosphorylation of Raf-1 on
Ser259, a modification that inhibits Raf-1 kinase activity.
Recently, Daaka et al. (11) suggested a switching
mechanism for ERK activation by which G
s-mediated
activation of PKA is required for activation of PTX-sensitive
G
i/o/t proteins and, subsequently, the ERK MAPK cascade.
However, neither 8-BrcAMP-induced stimulation of NKCC activity nor
isoproterenol-stimulated phosphorylation (activating) of Raf-1 on
Ser338 were PTX sensitive (Figs. 3 and 5). It appears that
parallel pathways, PTX-insensitive and PTX-sensitive, activate NKCC in the soleus muscle (Fig. 9). Taken together, our findings corroborate previous evidence that direct activation of
-AR is necessary for the
complete cascade of signaling events. Furthermore, we propose that
activation of PTX-sensitive proteins is a prerequisite for suppression
of inhibitory inputs on the ERK MAPK pathway and NKCC activity. Support
for the inhibitory influence on the Raf-1/ERK MAPK pathway comes from
our recent data that demonstrate an Akt-dependent inhibition of the ERK
cascade (and thus NKCC activity) (12). It has been shown
that active Akt markedly inhibits Raf-1 activity by inducing the
phosphorylation of Raf-1 on Ser259, preventing ERK
activation (33). Akt activity was increased, and
inhibition of Raf-1 prevailed in the PTX-treated soleus muscle (Fig.
5). This was accompanied by the inability to stimulate ERK1/2 and NKCC
activity (Figs. 3 and 4).
On the basis of these data, we propose that Gs-mediated PKA
activity is necessary, but not fully sufficient, for maximal
-AR-mediated stimulation of MAPK pathway-dependent NKCC in skeletal
muscle. In addition to PKA activation,
-AR activation in the soleus
muscle apparently prevents activation of Akt through PTX-sensitive
Gi/o/t proteins (Figs. 7 and 9). Active Akt can suppress
Raf-1/ERK MAPK pathway stimulation and subsequent NKCC activity. In the
predominantly fast-twitch plantaris muscle, PTX-sensitive mechanisms
are not involved in the regulation of MAPK pathway-dependent NKCC.
Together, these data demonstrate not only fundamental differences
between fast- and slow-twitch phenotypes for controlling K+
transport but also broader implications for differences in signaling that may be important in muscle plasticity and disease.
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ACKNOWLEDGEMENTS |
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We are grateful to L. A. Malinick for assistance with publication graphics.
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FOOTNOTES |
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This research was supported by an American Diabetes Association Research Award and American Heart Association Grant-In-Aid to D.B. Thomason.
Present address of J. A. Wong: New World Science and Technology, Inc., Silver Spring, MD 20910.
Address for reprint requests and other correspondence: D. B. Thomason, Dept. of Physiology, College of Medicine, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (E-mail: thomason{at}physio1.utmem.edu).
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.
May 29, 2002;10.1152/ajpcell.00096.2002
Received 4 March 2002; accepted in final form 23 May 2002.
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