Duality of G protein-coupled mechanisms for beta -adrenergic activation of NKCC activity in skeletal muscle

Aidar R. Gosmanov, Jennifer A. Wong, and Donald B. Thomason

Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Skeletal muscle Na+-K+-2Cl- cotransporter (NKCC) activity provides a potential mechanism for regulated K+ uptake. beta -Adrenergic receptor (beta -AR) activation stimulates skeletal muscle NKCC activity in a MAPK pathway-dependent manner. We examined potential G protein-coupled pathways for beta -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 beta -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


    INTRODUCTION
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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 beta -adrenergic agonist isoproterenol acts through receptors that couple to heterotrimeric GTP-binding proteins (G proteins, Galpha beta gamma ) 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 beta 2-adrenergic receptors (beta 2-AR). Depending on cell type, it is thought that the beta 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 beta 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) 110gamma /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 beta 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 beta 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.


    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 (10-5 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).

The mode of 86Rb uptake, and phosphorylation and activation of protein kinases upon stimulation, were evaluated by adding inhibitors of intracellular signaling pathways. For specific inhibition of PKA, we preincubated the muscle with H-89 in concentration 2 µM (7). To inhibit Raf-1 kinase, we preincubated the muscle with 1 µM GW5074 (19). To inhibit activation of PTX-sensitive Gi/o/t proteins, rats weighing ~125 g were injected with PTX (6 µg/rat ip) dissolved in sterile isotonic saline (0.5 ml) (24). Control rats received the same amount of vehicle buffer as PTX-treated rats. At 30 h after PTX injection, muscles were dissected and 86Rb transport was measured as described above.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Involvement of Galpha s-stimulated PKA in regulation of NKCC activity. As we have previously reported (12, 31), isoproterenol activation of soleus and plantaris muscle beta -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 Galpha s-coupled mechanism is an important element for signal transduction by the beta 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 beta -AR-stimulated NKCC activity but appears to be only partially sufficient in the predominantly slow-twitch soleus muscle.


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Fig. 1.   PKA activity was necessary, but not sufficient for, isoproterenol (Iso)-stimulated, bumetanide-sensitive 86Rb uptake. A: inhibition of PKA activity with H-89 prevented the Iso-stimulation of Na+-K+-2Cl- (NKCC) activity. Muscles were preincubated with the PKA inhibitor H-89 for 15 min before Iso stimulation and during the 10 min of Iso-stimulated 86Rb uptake. B: muscles stimulated with PKA-stimulating agents cholera toxin (CTX, 5 µg/ml), forskolin (20 µM), or 8-bromo-cAMP (8-BrcAMP) (0.5 mM) during the 10-min 86Rb uptake period could not evoke maximal NKCC activation. Data are means ± SE; n = 6-8 muscle pairs per point. *,dagger P < 0.05 relative to the basal level and Iso-stimulated value in soleus muscle, respectively.



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Fig. 2.   PKA activity was similar in both soleus (A) and plantaris (B) muscles. Whole muscle homogenates of soleus and plantaris muscles were subjected to stimulation with cAMP, 8-BrcAMP, or forskolin. The PKA activity in the homogenates phosphorylated the fluorescent kemptide. Inclusion of H-89 in the assay inhibited the PKA activity.

PTX sensitivity of stimulated NKCC activity. beta 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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Fig. 3.   Pertussis toxin (PTX) treatment inhibited Iso-stimulated, bumetanide-sensitive 86Rb uptake by the soleus muscle. PTX was injected into rats 30 h before the ex vivo experiments. The NKCC stimulators Iso and 8-BrcAMP were added during the 10-min 86Rb uptake period. PTX treatment abrogated the Iso-stimulated NKCC activity but did not affect the 8-BrcAMP-mediated NKCC activity in soleus muscle. Data are means ± SE; n = 10 muscle pairs per point. *,dagger P < 0.05 relative to the basal level and Iso-stimulated value in soleus muscle, respectively.

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 beta -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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Fig. 4.   PTX treatment prevented Iso-induced ERK1/2 phosphorylation and activation in the soleus muscle. PTX-pretreated muscles (see legend of Fig. 3) were stimulated for 10 min with 30 µM Iso. After treatment, muscles were immediately clamp frozen with liquid nitrogen and homogenized. A: equal amounts of protein (25 µg) were used for Western blot analysis with a phosphospecific ERK1/2 antibody, followed by stripping and reprobing with an anti-ERK-2 antibody. Data are the densitometric ratio of phospho-ERK to total ERK normalized to the basal mean value (taken as 1.0). B: an in vitro kinase assay was performed as described in METHODS, and phosphorylated Elk-1 substrate was detected by immunoblotting with phosphospecific Elk-1 antibody. Data are the densitometric ratio of phospho-Elk-1 to total ERK normalized to the basal mean value (taken as 1.0). Representative blots are shown. Data are means ± SE of 6 muscles. *,dagger P < 0.05 relative to the basal and Iso-stimulated ERK activity in the soleus muscle, respectively.

To establish whether PTX treatment affected ERK activation through modulation of Raf-1, we measured Raf-1 phosphorylation on Ser338 and Ser259. Phosphorylation of Raf-1 on Ser338 activates Raf-1 (23, 25, 31). Raf-1 activation is required for ERK stimulation (18). In contrast, Raf-1 phosphorylation on Ser259 inhibits kinase activity by attracting protein 14-3-3 (18). Therefore, one measure of the activation of Raf-1 is the relative degree of phosphorylation on these residues. Stimulation of the soleus muscle with isoproterenol induced a 6.5-fold increase in the Raf-1 phosphorylation on Ser338, whereas plantaris muscle exhibited a weak (1.5-fold over basal level) enhancement of Raf-1 phosphorylation (Fig. 5). PTX treatment, shown in Fig. 4 to inhibit ERK phosphorylation, did not alter isoproterenol-induced Raf-1 phosphorylation on Ser338 or Raf-1 expression (Fig. 5). The kinase activity of Raf-1 declines with phosphorylation on Ser259. Stimulation of the muscles with isoproterenol nearly doubled the level of Raf-1 phosphorylation Ser259 in the soleus muscle and nearly tripled the level of Ser259 phosphorylation in the plantaris muscle (Fig. 6). Remarkably, the inhibition of PTX-sensitive G proteins led to a robust eightfold increase in the inhibitory Raf-1 phosphorylation on Ser259 in the isoproterenol-stimulated soleus muscle (Fig. 6). PTX alone did not change the extent of Raf-1 Ser259 phosphorylation (Fig. 6).


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Fig. 5.   Iso stimulation of Raf-1 phosphorylation on Ser338 was not PTX sensitive. Soleus and plantaris muscles were incubated as described in METHODS. The supernatants of muscle lysates (100 µg of protein) were subjected to SDS-PAGE and immunoblotted with an anti-phospho-Ser338 Raf-1 antibody, followed by stripping and reprobing the blots with an anti-total Raf-1 antibody. Plotted data are the densitometric ratio of phospho-Raf-1 to total Raf-1 normalized to the basal mean value (taken as 1.0). Iso increased the phosphorylation of Raf-1 on Ser338 to a greater extent in the soleus muscle. PTX treatment did not affect the increase in Raf-1 phosphorylation. Data are means ± SE of 6 muscles. *P < 0.05 relative to the basal value of Raf-1 phosphorylation on Ser338.



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Fig. 6.   Effect of PTX treatment on Raf-1 phosphorylation on Ser259. Soleus and plantaris muscles were incubated as described in Fig. 5. The supernatants of muscle lysates (100 µg of protein) were subjected to SDS-PAGE and immunoblotted with an anti-phospho- Ser259 Raf-1, followed by stripping and reprobing the blots with anti-total Raf-1 antibody (representative blots are shown on Fig. 5). Plotted data are the densitometric ratio of phospho-Raf-1 to total Raf-1 normalized to the basal mean value (taken as 1.0). Iso increased the phosphorylation of Raf-1 on Ser259 to a greater extent in the plantaris muscle. Unlike the plantaris muscle, PTX treatment resulted in a robust increase of Raf-1 inhibition in the Iso-treated soleus muscle. Data are means ± SE of 6 muscles. *,dagger P < 0.05 relative to the basal and Iso-stimulated level of Raf-1 phosphorylation on Ser259, respectively.

Recent studies have identified a negative regulatory role for protein kinase B/Akt in ERK MAPK pathway activation (25, 33). A well-recognized possibility for ERK inhibition is via Akt-dependent Raf-1 phosphorylation on Ser259. To establish whether PTX treatment affected ERK activation through Akt modulation, we examined the Akt activation by assessing the phosphorylation of Akt on Ser473 and Akt kinase activity. Isoproterenol alone did not significantly change either Akt Ser473 phosphorylation or Akt activity in the soleus muscle (Fig. 7) or plantaris muscle (not shown). Akt activity was measured by in vitro kinase assay by using GSK-3 as the substrate. PTX treatment increased Akt phosphorylation on Ser473 1.5-fold in isoproterenol-stimulated soleus muscle, coincident with stimulation of Akt activity (Fig. 7). It is noteworthy that, in addition to increased isoproterenol-stimulated phosphorylation, Akt expression was 1.5-fold greater in the soleus muscle of PTX-treated rats (Fig. 7). Taken together, these data indicate that PTX may inhibit isoproterenol-stimulated ERK1/2 phosphorylation in the soleus muscle by Akt-mediated suppression of Raf-1 activity. Thus it appears that Raf-1 may act as a central control point for NKCC activation in the soleus muscle. If so, inhibition of Raf-1 should suppress isoproterenol-stimulated NKCC activity. GW5074, a potent Raf-1 kinase inhibitor (19), abolished the isoproterenol-stimulated NKCC activity in the soleus muscle (Fig. 8). Isoproterenol-stimulated NKCC activity in the plantaris muscle was not affected by GW5074 treatment (Fig. 8). A summary schematic of the proposed mechanism is shown in Fig. 9.


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Fig. 7.   Isoproterenol treatment induced Akt Ser473 phosphorylation and activation in PTX-treated soleus muscle. PTX-treated muscles (see legend of Fig. 3) were stimulated for 10 min with 30 µM Iso. A: supernatants of muscle lysates (100 µg of protein) were subjected to SDS-PAGE and immunoblotted with an anti-phospho- Ser473 Akt antibody, followed by stripping and reprobing the blots with an anti-total Akt antibody. Data are the densitometric ratio of phospho-Akt to total Akt normalized to the basal mean value (taken as 1.0). B: phosphorylation of GSK-3, an Akt substrate, on Ser9/21 was used to determine Akt activity. Plotted data are the densitometric ratio of phospho-Akt to total Akt normalized to the basal mean value (taken as 1.0). Representative blots are shown. These results demonstrate that PTX provoked a significant increase in Akt phosphorylation and activity in Iso-stimulated soleus muscle. Data are means ± SE of 6 muscles. *,dagger P < 0.05 relative to the basal and Iso-stimulated level of Akt activation, respectively.



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Fig. 8.   A specific inhibitor of Raf-1 kinase activity, compound GW5074, abrogated Iso-stimulated bumetanide-sensitive 86Rb uptake in the soleus muscle. Muscles were preincubated with the Raf-1 kinase inhibitor GW5074 for 15 min before Iso stimulation and during the 10 min of Iso-stimulated 86Rb uptake. Iso stimulation of bumetanide-sensitive 86Rb uptake in the plantaris muscle was not affected by GW5074 treatment. Data are means ± SE; n = 4-15 muscle pairs per point. *,dagger P < 0.05 relative to the basal and Iso-stimulated value, respectively.



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Fig. 9.   Schematic summary of muscle phenotype differences in the Iso-stimulated NKCC activity. Data presented here and previously (12, 31) indicate regulation of the ERK MAPK pathway through Gs- and Gi-coupled processes in the soleus muscle. Points of pharmacological inhibition are indicated with a circled cross. Feasible pathways not excluded by the data are marked as dashed arrows. beta -AR, beta -adrenergic receptor.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

beta -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 beta -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 beta -AR activation is the dissociation of the Galpha 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 beta -AR G protein-coupled mechanism. NKCC activity was stimulated only in the predominantly slow-twitch soleus muscle (Fig. 1B). Phenotype differences in beta -AR-stimulated PKA pathway components have been noted previously. In rat skeletal muscle, beta -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 beta -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 Galpha 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 beta -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 Galpha beta gamma subunits. Both Galpha i and Gbeta gamma 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 Galpha 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 Galpha s-mediated activation of PKA is required for activation of PTX-sensitive Galpha 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 beta -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 beta -AR-mediated stimulation of MAPK pathway-dependent NKCC in skeletal muscle. In addition to PKA activation, beta -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.


    ACKNOWLEDGEMENTS

We are grateful to L. A. Malinick for assistance with publication graphics.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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