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Hypotonic activation of volume-sensitive outwardly rectifying chloride channels in cultured PASMCs is modulated by SGK

Ge-Xin Wang,¹ Cian McCrudden,¹ Yan-Ping Dai,¹ Burton Horowitz,^2, Joseph R. Hume,¹ and Ilia A. Yamboliev¹

Departments of ¹ Pharmacology, and ² Physiology and Cell Biology, Center of Biomedical Research Excellence, University of Nevada School of Medicine, Reno, Nevada 89557-0270

Submitted 14 March 2003 ; accepted in final form 6 April 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The serum- and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase (PK) transcriptionally regulated by corticoids, serum, and cell volume. SGK regulates cell volume of various cells by effects on Na⁺ and K⁺ transport through membrane channels. We hypothesized a role for SGK in the activation of volume-sensitive osmolyte and anion channels (VSOACs) in cultured canine pulmonary artery smooth muscle cells (PASMCs). Intracellular dialysis through the patch electrode of recombinant active SGK, but not kinase-dead 60-SGK-K127M, heat-inactivated SGK, or active Akt1, partially activated VSOACs under isotonic conditions. Dialysis of active SGK before cell exposure to hypotonic medium significantly accelerated the activation kinetics and increased the maximal density of VSOAC current. Exposure of PASMCs to hypotonic medium (230 mosM) activated phosphatidylinositol 3-kinases (PI3Ks) and their downstream targets Akt/PKB and SGK but not PKC-. Inhibition of PI3Ks with wortmannin reduced the activation rate and maximal amplitude of VSOACs. Immunoprecipitated ClC-3 channels were phosphorylated by PKC- but not by SGK in vitro, suggesting that SGK may activate VSOACs indirectly. These data indicate that the PI3K-SGK cascade is activated on hypotonic swelling of PASMCs and, in turn, affects downstream signaling molecules linked to activation of VSOACs.

hypotonic cell swelling; Cl^– conductance; phosphatidylinositol 3-kinase; Akt/protein kinase B; protein kinase C-; pulmonary artery smooth muscle cells; volume-sensitive osmolyte and anion channels; serum- and glucocorticoid-inducible kinase

Various intracellular targets of SGK, including B-Raf (55) and the Forkhead transcription factor FHKRL1 (7), suggest that SGK is involved in regulation of gene transcription and mitogenic signaling. SGK mediates cell cycle progression (8, 11) and transmembrane ion and metabolite transport (30). SGK is involved in osmotic cell adaptation via regulation of epithelial Na⁺ channels in kidney-collecting duct (A6) cells (49). In human embryonic kidney (HEK293) cells, SGK mediates insulin-like growth factor-1-stimulated activation of K⁺ channels (22). The mechanisms of these effects of SGK remain poorly defined, although accumulating novel information suggests that channel regulation may be mediated by accessory proteins. For example, modulation of Na⁺-H⁺ exchanger in kidney cells was proposed to depend on interaction of SGK1 with the Na⁺-H⁺ exchanger regulatory protein NHERF2 (54). It was shown that interaction between SGK and NHERF2 is required for upregulation and activation of yet another ion channel, the renal outer medullary K⁺ channel ROMK1 (37, 38). Novel targets of SGK include astrocyte and hepatocyte amino acid transporter SN1 (4) and the glial glutamate transporter EAAT1 (3), and the regulatory mechanism may depend on association with the ubiquitin ligase Nedd4–2. SGK phosphorylation inhibits ubiquitin ligases of the Nedd4 family (12) and delays internalization and degradation of Na⁺ channels on the epithelial cell surface (13, 45). Because Na⁺ channels regulate salt and water balance of various cells, SGK might be a necessary factor in maintenance of body-fluid content and blood pressure (31).

Regulation of the water balance in mammalian cells is a complex process, which includes stringent control of the transmembrane traffic of ions other than Na⁺ (40). For example, hypotonic swelling of canine pulmonary artery smooth muscle cells (PASMCs) activates a volume-sensitive organic osmolyte and anion channel (VSOAC), characterized by a moderate outward rectification, and an anion permeability of I^– > Cl^– > aspartate (53). We have shown previously that PKC-catalyzed reversible phosphorylation may represent an important molecular link between change in cell volume and activity of VSOACs in canine PASMCs (57). This is reminiscent of the regulation of VSOACs in native cardiac myocytes and in NIH/3T3 cells transfected with guinea pig ClC-3, a candidate protein responsible for native VSOACs (15). Because SGK was shown to regulate various other ion channels, in this study, we hypothesized a role for SGK in the regulation of VSOACs in PASMCs. Our results revealed that hypotonic cell swelling elicited activation of PI3Ks and SGK, which is coupled to the regulation of native VSOACs. This is a novel mechanism of hypotonic activation of VSOACs in the pulmonary circulation, which links activation of the PI3K-SGK pathway to regulation of cell volume on osmotic fluctuations of blood plasma and smooth muscle cell environment.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Materials. D-Mannitol and wortmannin were purchased from Sigma (St. Louis, MO), and cell culture basal medium Eagle, culture medium M199, and newborn calf serum (NCS) were from GIBCO (GIBCO BRL, Gaithersburg, MD). Recombinant Akt1/PKB, polyclonal common, and phosphospecific anti-Akt/PKB antibodies were purchased from Cell Signaling Technology (Beverly, MA), anti-SGK antibody raised in sheep and recombinant active SGK1 (1–60, S422D) were from Upstate Biotechnology (UBI, Lake Placid, NY), SGK peptide substrate (Sgktide) was synthesized by Research Genetics (Huntsville, AL), and phospho-specific SGK antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary anti-rabbit, anti-sheep, and anti-mouse antibodies, conjugated to alkaline phosphatase, were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All commonly used reagents were from commercial sources.

Cell culture. Canine lung was obtained from adult mongrel dogs of either sex euthanized by barbiturate overdose, in conformity with Protocol for Animal Care and Use A01/02–23 issued by the University of Nevada, Reno. Lung was dissected to isolate second pulmonary artery branches, which was then cleaned from connective tissue and placed in Ca²⁺-free Hanks' solution, containing (in mM) 125 NaCl, 5.36 KCl, 15.5 NaHCO₃, 0.34 Na₂HPO₄, 0.44 KH₂PO₄, 10 glucose, 2.9 sucrose, and 10 HEPES at pH 7.4 and 37°C. Blood vessels were cut open longitudinally, endothelial cells were removed by a cotton swab, and the smooth muscle layer was minced and digested with 1 mg/ml type II collagenase, 0.1 mg/ml protease (P5147, Sigma), 2 mg/ml bovine serum albumin, 2 mg/ml trypsin inhibitor, and 0.3 mg/ml Na₂ATP at 37°C for 1.5 h. Cells were then recovered by three washes of the partially digested tissue with Ca²⁺-free Hanks' solution at 37°C. Dispersed cells were sedimented by centrifugation and resuspended in medium 199 (M199) cell culture medium supplemented with 10% NCS, 0.2 mM glutamine and antibiotics, plated onto 75 cm² cell culture flasks coated with type I rat tail collagen, and grown to 80% confluence. These primary cells were trypsinized, passage onto culture dishes, and grown to 90–95% confluence in M199 supplemented with 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenious acid, 5.35 µg/ml linoleic acid (ITS+), and 10% NCS.

Preparation of PASMC lysates. Primary cells used for the kinase activity assays, immunoprecipitation of cytosolic protein, and Western blot analysis were growth-arrested in M199/0.1% NCS for 24–36 h before the assay. This M199 culture medium has an osmolarity of 290 mosM. The medium was diluted with sterile deionized water to obtain the 230 mosM (23%) hypotonic solutions for use in our experiments. The 230 mosM culture medium was then supplemented with D-mannitol until the osmolarity was recovered to 290 mosM (isotonic). All PASMCs were treated with these solutions for the time span and conditions outlined in the individual experimental protocols. Cells were then washed twice with ice-cold PBS [(in mM) 10 Na₂HPO₄, 1.8 KH₂PO₄, 2.6 KCl, and 137 NaCl, pH 7.4] and lysed with Ripa buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM Na₂EDTA, 0.5% NP40 (vol/vol), 0.5% Triton X-100 (vol/vol), 1 mM NaF, 1 µM leupeptin, and 1 µM AEBSF]. Lysates were passaged 20 times through a 25-gauge needle and incubated on ice for at least 30 min to increase protein yield. Unless otherwise specified, cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C, and supernatants were transferred into clean Eppendorf tubes. Protein concentrations were assayed by the Micro BCA Protein Assay (Pierce, Rockford, IL), and the supernatants were frozen at –20°C until use.

Bacterial expression and purification of kinase-dead SGK-K127M protein. Mammalian expression construct (pcDNA3, Invitrogen) carrying His-tagged kinase-dead rat SGK-K127M was kindly provided by Dr. Gary Firestone (University of California, Berkeley). After linearization, SGK-K127M was amplified with a forward PCR primer, which introduced a 5' Kpn I cloning site, a Kozak sequence, and an in-frame Flag-tag sequence (5'-AGGGGTACCTCCACCATGGATGATTACAAGGATGACGACGATAAGTCCCAACCTCAGGAG-3'). The COOH-terminal portion of this primer is complementary to a 12-bp nucleotide sequence downstream of base pair 181 of the SGK gene, thus truncating the first 60 NH₂-terminal amino acids of the translated protein (referred to as 60-SGK-K127M). This approach reduces targeting of SGK to intracellular structures and proteasomal degradation and increases the cytosolic stability of SGK (6). The reverse primer (5'-ACACGCGGCCGCTCAGAGGAAGGAGTCCAT-3') introduced a 3' Not I cloning site. Amplified PCR products were digested with restriction nucleotidases and subcloned into the Kpn I and Not I sites of the bacterial expression vector pET-30a(+) (Novagen), followed by transformation into BL21(DE3)pLysS Escherichia coli strain for high-level expression of recombinant Flag-tagged 60-SGK-K127M protein. Synthesized protein was purified by affinity chromatography (ANTI-FLAG M2-Agarose mouse affinity gel), followed by elution of the retained protein with 3X FLAG Peptide solution, as recommended by the manufacturer (Sigma). The yield was quantitated by densitometry of SDS-PAGE-separated Coomassie brilliant blue-stained SGK bands, using a dilution series of bovine serum albumin protein standards. Lack of kinase activity was verified by in vitro phosphorylation of Sgktide, relative to catalytic activity of recombinant active SGK, as outlined below.

SGK immunoprecipitation and activity assay in vitro. PASMCs grown in 100-mm petri dishes were treated according to the experimental protocol, and cell lysates were prepared as described. SGK was immunoprecipitated from 500 µg of total cell protein for 1 h at 4°C, using an anti-sheep primary antibody (Upstate Biotechnology). Immune complexes were harvested with protein A/G agarose plus beads (Santa Cruz Biotechnology) and washed two times with 0.5 ml of RIPA buffer and two times with 0.5 ml of SGK assay buffer (50 mM Tris·HCl, pH 7.4, 10 mM MgCl₂). SGK activity was assayed by in vitro phosphorylation of a previously described synthetic peptide substrate, Sgktide (KKRNRRLSVA) (1). The phosphorylation reaction was run for 15 min at 30°C in a reaction volume of 40 µl, containing 30 µl of SGK immunoprecipitate, 0.1 mM Sgktide, 1 µM PKA inhibitory peptide (Sigma), 0.1% -mercaptoethanol, 250 µM ATP, and 10 µCi [-³²P]ATP (ICN Biomedicals, Costa Mesa, CA). Phosphorylation was stopped by cooling on ice, 20 µl of reaction were spotted onto P81 filter paper (Whatman, Maidstone, UK), and excess radioactivity was removed from the filters by 5 x 5-min washes with 0.85% orthophosphoric acid, followed by 1 x 1-min wash with 95% ethanol. The radioactivity incorporated into the Sgktide was assayed by scintillation counting. Nonspecific radioactive signal was assayed in control reactions, lacking the substrate. To obtain the net SGK activity, the nonspecifically bound radioactivity was subtracted from the counts of substrate-containing reactions. Finally, kinase activity of osmotic stress-treated cells was calculated and presented relative to cell controls.

Phosphorylation of immunoprecipitated ClC-3 in vitro. Lysed PASMCs were first centrifuged at 2,000 rpm for 10 min at 4°C to sediment nuclei, nonlysed cells, and cell debris. The supernatants were fractionated by a high-speed centrifugation at 100,000 g for 1 h at 4°C in a Beckman TL-100 Ultracentrifuge (Beckman-Coulter, Fullerton, CA). The obtained P100 membrane fraction was resuspended in Ripa buffer, and ClC-3 channel was precipitated with a rabbit polyclonal antibody, raised against a peptide mapping the COOH-terminal amino acid sequence 670–687 of guinea pig ClC-3 (U83464 [GenBank] ). Immune complexes were harvested and washed as described for SGK, and immunoprecipitated ClC-3 was phosphorylated with active SGK and PKC- in vitro in a final volume of 50 µl, containing 100 µM ATP/10 µCi [-³²P] ATP per reaction for 1 h at 30°C. The reactions were stopped by addition of x4 SDS sample buffer to produce final concentractions of 0.06 M Tris·HCl (pH 6.8), 2% SDS, 10% glycerol, 1 mM DTT, and 0.03% bromphenol blue. After boiling for 5 min, protein was separated by SDS-PAGE (10% acrylamide), washed, stained with Coomassie brilliant blue, and completely destained to remove the nonspecifically bound radioactivity. Protein phosphorylation was assayed by phosphorimaging, using a Bio-Rad model 525 Molecular Imager (Bio-Rad, Hercules, CA).

Western blot analysis. Western blot analysis was applied to assay protein expression and phosphorylation of ClC-3, SGK, and Akt/PKB. Equal amount of total or immunoprecipitated protein (usually 30 µg) was resolved by SDS-PAGE and transferred onto nitrocellulose membranes for 1.5 h at 24 V and 4°C (Genie blotter, Idea Scientific, Minneapolis, MN). Membranes were blocked for 2 h with 5% skim milk in PBS. Protein levels of SGK were assayed with an anti-SGK antibody raised in sheep (Upstate Biotechnology) and diluted 1:500 in 5% milk/PBS. Ser422-phosphorylated SGK was assayed with a goat anti-phospho-Ser422-SGK antibody (Santa Cruz Biotechnology), and total and phospho-Ser473-Akt/PKB were assayed with rabbit polyclonal and mouse monoclonal antibodies, respectively (Cell Signaling Technology), diluted 1:1,000 with 5% milk/PBS. Expression of ClC-3 was detected with a rabbit polyclonal antibody as described. Incubation with the primary antibodies took place for 2 h at room temperature or overnight at 4°C. Excess primary antibody was removed by 3 x 5-min washes with 2,4,6-trinitrotoluene buffer (100 mM Tris, pH 7.5, 0.1% Tween-20, 150 mM NaCl), followed by a 1-h incubation with secondary alkaline phosphatase-conjugated antibodies, diluted 1:10,000 with 5% milk/PBS. Excess secondary antibody was removed by 3 x 5-min washes with 2,4,6-trinitrotoluene, and color was developed with the 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium alkaline phosphatase substrate. Blots were scanned with a UMAX Powerlook flatbed scanner (Bio-Rad) to obtain images, and the immunoreactive bands were analyzed by scanning densitometry using Quantity One software (Bio-Rad). The relative increase of protein content or phosphorylation was calculated by dividing the density of immunoreactive bands of PASMCs incubated with anisotonic solutions by the band density of control cells bathed in isotonic culture media.

PI3K activation assay. PASMCs were grown in 100-mm petri dishes to 90% confluence and were growth-arrested for 24 h. Cells were incubated with isotonic or hypotonic (230 mosM) media before or after a 60-min incubation with the PI3K inhibitor wortmannin (300 nM). Cells were then scraped with lysis buffer composed of 20 mM Tris·HCl (pH 7.5), 2 mM EDTA, 10 mM EGTA, and 0.1% basal medium Eagle (44). Lysates were incubated on ice for 45 min and then centrifuged at 14,000 g for 15 min at 4°C. PI3K- was immunoprecipitated with a rabbit polyclonal antibody (Upstate Biotechnology) and protein A/G agarose plus beads (Santa Cruz Biotechnology). Immune complexes were washed two times with 0.5 ml of RIPA buffer and two times with 0.5 ml of PI3K assay buffer: 20 mM Tris·HCl, pH 7.5, 0.25 mM EDTA, 50 mM KCl, and 4 mM MgCl₂ (44). The immunoprecipitates were used to verify immunoprecipitation of PI3K by Western blot analysis and for activity assay by in vitro phosphorylation of phosphatidylinositol (Avanti, Alabaster, AL). Phosphorylation reaction contained PI3K immunoprecipitate, 10 µg of phosphatidylinositol substrate, and 0.25 mM ATP/10 µCi [-³²P]ATP in a final volume of 40 µl. Phosphorylation was carried out at 60°C for 30 min and stopped by addition of 200 µl of 1 N HCl and cooling on ice. Lipids were extracted with 0.5 ml of a 1:1 mix of CHCl₃ and MeOH, and extracts were transferred into clean tubes and evaporated under a stream of N₂. Dry residues were recovered in 10 µl of CHCl₃, and the whole volume was spotted on thin-layer chromatography (TLC) plates (Silica gel HL, Analtech, Newark, DE) and developed with a mobile phase composed of CHCl₃, MeOH, 50% ammonium hydroxide, and water at a volume ratio of 130:80:8:10. The radioactive bands of phosphorylation product phosphatidylinositol 3-phosphate were identified by iodine staining using PI4P as TLC standard (Avanti). The TLC plates were then exposed to a phosphorimaging screen, and radioactive spot densities were assayed using a Bio-Rad model 525 Molecular Imager (Bio-Rad). Radioactive signals were normalized to the respective band densities derived from the immune blots, and kinase activities were expressed relative to controls.

PKC- activation assay. PKC- activation was assayed by phosphorylation of a synthetic peptide substrate (ERMRPRKRQGSVRRRV) in vitro using a PKC- assay kit and protocol of Upstate Biotechnology. The phosphorylation reaction took place at 30°C for 60 min in a reaction volume of 40 µl, containing 30 µl of cell lysate, 10 µg of substrate peptide, lipid activator, and [-³²P]ATP as recommended by the manufacturer. Phosphorylation reactions were stopped by cooling on ice, and 10 µl of reaction were spotted onto P81 filter strips (Whatman, Maidstone, UK). Excess radioactivity was removed by three washes with 0.85% orthophosphoric acid, followed by one wash with 95% ethanol. The phosphate, incorporated in the substrate peptide, was quantitated by radiography and densitometry. The individual filters were then separated and subjected to scintillation counting. Nonspecific radioactive signal was assayed in control reactions, lacking enzyme or substrate, and then subtracted from the signal of substrate-containing reactions to determine the net PKC--dependent peptide phosphorylation. Finally, PKC- activation of treated cells was calculated and presented relative to cell controls incubated in isotonic medium.

Electrophysiology. For the electrophysiological measurements, primary cultured cells were trypsinized, transferred onto glass cover slips, and allowed to attach overnight at 37°C in M199 culture medium without NCS. Membrane currents were measured using a whole cell voltage-clamp technique. Patch pipettes were made from borosilicate glass capillaries (Sutter Instrument) and had a tip resistance of 1.5–2.5 M when filled with pipette solutions. The bath and pipette solutions were connected via Ag/AgCl wires to a patch-clamp amplifier (3900A Integrating Patch Clamp, Dagan, Minneapolis, MN). A 3 M KCl-agar salt bridge between the bath and Ag/AgCl reference electrode was used to minimize changes in liquid junction potential. To follow the time course of change in membrane currents, repetitive voltage clamp steps to ±80 mV were applied every 30 s from a holding potential of –40 mV. Current-voltage relations were obtained by measuring membrane currents elicited by 400-ms pulses to potentials ranging from –100 to +120 mV in 20-mV increments applied from a holding potential of –40 mV every 5 s. Current densities were calculated by dividing the whole cell membrane current by cell capacitance. All bath and pipette solutions were chosen to facilitate VSOAC current recording. The hypotonic bath solution contained (in mM) 107 N-methyl-D-glutamine, 107 HCl, 1.5 MgCl₂, 2.5 MnCl₂, 0.05 GdCl₃, 10 glucose, and 10 HEPES (pH 7.4, 230 mosM). Appropriate amounts of D-mannitol were added to make the isotonic (310 mosM) and hypertonic (350 mosM) bath solutions. The pipette solution contained (in mM) 95 CsCl, 20 TEA-Cl, 5 ATP-Mg, 5 EGTA, 60 D-mannitol, and 5 HEPES (pH = 7.2, 300 mosM). External solution with osmolarity of 230 mosM has previously been shown to elicit significant increase in PASMC volume (53). Anion permeability was determined by substitution of 100 mM Cl^– in bath solutions with equimolar concentrations of I^– or aspartate. Modified Goldman-Hodgkin-Katz equation was used to calculate permeability ratios. For the experiment with wortmannin, PASMCs were first incubated with 300 nM wortmannin for 30 min. Then membrane currents were measured with patch electrodes filled with pipette solution containing 300 nM wortmannin. For the experiments with SGK and Akt, the kinases were added in pipette solutions. All experiments were conducted at room temperature (22–24°C).

Statistical methods. Results are presented as means ± SE. The n values refer to the number of parallel experiments. Student's t-test for paired and unpaired data, Dunnett's test, or one-way ANOVA was applied to test for differences between treatment means as appropriate. Values of P < 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Biophysical characterization of native VSOAC currents in cultured PASMCs. Previous studies of Yamazaki et al. (53) showed that hypotonic swelling induces characteristic VSOAC currents in freshly dispersed canine PASMCs. VSOAC currents with similar features were also observed in first-passage canine-cultured PASMCs. In isotonic solutions, voltage steps from a holding potential of –40 mV to potentials ranging from –100 to +120 mV elicited only small basal membrane currents. Exposure of cells to hypotonic medium (230 mosM) gave rise to robust membrane currents at all tested potentials (Fig. 1A). The swelling-activated current displayed moderate outward rectification and slight inactivation at very positive potentials (60 mV). Application of 100 µM DIDS to the bath solution significantly inhibited the currents under hypotonic conditions (Fig. 1, A and B). Consistent with other reports, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) more effectively inhibited the outward than the inward VSOACs. In contrast, 10 µM tamoxifen caused strong inhibition of both the outward and inward currents (Fig. 1, C and D). The swelling-induced currents reversed at a potential close to 0 mV, i.e., close to the calculated Cl^– equilibrium potential. Substitution of 100 mM extracellular Cl^– by equimolar concentration of I^– or aspartate shifted the reversal potentials to more negative or more positive directions, respectively, indicating an anion permeability sequence of I^– > Cl^– > aspartate (Fig. 1E). These biophysical and pharmacological characteristics are identical to those of VSOACs previously described in freshly dispersed PASMCs (53), implying that cell culturing had little effect on the functional properties of VSOACs.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Characterization of swelling-induced Cl– current in cultured pulmonary artery smooth muscle cells (PASMCs). Top: step-pulse protocols used to elicit membrane current as shown in A–D. A and B: representative current traces (A) and current-voltage relation curves (B) showing basal membrane current and swelling-induced volume-sensitive osmolyte and anion channel (VSOAC) current obtained from a cell under isotonic and hypotonic conditions and the inhibitory effects of 100 µM DIDS on VSOACs. C and D: representative current traces (C) and current-voltage relation curves (D) showing basal membrane current and swelling-induced VSOAC obtained from another cell and the inhibitory effects of 10 µM tamoxifen on VSOACs. E: representative swelling-induced VSOAC current traces showing change of reversal potential caused by substitution of Cl– (100 mM) in the bath solution by equimolar concentration of I– or aspartate. The current, elicited by repetitive ramp pulses (inset), exhibited anion permeability I– > Cl– > aspartate.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Active, but not kinase-dead, serum- and glucocorticoid-inducible kinase (SGK) stimulates VSOACs in cultured PASMCs. Membrane currents were elicited by voltage pulses to ±80 mV from a holding potential of –40 mV every 30 s. A–C: representative traces showing time course of change in current amplitudes measured at +80 mV () and –80 mV () under isotonic, hypotonic, and hypertonic conditions, as indicated by the bars above the traces. Cells were dialyzed with either vehicle (A), 7.5 µg/ml active SGK (B), or 7.5 µg/ml kinase-dead SGK (C) via patch pipettes. D: summarized data showing increase of current densities at +80 mV caused by intracellular dialysis with active SGK. Current densities were measured immediately (0 min) and 10 min after establishment of whole cell configuration. *P < 0.05 compared with isotonic 0 min. E: active SGK shortens the time for half-maximal activation of VSOAC under hypotonic conditions *P < 0.05 vs. buffer control.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Biophysical and pharmacological properties of SGK-induced Cl– currents in cultured PASMCs. Cells were intracellularly dialyzed with 7.5 µg/ml active SGK via patch pipettes. A and B: inhibition of SGK-induced currents by 100 µM DIDS and 10 µM tamoxifen, respectively. Left: representative time courses of change in the current amplitude measured at ±80 mV. The original current traces elicited by voltage steps ranging from –100 to +100 mV at 20-mV increments under isotonic and isotonic plus drug conditions are shown at middle and right, respectively. C: anion permeability of the SGK-stimulated channels. Currents were elicited by ramp pulses from –80 to +80 mV, and changes of reversal potential caused by substitution of Cl– in the bath solution by I– or aspartate were used to calculate the anion permeability ratio (PX/Cl). PI/Cl and Paspartate/Cl obtained from four cells were 1.33 ± 0.09 and 0.35 ± 0.05, respectively.

Native ClC-3 channels are directly phosphorylated by PKC- but not by SGK.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Native ClC-3 channels of PASMCs are phosphorylated by recombinant active protein kinase (PK) C- (rPKC-) but not recombinant SGK (rSKG) in vitro. ClC-3 was immunoprecipitated with a polyclonal antibody raised against an intracellular COOH-terminal epitope of the channel (immunoblot, top left) and immunoprecipitate (IP) was used as a substrate for rPKC- and rSGK (Upstate Biotechnology) in phosphorylation reactions in vitro. Protein phosphorylation was visualized after SDS-PAGE and radiography (top right). In parallel control experiments, rSGK phosphorylated the synthetic peptide substrate Sgktide in vitro (bottom). Phosphorylation reaction was spotted onto P81 filters, and filters were washed and counted on a scintillation counter to assay the radioactivity [in counts/min (CPM)] incorporated in the substrate Sgktide.

SGK is activated during hypotonic swelling of PASMCs. Because dialysis of active but not kinase-dead SGK led to isotonic activation of VSOACs (Fig. 2), the link between these proteins may have functional importance if hypotonic swelling causes activation of native SGK. To test this notion, we exposed cultured PASMCs to hypotonic media, immunoprecipitated SGK, and assayed its catalytic activity by in vitro phosphorylation of a synthetic peptide substrate, Sgktide, which contains the consensus phosphorylation motif of SGK (1). Exposure of PASMCs to hypotonic medium (230 mosM) triggered a transient activation of SGK, which was maximal at 10 min (2.73 ± 0.69-fold the activity of SGK in control cells, incubated in isotonic medium; Fig. 5A). As a positive control, we incubated cells for 30 min with pervanadate before SGK activation assay. Pervanadate is recognized as a highly efficient nonselective phosphatase inhibitor and has been shown to potently and nonspecifically activate SGK in other cells (39). Exposure of PASMCs to pervanadate stimulated a significant, more than threefold increase of the catalytic activity of SGK compared with cell controls (Fig. 5A).

View larger version (29K): [in this window] [in a new window]   Fig. 5. Hypotonic cell swelling activates SGK in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. A: first-passage PASMCs, untreated or incubated for 30 min with pervanadate or wortmannin (WM; 300 nM) before incubation with hypotonic (230 mosM) culture medium, were lysed, SGK was immunoprecipitated from equal amounts of total cell protein (inset), and kinase activity was assayed by in vitro phosphorylation of the peptide substrate Sgktide. The incorporated radioactivity was quantified by scintillation counting. The activation of SGK was expressed relative to cell controls incubated in isotonic medium (open bar, n = 5). Statistical significance was evaluated by the Dunnett's test. *P < 0.05 compared with controls incubated in isotonic (300 mosM) culture medium. B: time course of hypotonic stress-activated SGK, determined by Western blot analysis with a phosphospecific antibody, recognizing SGK, phosphorylated on Ser422. Immunoreactive bands were quantified by scanning densitometry, and activation was presented relative to cell controls, incubated in isotonic solution (C; n = 4). C: control time-course experiment depicting lack of SGK activation by environmental factors in isotonic culture medium (290 mosM) and time-dependent activation by hypotonic (230 mosM) treatment of PASMCs. SGK activation was assayed by Western immunoblotting with a phospho-specific antibody, recognizing Ser422-phosphorylated SGK. Immunoreactive bands were quantified by densitometry and activation was presented relative to zero-time cell controls (n = 4).

It was previously shown that environmental factors, including heat-shock, oxidative stress, and ultraviolet irradiation, could activate SGK (reviewed by Firestone et al. 21a). To account for accidental environmental or procedural activation of SGK, in a set of experiments, we incubated PASMCs in isotonic (290 mosM) and hypotonic (230 mosM) media, lysed cells at different times up to 30 min, and assayed phosphorylation of S422 by immunoblotting. Similar to the data presented in Fig. 5A, exposure to hypotonic media consistently induced transient phosphorylation of SGK, whereas incubation in isotonic media did not change SGK phosphorylation (Fig. 5C). Together, our data indicate that exposure to hypoosmotic solutions leads to a time-dependent activation of SGK in canine-cultured PASMCs.

Hypotonic activation of SGK is mediated by PI3Ks. Although PI3Ks have been identified as key upstream kinases of SGK in H4IIE and Rat2 cells (28), other kinases, including PKA (42) and the big mitogen-activating PK (BMK)-1 (24), can also activate SGK. To determine the PK that mediates the hypotonic activation of SGK, we incubated PASMCs with cell permeable inhibitors, selective for the aforementioned PKs: wortmannin for PI3K, PKA inhibitor peptide, and PD98059 for the upstream activator of BMK-1, MKK1. Treatment with the inhibitors took place for 30 min before and during a 10-min exposure of PASMCs to hypotonic solutions. The results of these experiments indicated that inhibition of PKA and BMK-1 was without effect, whereas inhibition of PI3K with wortmannin eliminated the hypotonic activation of SGK (Fig. 5A). Therefore, hypotonic stress of PASMCs leads to a wortmannin-dependent activation of SGK, suggesting that PI3K may also be activated on hypotonic cell stress. To test this possibility, we measured activation of this kinase by phosphorylation of phosphatidylinositol in vitro. The results of these experiments revealed that indeed hypotonic exposure of PASMCs leads to activation of PI3K compared with cells incubated in isotonic media (Fig. 6A). These observations independently confirmed that hypotonic swelling of PASMCs is associated with activation of PI3Ks, which further leads to activation of SGK. Our experiments, therefore, indicate that in canine-cultured PASMCs SGK is a component and functions downstream of PI3Ks.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Hypotonic treatment stimulated PI3Ks and enhanced phosphorylation of Akt/PKB but did not activate PKC-. Hypotonic activation of PI3K and Akt/PKB was inhibited in PASMCs, preincubated with wortmannin (Wm; 300 nM). A: first-passage PASMCs were incubated in hypotonic medium (230 mosM) for different times, without or in the presence of 300 nM wortmannin. PI3K- was immunoprecipitated and activation was assayed by phosphorylation of phosphatidylinositol in vitro. Reaction products were separated by thin-layer chromotography (TLC), and plates were subjected to radiography. Radioactive bands of the reaction product phosphatidylinositol 3-phosphate (PI332P; top) were scanned, and enzyme activation was expressed relative to nontreated cell controls (bottom; n = 3). *P < 0.05. B: phosphorylation of Akt/PKB was analyzed by Western immunoblotting, using a phosphospecific-Ser473-Akt/PKB antibody (top; Cell Signaling Technology). Immunoreactive band densities of phospho-Akt/PKB were normalized to band densities of Akt/PKB and presented relative to Akt/PKB phosphorylation of cell controls incubated in isotonic medium (290 mosM, open bar, n = 7). Statistical significance was evaluated by Dunnett's test. *P < 0.05 compared with cell controls. C: control PASMCs and cells incubated in hypotonic medium (230 mosM) without or in the presence of wortmannin (300 nM) were lysed and centrifuged. PKC- was immunoprecipitated from supernatants and used to phosphorylate a synthetic substrate peptide (ERMRPRKRQGSVRRRV, UBI) in vitro. Reactions were spotted onto P81 paper strips and analyzed by radiography (top). Bottom: spot densities from two parallel experiments with different batches of treated PASMCs (black bars) were plotted relative to nontreated cell controls (gray bars).

Akt/PKB has been shown to modulate L-type Ca²⁺ (2) and human ether-a-go-go-related gene potassium channels (56). To test whether Akt/PKB is involved in activation of VSOACs, we dialyzed through the patch pipette recombinant active Akt1 (20 U/ml) into cultured PASMCs. In contrast to active SGK, intracellular dialysis with active Akt1 neither induced VSOAC activation under isotonic conditions nor affected current densities and activation rate of VSOACs on hypotonic challenge. At +80 mV, current densities measured at 0 min, 10 min after Akt1 dialysis under isotonic conditions, and at the steady state under hypotonic conditions were 5.23 ± 1.39, 5.84 ± 2.98, and 131.16 ± 8.28 pA/pF (n = 5), respectively. The time for half-maximum activation of VSOAC induced by hypotonic exposure amounted to 5.96 ± 0.80 min. These data are not significantly different from the measurements in control cells. These results indicate that, although hypotonic swelling activates PI3K and its downstream effectors Akt and SGK, only SGK activity is involved in activation of VSOACs.

PKC- activity is independent of hypotonic swelling or PI3K activation. PKC- was shown previously to regulate VSOACs in PASMCs (57). Also, activation of PI3K and phosphatidylinositol-dependent PK was shown to regulate a number of PKC isozymes, including PKC- (14, 33). Therefore, we tested whether PKC- is activated on hypotonic swelling of PASMCs and whether activation depends on PI3K. For these experiments, we incubated cells with wortmannin for 1 h before hypotonic (230 mosM) or isotonic (controls) exposure, then lysed cells, and assayed activity of PKC- by in vitro phosphorylation of a synthetic peptide substrate. Neither hypotonic exposure activated nor cell incubation with wortmannin led to inhibition of PKC- activity (Fig. 6C). It seems, therefore, that hypotonic swelling-induced activation of PI3Ks dissociates from activation of PKC-.

PI3K activity is coupled to activation of VSOACs. Because SGK activity is linked to modulation of VSOACs and PI3K activity is required for activation of SGK, PI3K should also be coupled to VSOACs. To test this possibility, we measured swelling-induced VSOAC currents in cultured PASMCs incubated with 300 nM wortmannin in isotonic solutions for 30 min before the hypotonic challenge (230 mosM). Figure 7, A and B, depicts two representative activation time courses of VSOAC currents obtained from a control cell and a cell pretreated with wortmannin. Unlike in the controls, preincubation with wortmannin not only reduced the maximal amplitude but also decreased the activation rate of the swelling-induced VSOAC current (Fig. 7, C and D). The preincubation with wortmannin produced a significant 40% attenuation of the maximal amplitude of the hypotonic swelling-activated VSOAC currents (from 164.8 ± 17.6 to 96.4 ± 10.3 pA/pF at +80 mV). Normalization of the current amplitudes at different time points to the respective maximal amplitudes revealed that inhibition of PI3K signaling also caused a distinct reduction of the activation rate of VSOAC: the time for half-maximal activation of VSOACs increased from 4.92 ± 0.38 min in controls to 9.01 ± 1.41 min in wortmannin-treated cells (Fig. 7B). These results indicate that hypotonic activation of PI3Ks is coupled to VSOACs and modulates their activation kinetics.

View larger version (35K): [in this window] [in a new window]   Fig. 7. PI3K activity is coupled to VSOAC in cultured PASMCs. Membrane currents were elicited by voltage pulses to ±80 mV from a holding potential of –40 mV every 30 s. A and B: representative traces showing time course of change in current amplitudes measured at ±80 mV in cells preincubated for 30 min with either control isotonic solution (A) or 300 nM wortmannin (B) before hypotonic challenge. Ten micromolar tamoxifen was used to inhibit the swelling-induced Cl– currents. C: comparison of Cl– current densities between control cells and cells treated with 300 nM wortmannin for 30 min under isotonic and hypotonic conditions. Inward current densities measured at –80 mV are shown as downward bars, whereas outward current densities measured at +80 mV are shown as upward bars. *P < 0.05. D: comparison of activation time course of VSOAC currents induced by hypotonic cell swelling between control cells and cells pretreated with 300 nM wortmannin for 30 min. Current amplitudes measured at different time points are normalized to the respective maximum current amplitudes that were arbitrarily taken as 100%.

	DISCUSSION

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PI3Ks are key enzymes for a number of intracellular functions that include cell proliferation and growth, cell motility, maintenance of the glucose balance, and cell survival [reviewed by Vanhaesebroeck et al. (47)]. These functions are mediated by phosphorylated lipid substrates of PI3K that serve as second messengers and activate proteins that operate downstream of PI3Ks, such as monomeric G proteins, PKs Akt/PKB, p70 ribosomal S6 kinase, and enzymes of the PKC family (52). The notion that PI3Ks and downstream signal transduction molecules are involved in regulation of cell volume has been explored in previous studies. For example, studies in HTC hepatoma cells propose that PI3Ks mediate regulatory volume decrease by release of intracellular ATP and activation of extracellular P2 receptors and subsequently Cl^– channels (20). Interestingly, inhibition of PI3Ks was associated with delayed recovery of cell volume, suggesting PI3K activity is necessary for rapid activation of Cl^– channels after cell swelling. Inhibition of PI3Ks in our experiments also altered the pattern of Cl^– current by delaying the activation kinetics and reducing the maximal amplitude of VSOAC during hypotonic inflation (Fig. 7). Although these results suggest PI3Ks are important for regulatory volume decrease in PASMCs, downstream mechanisms that mediate this effect may not include activation of P2 receptors because application of exogenous ATP directly inhibits VSOACs through a P2-independent interaction with the channel (53). Therefore, our results suggest that other signal transduction pathways downstream of PI3Ks modulate VSOACs in PASMCs.

We focused on three PI3K-dependent signal transduction pathways, i.e., Akt/PKB, PKC-, and SGK. Previous studies have identified major roles of Akt/PKB in cell proliferation and survival and in insulin signaling (5, 32). Although information regarding role of Akt/PKB in activation of ion channels is sparse (2, 56), Akt/PKB was activated during hypotonic swelling of PASMCs (Fig. 6B) and hence was considered a potential regulatory protein upstream of VSOACs. Dialysis of active Akt1, however, neither affected the isotonic VSOAC nor changed the activation kinetics or the maximal amplitude of swelling-activated VSOAC. Hypotonic activation of Akt/PKB, therefore, may be required for other cellular processes. PKC- was shown to regulate VSOACs in PASMCs (57) and to be activated in a PI3K-dependent manner (47, 52). Despite the activation of PI3K, exposure of PASMCs to hypotonic medium did not enhance activity, and inhibition of PI3Ks with wortmannin did not reduce the basal activity of PKC- (Fig. 6C). Based on these results, we exclude Akt/PKB and PKC- as essential hypotonic activators of VSOACs of canine PASMCs.

SGK is a relatively newly identified kinase downstream of PI3K (39). Other kinases can also phosphorylate and activate SGK. For example, the cAMP-dependent PK (PKA) phosphorylates Thr369 of SGK, but phosphorylation of this site is sufficient for activation only when another amino acid, Ser422, is phosphorylated in a PI3K-dependent manner (42). BMK-1 and extracellular regulated kinase 5 can phosphorylate Ser78 and activate SGK in a mode apparently independent of PI3Ks (24). However, BMK-1 was identified as a redox-responsible enzyme, and it remains to be determined whether it is activated during osmotic disbalance. Thus PI3K seems to be the major activator of SGK on various cell treatments, including hypotonic swelling of PASMCs. This notion is also consistent with the observed wortmannin-mediated inhibition of hypotonic activation of SGK (Fig. 5A).

Functional roles of SGK in vascular smooth cells are poorly characterized, but in other cells SGK was shown to regulate gene transcription and mitogenic signaling (7, 34), cell proliferation (24), cell cycle progression (8), and transmembrane ion (10, 21, 43) and metabolite transport (30). SGK plays a role in maintenance of the body water balance and osmotic cell adaptation via regulation of epithelial Na⁺ channels and water uptake in kidney collecting duct (A6) cells (10, 45, 49). SGK could also activate K⁺ channels in HEK293 cells (22). To this list of functions, our study adds another novel and potentially important SGK-dependent function, which includes modulation of VSOACs that may be necessary for regulatory volume decrease after hypotonic cell swelling. This notion is based on the observation that intracellular dialysis of active SGK not only triggered activation of VSOACs under isotonic conditions but also augmented the swelling-induced activation rate and maximal amplitude of VSOAC. Because dialysis of kinase-dead and heat-inactivated SGK failed to promote similar events, isotonic activation of VSOACs seems to depend on kinase activity rather than on protein-protein interactions of SGK. It is worthwhile to note, however, that although wortmannin prevented hypotonic activation of SGK (Fig. 5A), it only partially inhibited VSOAC (Fig. 7). These data indicate that PI3K-SGK signaling is likely to be an alternative mechanism for activation of VSOAC, whereas PKC- may be the primary regulator of these channels (see below).

Identifying the exact mechanism by which SGK stimulates VSOACs may be a complicated task, primarily due to the lack of consensus regarding the molecular identity of VSOACs (23, 26, 36, 46). Novel data have provided strong evidence that the swelling-activated Cl^– current in canine PASMCs may in fact be due to the voltage-gated Cl^– channel ClC-3 (48). This notion is based on the presence of ClC-3 transcripts (53) and channel protein (48), as well as on various functional observations. For example, dialysis of two antibodies raised against peptides, mapping unique motifs of the ClC-3 protein, completely inhibited the hypotonic swelling-activated Cl^– current, thus suggesting that the ClCn3 gene may indeed encode VSOACs in canine PASMCs. Analogous results were also produced in Xenopus oocytes, bovine epithelial cells, native cardiac myocytes, and human gastric epithelial cells (16, 17, 25, 27, 50). These data raise the question of whether the regulatory effects of SGK might be the result of a direct phosphorylation of ClC-3. The amino acid sequence of the ClC-3 protein in PASMCs is unknown; however, the guinea pig ClC-3 contains a consensus phosphorylation sequence for SGK (CRRRKST₃₆₃K), located in the intracellular loop spanning the transmembrane domains J and K (18). Alternatively, SGK might phosphorylate Ser51, located within a PKC consensus sequence, in the cytosolic NH₂-terminus of the ClC-3 channel (29, 39). PKC-mediated phosphorylation of Ser51 was previously shown to play a critical role for inactivation of ectopically expressed ClC-3 in NIH 3T3 cells (15). Although we were able to demonstrate a direct phosphorylation of ClC-3 protein by PKC- in vitro, our attempts to phosphorylate ClC-3 with active SGK remained without success (Fig. 4). Therefore, the observed isotonic activation by active SGK, as well as the faster and more potent activation of Cl^– current on hypotonic stress, is most likely the result of an indirect functional link between SGK and ClC-3 channels. Despite the notion that voltage-gated Cl^– channels form functional dimers and might operate independent of other binding partners (35), evidence for modulation by accessory proteins has also been published. For example, barttin was identified as an accessory protein, which functions as an essential subunit of renal tubule ClC-Ka and ClC-Kb channels (19). Identification of regulatory accessory proteins of ClC-3 channels, therefore, may be a promising direction for future research.

The nature of the signal transduction event that governs activation of VSOACs is under extensive study. It is likely that signals that translate cell volume perturbations into activation of VSOACs are subject to multilevel coordination. Mechanisms based on phosphorylation by PKs, including PKC, PKA, and tyrosine kinase may be involved in the regulation of VSOACs [reviewed by Nilius et al. (36)]. More recent data suggest a role of the monomeric RhoA GTPase as a permissive factor in activation of swelling-activated Cl^– channels in vascular endothelial cells (9) and in NIH 3T3 fibroblasts (41). In PASMCs, PKC- seems to play an essential role in regulation of VSOACs (57). PKC- activity supplies an inhibitory signal on VSOACs, whereas inhibition of the enzyme with partially selective pharmacological inhibitors or with the highly selective peptide inhibitor that interferes with anchoring of active PKC-, i.e., V1–2, eliminates the inhibitory effect and causes constitutive activation of VSOAC in isotonic milieu (57). Because dialysis of active SGK activates VSOACs under the same experimental conditions (in isotonic media), the stimulating effect of SGK may override and promote channel activation despite the inhibitory effect of PKC-. A future challenge may be to examine whether PKC--dependent phosphorylation of the ClC-3 protein is necessary to maintain VSOACs in a closed conformational state and to distinguish events leading to the release of this inhibitory effect in vivo.

In conclusion, in this study, we have delineated a signal-transduction pathway that is activated in response to hypotonic swelling of PASMCs and leads to activation of PI3Ks and subsequently to activation of SGK. The PI3K-SGK cascade modulates activation of native VSOACs and hence may contribute to volume regulation and homeostasis of the vasculature.

	GRANTS

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This study was supported by National Institutes of Health Grants P20 RR-15581 (National Center for Research Resources) and HL-49254.

	ACKNOWLEDGMENTS

 
The authors acknowledge Dr. William T. Gerthoffer for constructive discussion and suggestions. The authors also acknowledge Lisa Miller and Shaner Bongalon for excellent work in cloning and production of the kinase-dead SGK-K127M mutant. Shanti Rawat is acknowledged for providing excellent technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: I. A. Yamboliev, Dept. of Pharmacology, MS 318, Univ. of Nevada School of Medicine, Reno, NV 89557-0270 (E-mail: yambo{at}med.unr.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.

Deceased 19 December 2003.

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