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Am J Physiol Heart Circ Physiol 289: H2566-H2574, 2005. First published July 22, 2005; doi:10.1152/ajpheart.00292.2005
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Regulation of cardiac volume-sensitive chloride channel by focal adhesion kinase and Src kinase

Kenneth B. Walsh and Jining Zhang

Department of Pharmacology, Physiology, and Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina

Submitted 24 March 2005 ; accepted in final form 18 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The volume-sensitive chloride current (ICl,swell) is found in the mammalian myocardium and is activated by osmotic swelling. The goal of this study was to examine the importance of the tyrosine kinases focal adhesion kinase (FAK) and Src kinase in cardiac ICl,swell regulation. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing {beta}-galactosidase (AdLacZ), FAK, or FAK-related nonkinase. FAK-related nonkinase (FRNK) is an endogenous cardiac protein, which functions as an inhibitor of FAK. Whole cell patch-clamp recordings demonstrated that osmotic swelling was associated with the activation of an outward rectifying current in uninfected and AdLacZ-infected cells. Consistent with the properties of ICl,swell, this current displayed a reversal potential close to the equilibrium potential for Cl–; was inhibited by the Cl– channel blockers 4,4'-dinitrostilbene-2,2'-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and tamoxifen; and was eliminated in hypertonic solution. In addition to activating ICl,swell, hypotonic swelling enhanced the tyrosine phosphorylation of multiple cardiac proteins including those in the range of 68–70 and 120–130 kDa. Pretreatment of the cells with the drug 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of FAK and Src, diminished swelling-induced phosphorylation of these proteins but paradoxically increased ICl,swell. Furthermore, overexpression of FRNK but not FAK caused a twofold augmentation in ICl,swell and increased the rate of current activation. Thus the tyrosine kinases FAK and Src contribute to the regulation of ICl,swell.

cardiac myocytes

THE VOLUME-SENSITIVE CHLORIDE CURRENT(ICl,swell) is found in mammalian cardiac myocytes and is important for the regulatory volume decrease that follows osmotic swelling (8, 24). Although initially discovered in canine atrial (22) and ventricular (32) myocytes, ICl,swell has been identified in the myocardium of a number of species including the rabbit (6), guinea pig (34), chick (38), and human (18). The cardiac ICl,swell displays outward rectification under both physiological and symmetrical Cl– concentration gradients and, in general, has an anion selectivity sequence of I– > Br– > Cl– > F– (22, 34). This current is blocked by stilbene disulfonates such as 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, as well as the arylaminobenzoate compound 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and the estrogen antagonist tamoxifen (6, 23, 32, 34).

A number of signaling pathways have been proposed to couple cell volume expansion to ICl,swell activation. Whereas both protein kinase A (PKA) and protein kinase C (PKC) can regulate the activity of cardiac ICl,swell, neither enzyme is directly responsible for the activation of the current (6, 22, 32). For this reason, attention has focused on other signaling molecules such as tyrosine kinases. Genistein and herbimycin A, two nonspecific tyrosine kinase inhibitors, prevent swelling-induced ICl,swell activation in canine atrial cells (25). In addition, tyrosine kinase inhibitors decrease the response of ICl,swell to hypotonic swelling in noncardiac cells including human intestinal (19), bovine endothelial (35), and human prostate cancer (20) cells. However, a mechanistic relationship between the stimulation of tyrosine kinases and the activation of ICl,swell has not been firmly established. On the contrary, in some tissues, tyrosine phosphorylation is associated with an inhibition of ICl,swell. For example, drug-induced inhibition of soluble tyrosine kinases enhances ICl,swell in human atrial (4) and rabbit ventricular (13) myocytes. In addition, when targeted to lipid rafts, the tyrosine kinase Src represses ICl,swell in pulmonary endothelial cells (31).

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that is localized to regions of cardiac myocyte-extracellular matrix contact (termed focal adhesions) (10, 33). FAK undergoes autophosphorylation at Tyr397 in response to cell adhesion, integrin clustering, and growth factor stimulation (2). Phosphorylation of this site creates a high-affinity binding site for the Src homology 2 domain of the Src-family kinases that phosphorylate additional tyrosine residues including Tyr576 and Tyr577. After being activated, both FAK and Src can stimulate a vast array of "downstream" effector molecules including small G proteins, MAP kinases, paxillin, and cytoskeletal proteins (10, 33). Recent experiments have linked FAK activation, triggered by mechanical stretch, to the regulation of an outward rectifying Cl– current in the heart (1).

In the current study, we tested the hypothesis that FAK and Src play a role in regulating the cardiac ICl,swell. Neonatal rat ventricular myocytes were infected with adenovirus expressing {beta}-galactosidase (AdLacZ), FAK (AdFAK), and FAK-related nonkinase (AdFRNK). FRNK is an endogenous cardiac protein consisting of the noncatalytic terminal of FAK, which functions as an inhibitor of FAK (5, 27). Exposure of control myocytes to hypotonic solution resulted in the activation of a Cl– current with properties consistent with those of ICl,swell. In addition, hypotonic swelling induced the tyrosine phosphorylation of several cardiac proteins including FAK and paxillin. Surprisingly, ICl,swell increased after treatment of the cells with the FAK and Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and during overexpression of FRNK. It is concluded that the tyrosine kinases FAK and Src contribute through a complex mechanism to the regulation of the cardiac ICl,swell.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and culture of cardiac ventricular myocytes. Animal protocols were approved and monitored by the Institutional Animal Care and Use Committee of the University of South Carolina, Columbia. Neonatal rat ventricular myocytes were isolated and cultured as described previously (21, 36). In brief, heart ventricles were removed from neonatal pups (age, 3–4 days), minced into 1-mm3 pieces, and subjected to collagenase (type B; Boehringer Mannheim Biochemicals) dissociation (21). For the preparation of the aligned myocytes, collagen (type I; Celtrix) was applied to culture dishes or plastic coverslips. While the dish was tilted, a small sterile scraper was used to draw the collagen solution across the dish. Excess collagen was then aspirated, and the culture dish was transferred to a 37°C incubator. When plated on this oriented or aligned collagen substrate, the cells exhibit an in vivo-like phenotype with a rod-shaped appearance (21, 36). Myocytes were cultured in DMEM (GIBCO) supplemented with 10% horse serum (Flow Laboratories) and maintained in a humidified atmosphere of 5% CO2 at 37°C.

Adenovirus infection. On day 1 of cell culture, myocytes were infected with either AdFAK or AdFRNK. AdFAK and AdFRNK were generously supplied by Dr. Joseph Loftus (Mayo Clinic Scottsdale). Control experiments were performed on myocytes infected with AdLacZ. This virus was constructed with the use of the pAdEasy system (Stratagene), and viral titer was determined on all stocks with the use of the Adeno-X kit (BD Biosciences). Myocytes were infected at matched multiplicities of infection of either 20 or 50, and expression was confirmed by immunoblot analysis. Patch-clamp recordings and Western blot analysis were performed 2 to 3 days after infection.

Recording procedure and measurement of ICl,swell The patch-clamp method (7) was used to record the whole cell ICl,swell with the use of the L/M EPC-7 (Adams and List) and Axopatch 200 (Axon Instruments) amplifiers. Pipettes were made from Prism glass capillaries (Dagan) and had resistances of 1–2 M{Omega} when filled with internal solution. All experiments were conducted on isolated, noncoupled myocytes at room temperature (22°-24°C). For the measurement of ICl,swell, cells were first placed in an isotonic external solution consisting of (in mM) 132 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 0.1 CdCl2, 1 BaCl2, 5 dextrose, 5 HEPES, pH 7.4 (with NaOH; 280 mosM). Cd2+ was present in the solution to block the L-type Ca2+ current, whereas Ba2+ was included to inhibit the inward rectifier K+ current. The Na+ current was blocked with TTX (10 µM), and the Na+ channels were inactivated by maintaining the myocytes at a holding potential of –40 mV. To activate ICl,swell, the external solution was replaced with hypotonic solution consisting of (in mM) 90 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 0.1 CdCl2, 1 BaCl2, 5 dextrose, 5 HEPES, pH 7.4 (with NaOH; 196 mosM). The normal internal solution consisted of (in mM) 60 CsCl, 50 Cs-aspartate, 2 MgCl2, 1 CaCl2, 11 EGTA, 3 ATP, 10 HEPES, pH 7.3 (with CsOH; 280 mosM). The osmolarity of the solutions was checked before experimentation with the use of a vapor pressure osmometer (Wescor).

Membrane currents were recorded with 12-bit analog-to-digital converters (Axon Instruments). Data were sampled at 2 kHz and filtered at 0.5 kHz with a low-pass Bessel filter (Frequency Devices). The series resistance was compensated to give the fastest possible capacity transient without producing oscillations. With this procedure, >70% of the series resistance could be compensated. At the end of each experiment, cells were perfused with hypertonic solution consisting of (in mM) 150 mannitol, 90 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 0.1 CdCl2, 1 BaCl2, 5 dextrose, 5 HEPES, pH 7.4 (with NaOH; 356 mosM). The ICl,swell was then determined by subtracting the currents recorded in the hypertonic solution from those measured in the hypotonic solution. The reversal potential (Erev) for ICl,swell was defined as the potential where the swelling-induced current was zero. In those cases in which the exact zero current was not recorded, Erev was determined by fitting a line through the points on the current versus voltage curve directly above and below this potential.

Preparation of cardiac cell lysates and Western blot analysis. To prepare cell lysates for Western blot analysis, cardiac myocyte cultures were placed into a lysis buffer [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 50 mM 1,4-dithiothreitol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethysulfonyl fluoride, 1 mM EGTA, 0.25% sodium deoxycholate, 2 µg/ml aprotinin, and protease inhibitor cocktail (Roche)]. The lysates were immediately transferred to precooled microfuge tubes and sonicated to reduce viscosity. The protein content of the cell preparations was determined with the use of a protein assay kit (Pierce). For Western blot analysis of tyrosine phosphorylation, proteins (120–150 mg/lane) were separated by electrophoresis on 10% SDS-polyacrylamide gels with the use of a standard cell (Bio-Rad) run overnight. The running buffer contained 25 mM Tris, 193 mM glycine, pH 8.3, and 0.1% SDS. Proteins were transferred to polyvinylidene difluoride membranes with the use of a Trans-Blot apparatus (Bio-Rad). The transfer buffer contained 25 mM Tris, 192 mM glycine, pH 8.5, and 20% methanol. For immunodetection, membranes were first blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20, bovine serum albumin, and 0.025% Na azide for 60 min at room temperature. Anti-phosphotyrosine (1:2,000) (Cell Signaling Technology) antibody was incubated with the membranes overnight at 4°C. After primary antibody treatment, the membranes were washed with TBS-0.1% Tween 20 and incubated with a secondary antibody (horseradish peroxidase-conjugated mouse IgG; Cell Signaling Technology). Immunoreactive bands were visualized on X-ray film (Kodak) with the use of the enhanced chemiluminescence method (Pierce) and quantified by densitometry (Bio-Rad Molecular Imager). FAK, paxillin, and Src were detected with the use of phospho (p)-specific antibodies (1:1,000 for pFAK-Tyr576/577, 1:1,000 for pPaxillin-Tyr118, and 1:1,000 for pSrc-Tyr527; Cell Signaling Technology and Upstate Biotechnology) according to procedures described previously (3). Each immunoblot result was confirmed on three separate myocyte cultures.

Measurement of cell area and statistical analysis. The measurement of the cell area provides a good estimate for determining changes in cell volume (15). Cell images were obtained with the use of an ORCA ER 1394 digital camera (Hamamatsu), and cell area was calculated with the use of Image Pro Plus software (Media Cybernetics). The averaged cell area and ICl,swell values presented are means ± SE. Multiple comparisons of mean values were performed by one-way ANOVA with post hoc Bonferroni tests. Student's t-test was used for comparing the two groups. Values of P < 0.05 were considered significant.

Drugs and chemicals. Isoproterenol, tamoxifen, and anthracene-9-carboxylic acid (A-9-C) were purchased from Sigma Chemical (St. Louis, MO). TTX, DNDS, NPPB, PP2, and 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3) were obtained from CalBiochem (San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activation of ICl,swell in neonatal rat ventricular myocytes. Figure 1, left, shows an example of swelling-induced currents recorded from a neonatal rat ventricular myocyte infected with AdLacZ. Internal and external solutions were designed in these experiments to eliminate Ca2+, Na+, and K+ currents (see MATERIALS AND METHODS). Under these conditions, negligible background currents were measured when the cells were bathed in isotonic external solution. However, application of hypotonic external solution resulted in the activation of a time-independent, outward rectifying current. The swelling-induced current reached a peak amplitude within 10–15 min of addition of the hypotonic solution and declined more rapidly back to baseline levels on addition of hypertonic solution (Fig. 1, top right). In 12 myocytes examined, the peak current amplitude was –7 ± 1 pA/pF (at –100 mV) and 17 ± 2 pA/pF (at +60 mV). The swelling-induced current was also present in uninfected myocytes (not shown).



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  		Fig. 1. Measurement of volume-sensitive chloride current (ICl,swell) in neonatal rat ventricular myocytes. Left: currents recorded in isotonic, hypotonic, and hypertonic external solutions during voltage steps, given in 20-mV increments, to potentials ranging from –100 to +60 mV. Top right: time course of changes in ICl,swell recorded at +60 mV in hypotonic and hypertonic external solutions. Currents were normalized to peak current measured in hypotonic solution. Bottom right: average current versus voltage relationship for volume-sensitive current measured in NaCl (n = 12 cells) and Na aspartate (n = 5 cells) hypotonic external solutions.

 
To identify the ionic basis of the swelling-induced current in the neonatal myocyte cultures, the Erev of the current was measured under conditions where external and internal concentrations of Cl– were varied. Figure 1, bottom right, displays the current-voltage relation for the swelling-induced current measured with an extracellular Cl– concentration ([Cl–]o) of 101 mM and an intracellular Cl– concentration ([Cl–]i) of 66 mM. The Erev in these experiments (–9 mV, n = 12) was close to the Cl– equilibrium potential (ECl = –11 mV). When the [Cl–]o was reduced to 11 mM by substitution of Cl– with aspartate, the Erev shifted to more positive potentials (21 mV, n = 5; Fig. 1) as predicted from the ECl (45 mV). With nearly symmetrical concentrations of Cl– across the plasma membrane ([Cl–]o = 101 mM; [Cl–]i = 100 mM), the Erev was –1 mV (ECl = 0 mV; results not shown). Furthermore, under each experimental condition described above, the swelling-induced current displayed an outward rectifying current-voltage relation (Fig. 1).

It was also determined whether the swelling-induced current could be activated in the absence of hypotonic solution through stimulation of either cAMP-dependent PKA or PKC. An alternatively spliced variant of the cystic fibrosis transmembrane conductance regulator (CFTR) is present in the myocardium of a number of mammalian species (8, 24). CFTR functions primarily as a PKA-activated Cl– channel (14). However, application of the {beta}-adrenergic receptor agonist isoproterenol (1 µM), to stimulate PKA, failed to activate a background current (n = 8 cells; results not shown). This concentration of isoproterenol produces a fivefold increase in cAMP levels and augments the L-type Ca2+ current in the neonatal myocytes (36). In addition, no evidence for activation of the current was obtained during 10 min treatment with the phorbol ester phorbol 12,13-dibutyrate (100 nM). Thus the properties of swelling-induced current described in this study are consistent with those of ICl,swell previously identified in mammalian cardiac myocytes (6, 22, 32).

To further confirm the presence of ICl,swell in the myocytes, the effect of several organic Cl– channel blockers was examined on the swelling-induced current. The results of these experiments are summarized in Fig. 2. The stilbene compound DNDS (200 µM) blocked the current in a voltage-dependent manner with strong inhibition of the outward currents and no inhibition of the inward currents (Fig. 2). A similar voltage-dependent block of ICl,swell by DNDS has previously been described (6, 23). Block of the channel by the anti-estrogen tamoxifen (50 µM) also occurred in a voltage-dependent manner. However, with tamoxifen there was also significant inhibition of the inward currents. In contrast to the results with DNDS and tamoxifen, the arylaminobenzoate NPPB (20 µM) acted in a voltage-independent manner to block ICl,swell. Finally, no inhibition of the channel was observed during application of A-9-C, even with a drug concentration as high as 500 µM (Fig. 2).



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  		Fig. 2. Effect of chloride channel blockers on ICl,swell. Top: currents recorded in hypotonic external solution during voltage steps, given in 20-mV increments, to potentials ranging from –100 to +60 mV. Currents were measured before and after addition of 200 µM 4,4' dinitrostilbene-2,2'-disulfonic acid (DNDS). Bottom: percent decrease in ICl,swell measured at –100 and +60 mV with DNDS (200 µM), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 20 µM), tamoxifen (50 µM), and anthracene-9 carboxylic acid (A-9-C; 500 µM). Each bar represents mean ± SE change in current measured in 4 to 6 myocytes. *P < 0.05 vs. decrease at –100 mV.

 
Regulation of ICl,swell via tyrosine phosphorylation. Previous studies suggest that tyrosine kinases play a role in the activation of the cardiac ICl,swell (4, 25). As shown in Fig. 3, anti-phosphotyrosine immunoblot analysis was performed to determine if hypotonic swelling stimulates tyrosine kinase activity in the neonatal myocytes. Hypotonic stress enhanced the tyrosine phosphorylation of multiple cardiac proteins including those with molecular sizes of 68–70, 80–85, and 120–130 kDa. Increased phosphorylation of the proteins could be measured within 2 min of hypotonic media exposure and peaked within 5 min (Fig. 3). In the continued presence of the hypotonic solution, phosphorylation returned to isotonic levels by 20 min (Fig. 3). Pretreatment of the cells with the drug PP2 (10 µM), a selective inhibitor of FAK and Src kinases, diminished swelling-induced phosphorylation of these proteins (Fig. 3). The phosphotyrosine proteins with molecular sizes in the range of 68–70 and 120–130 kDa may correspond to the focal adhesion proteins paxillin and FAK, respectively. To confirm this possibility, Western blot analysis was performed with the use of antibodies specific for the phosphorylated forms of the two proteins. As predicted, the anti-pPaxillin and anti-pFAK antibodies recognized proteins of ~68 kDa and 120 kDa (Fig. 3), respectively. Furthermore, exposure of the cells to hypotonic media for 5 min increased the intensity of both bands. Changes in the phosphorylation of Src kinase were also determined. Phosphorylation of Src at Tyr527 inhibits enzyme activity, whereas phosphorylation at Tyr416 stimulates Src (28). As shown in Fig. 3, osmotic swelling caused no change in levels of pSrc (Tyr527). Changes in the phosphorylation of Tyr416 were also tested; however, immunoblot signals could not be detected under either isotonic or hypotonic conditions.



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  		Fig. 3. Changes in tyrosine phosphorylation induced by hypotonic swelling. Top: cardiac cell lysates were prepared from myocytes cultured in isotonic or hypotonic media (for 5 min or indicated times) and cellular proteins separated by SDS-PAGE. Polyvinylidene difluoride membranes were probed with an anti-phosphotyrosine antibody (Ab) as described in MATERIALS AND METHODS. In some experiments, myocytes were pretreated for 10 min with 10 µM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) before hypotonic media exposure. Bottom: immunoblots obtained with phospho (p)-specific anti-focal adhesion kinase (pFAK), anti-pPaxillin, and anti-pSrc Abs. Myocytes were cultured in isotonic or hypotonic media for indicated times.

 
The results shown in Fig. 3 indicate that hypotonic swelling causes an enhanced tyrosine phosphorylation of several cardiac proteins including FAK. Thus it was hypothesized that activation and/or maintenance of ICl,swell may be dependent on stimulation of tyrosine kinases such as FAK and Src. However, addition of PP2 during the peak activation of ICl,swell produced a small yet significant increase in the current (36 ± 5% at +60 mV; n = 6 cells). No significant increase in ICl,swell was measured after the addition of PP3 (4 ± 3% at +60 mV; n = 6 cells), a structural analog of PP2 that does not inhibit tyrosine kinases. The augmenting effect of PP2 required several minutes (5–6 min) of cell treatment and was completely reversed on washout of the drug. Although PP2 enhanced the peak amplitude of ICl,swell, it is possible that pretreatment of the myocytes with the drug might prevent the activation of the current by inhibiting tyrosine phosphorylation (as shown in Fig. 3). Contrary to this prediction, 10 min of pretreatment of the cells with PP2 increased the peak steady-state current measured in the presence of hypotonic solution (Fig. 4). In addition, when compared with the control and PP3-treated myocytes, the rate of ICl,swell activation was enhanced after PP2 treatment (Fig. 4).



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  		Fig. 4. Regulation of ICl,swell by PP2. Left: currents recorded in isotonic, hypotonic, and hypertonic external solution during voltage steps, given in 20-mV increments, to potentials ranging from –100 to +60 mV. Cell was cultured in isotonic media containing 10 µM PP2 for 10 min before start of experiment. Top right: time course of changes in ICl,swell recorded from a PP2-reated cell at +60 mV in hypotonic and hypertonic external solutions. Currents were normalized to peak current measured in hypotonic solution. Bottom right: peak amplitude of ICl,swell measured in myocytes pretreated for 10 min with PP2 or 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3; n = 6 cells each) and cells infected with either adinovirus expressing {beta}-galactosidase (AdLacZ; n = 12 cells), AdFAK (n = 6 cells), or FAK-related nonkinase (AdFRNK; n = 9 cells). *P < 0.05 vs. PP3; {dagger}P < 0.05 vs. LacZ; {ddagger}P < 0.05 vs. FAK.

 
PP2 inhibits FAK as well as Src family kinases including Src, Fyn, and Hck. To determine a specific role of FAK in ICl,swell regulation, myocytes were infected with AdFAK and AdFRNK. Overexpression of FAK produced no significant change in the size of ICl,swell compared with that measured in the AdLacZ-infected cells (Fig. 4). In contrast, myocytes infected with AdFRNK displayed a twofold increase in ICl,swell (Figs. 4 and 5). Furthermore, overexpression of FRNK caused ICl,swell to activate more rapidly when compared with myocytes infected with AdLacZ and AdFAK (Fig. 5). To quantify this effect, the onset of current activation in the presence of hypotonic solution was fit with a sigmoidal function. In control AdLacZ myocytes, the time to 50% activation during hypotonic solution exposure (t) was 7 ± 1 min (n = 5 cells). In cells overexpressing FRNK, the t decreased to 4 ± 1 min (n = 5 cells). These values were significantly different (P < 0.05). In comparison, the t in the PP2- (n = 4 cells) and PP3-treated (n = 4 cells) myocytes was 4 ± 1 and 8 ± 2 min, respectively (P > 0.05). Thus either treatment of the myocytes with PP2 or infection with AdFRNK enhanced the size of ICl,swell.



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  		Fig. 5. Effect of FRNK overexpression on ICl,swell. Left: currents recorded in isotonic, hypotonic, and hypertonic external solution during voltage steps, given in 20-mV increments, to potentials ranging from –100 to +60 mV. Cell was infected with AdFRNK. Top right: time course of changes in ICl,swell recorded from an AdFRNK-infected cell at +60 mV in hypotonic and hypertonic external solutions. Currents were normalized to peak current measured in hypotonic solution. Bottom right: average current versus voltage relationship for volume-sensitive current measured in hypotonic external solutions during overexpression of FRNK (n = 9 cells).

 
If FRNK acts through a mechanism similar to PP2 in regulating ICl,swell, a comparable inhibition in swelling-induced tyrosine phosphorylation might be expected during overexpression of this protein. When compared with myocytes infected with AdLacZ, overexpression of FRNK caused a strong depression in both unstimulated and swelling-stimulated tyrosine phosphorylation (Fig. 6). In addition, AdFRNK infection inhibited paxillin phosphorylation when the myocytes were exposed to hypotonic media (Fig. 6). Figure 6, middle, summarizes the results measured in the phosphotyrosine immunoblot experiments. Swelling-induced increases in the protein phosphorylation of the 68- and 120-kDa proteins are plotted for control (uninfected and AdLacZ-infected), AdFRNK-infected, and PP2-treated myocytes. On the basis of densitometric analysis, hypotonic swelling of control myocytes caused 4-fold and 2.5-fold increases in the intensity of the 120- and 68-kDa proteins, respectively. In contrast, the AdFRNK-infected and PP2-treated cells displayed average increases of <30% for the proteins.



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  		Fig. 6. Summary of changes in tyrosine phosphorylation and cell size. Top: cardiac cell lysates were prepared from myocytes infected with either AdLacZ or AdFRNK and cultured in isotonic or hypotonic media (5 min). Polyvinylidene difluoride membranes were probed with either anti-phosphotyrosine or anti-pPaxillin Abs. Middle: summary of swelling-induced increases in tyrosine phosphorylation of the 120- and 68-kDa proteins measured with phosphotyrosine immunblot analysis. Intensity of each band was determined by densitometic analysis, and change was expressed as a value relative to intensity calculated from cells in isotonic media. Relative change in band intensity is plotted for control (uninfected and AdLacZ infected), AdFRNK-infected (FRNK), and PP2-treated (10 µM) myocytes. *P < 0.05 vs. PP2 and FRNK. Bottom: summary of changes in cell area during hypotonic stimulation. Cell area was quantified either under cell culture conditions (isotonic and hypotonic media) or during patch-clamp analysis (holding potential of –40 mV; isotonic and hypotonic Tyrode solution), and changes in cell area were determined relative to time 0 (before solution change). *P < 0.05 vs. corresponding isotonic time point; {ddagger}P < 0.05 vs. 5-min hypotonic time point.

 
Because swelling-induced changes in tyrosine phosphorylation were measured in hypotonic media, whereas activation of ICl,swell was stimulated in hypotonic Tyrode solution, it was important to determine if the changes in cell volume were the same under both experimental conditions. As displayed in Fig. 6, bottom, hypotonic-induced changes in cell volume were the same in myocytes undergoing patch-clamp analysis (in Tyrode solution) and biochemical analysis (in media). No changes in cell volume were observed in myocytes maintained in isotonic solutions (Fig. 6).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Identification of ICl,swell in neonatal rat ventricular myocytes. In the present study we tested the hypothesis that the tyrosine kinases FAK and Src are involved in regulating the cardiac ICl,swell. Hypotonic swelling of cultured neonatal rat ventricular myocytes enhanced the tyrosine phosphorylation of several proteins including those in the size range of 68–70 and 120–130 kDa. This phosphorylation was strongly reduced by pretreatment of the myocytes with the tyrosine kinase inhibitor PP2. However, cell treatment with PP2 or overexpression of the FAK inhibitor FRNK augmented the size and increased the activation rate of ICl,swell. Thus the tyrosine kinases FAK and Src contribute through a complex mechanism to the regulation of the cardiac ICl,swell.

The cardiac ICl,swell has been identified in the myocardium of a number of species including the canine (22, 32), rabbit (6), guinea pig (34), chick (38), and human (18). Although one previous study (29), with the use of a radiolabeled I– efflux assay, identified a volume-sensitive anion channel in neonatal rat ventricular myocyte cultures, the present study is the first to characterize the electrophysiological properties of ICl,swell in this preparation. The addition of hypotonic external solution to the neonatal myocytes resulted in the activation of a current with properties consistent with those of the cardiac ICl,swell (6, 22, 32). The swelling-induced current had a Erev close to the ECl, displayed an outward rectifying current versus voltage relationship under both asymmetrical and symmetrical concentrations of transmembrane Cl–, and was blocked in a voltage-dependent manner by the drugs DNDS and tamoxifen. Furthermore, activation of the current could be completely reversed on addition of hypertonic external solution. Consistent with previous studies, this deactivation of the current in hypertonic solution occurred more rapidly than did the activation phase in hypotonic solution (9). Despite these similarities, it should be noted that the sensitivity of the neonatal ICl,swell for tamoxifen and A-9-C was less than that reported for the channel in adult myocytes (4, 23, 34).

Signaling pathways involved in ICl,swell activation. Several previous investigations have examined the role of protein kinases in ICl,swell activation. It is generally accepted that neither PKA- nor PKC-mediated phosphorylation is required for the activation of ICl,swell (8, 24). The cardiac volume-sensitive chloride channel can be activated in the presence of the protein kinase inhibitors H-7 and H-8 (6, 32) as well as during cell dialysis with a peptide inhibitor of PKA (6). The activation of ICl,swell in pulmonary endothelial cells also does not involve PKA or PKC (26). The inability of isoproterenol and phorbol 12,13-dibutyrate to activate ICl,swell in the present study is consistent with these findings. Although a previous study (12) identified the CFTR Cl– channel in the neonatal rat ventricular myocytes, we found no evidence for the activation of a PKA-dependent Cl– current under the conditions of our experiments. This discrepancy may be accounted for by the different ages of the neonates (3 to 4 days old, current study; 1 day old, Ref. 12) or the length of the myocyte culture (3–4 days, current study; up to 14 days, Ref. 12) in the two studies.

Because nonreceptor tyrosine kinases are activated during mechanical and osmotic stimulation (16, 17), it was suggested that these kinases might function in ICl,swell activation (30). Sadoshima et al. (17) demonstrated that osmotic swelling of neonatal ventricular myocytes is associated with the tyrosine phosphorylation of several cardiac proteins including those with molecular sizes of ~70, 85, 120, and 130 kDa. For the majority of proteins, tyrosine phosphorylation occurred within the first minute of hypotonic media exposure and reverted back to control levels during 5–10 min of treatment with hypotonic media (17). In our experiments, increased phosphorylation occurred within 2 min of hypotonic solution addition and reached a peak within 5 min (Fig. 3). Thus the kinetics of tyrosine phosphorylation were consistent with those of the initial activation of ICl,swell. However, protein phosphorylation declined below the peak level after 10 min of hypotonic solution exposure and returned to control levels by 20 min. Therefore, at a time when ICl,swell was still activating (Fig. 1), tyrosine phosphorylation was declining back to control levels.

We identified FAK and paxillin as targets of hypotonic swelling-induced tyrosine phosphorylation. Whereas phosphorylation of FAK was previously shown to increase during osmotic swelling (17), this is the first report of swelling-induced increases in the tyrosine phosphorylation of paxillin. Both FAK and paxillin are located within cardiac focal adhesions where they form a structural link between the extracellular matrix and the actin cytoskeleton. In addition, paxillin serves as a docking site for a number of tyrosine kinases including Src, FAK, and proline-rich tyrosine kinase 2 (pyk2) (10, 33). Phosphorylation of tyrosine residues on paxillin by these kinases recruits other signaling molecules, including Crk and p130Cas, which, along with FAK, stimulate the ERK, p38, and JNK MAP kinases (10, 33). Through recruitment of ERK, pyk2, and other kinases, phosphorylation of paxillin may be an important initial step in volume-induced changes in cardiac electrical activity.

Previous studies have demonstrated that the effects of tyrosine kinase inhibition on the cardiac ICl,swell are dependent on the chemical inhibitor employed and the cardiac cell under investigation. For example, when applied before or during hypotonic stimulation, the broad-spectrum tyrosine kinase inhibitor genistein inhibits the activation of ICl,swell in dog atrial (25) and embryonic chick heart (37) cells. In contrast, when applied after ICl,swell activation, genistein enhances the current in human atrial (4) and rabbit ventricular (13) myocytes. In addition, the selective FAK and Src kinase inhibitor PP2 augments ICl,swell in human atrial (4) and rabbit ventricular (13) myocytes. Conversely, inhibition of the epidermal growth factor receptor kinases suppresses ICl,swell in these cardiac cells (4, 13). On the basis of the later two findings, it was proposed that receptor-coupled and nonreceptor families of tyrosine kinases may have opposite actions on the volume-sensitive chloride channel (4).

In the present study, pretreatment of the neonatal myocytes with PP2 diminished tyrosine phosphorylation in the presence of hypotonic solution. Therefore, it was anticipated that PP2 would inhibit the activation of ICl,swell. However, application of PP2 or overexpression of FRNK, which also inhibited tyrosine phosphorylation, increased the size of ICl,swell and enhanced the rate of current activation. This suggests that there is not a simple correlation between cellular tyrosine phosphorylation (as measured in Fig. 3) and the activation of ICl,swell in the neonatal myocytes. This conclusion is supported by two additional findings. First, decreases in the basal tyrosine phosphorylation state of the myocyte, measured before addition of hypotonic media, did not result in the activation of ICl,swell. When compared with the control myocytes (untreated or AdLacZ infected), treatment with PP2 or overexpression of FRNK decreased the amount of basal tyrosine phosphorylation (Figs. 3 and 6) but did not activate ICl,swell in the absence of hypotonic swelling (Figs. 4 and 5). Second, PP2 was able to augment the fully activated, steady-state ICl,swell at a time point (15–20 min after hypotonic solution addition) when tyrosine phosphorylation had already declined back to control levels. However, because of the limitations of the phosphotyrosine immunoblot analysis, it cannot be ruled out that both osmotic swelling and FAK inhibition modified the tyrosine phosphorylation of an important target protein that was not detected in our study. Significant changes in tyrosine phosphorylation may also have occurred within several seconds of hypotonic media addition that were not assayed with our procedure. Finally, it is likely that other proteins, including MAP kinases and Rho GTPases that regulate volume-sensitive channels (9), are stimulated during cardiac cell swelling (17). The role of these signaling molecules in the regulation of the cardiac ICl,swell will require further study.

Limitations of study. There were a number of technical limitations in the present study. The ICl,swell recordings were obtained at room temperature. It is expected that reducing the temperature of the external solutions from physiological to room temperature would alter cellular tyrosine phosphorylation, as well as other signaling events involved in ICl,swell activation. To limit the number of solution changes in ICl,swell recording procedure, exposure of the myocytes to hypotonic solution involved a reduction in the external NaCl concentration from 132 to 90 mM. Reductions in extracellular sodium, by reversing the direction of the sarcolemmal Na+/Ca2+ exchanger, increase intracellular Ca2+ in rat ventricular myocytes (11). Reducing extracellular sodium from 145 to 70 mM enhances KCl-induced increases in intracellular Ca2+ by ~20 nM in these myocytes (11). The reduction in the NaCl concentration also decreased the ionic strength of the external solution. Decreases in intracellular ionic strength activate ICl,swell in endothelial cells, independent of changes in cell volume (9). If changes in ionic strength shift the set point of a hypothetical volume sensor, it is possible that reductions in extracellular ionic strength might also affect ICl,swell activation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-45789 and grants from the American Heart Association.


   ACKNOWLEDGMENTS
 
We thank Kathryn J. Long for preparing the neonatal myocytes used in the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. B. Walsh, Dept. of Pharmacology, Physiology, and Neuroscience, School of Medicine, Univ. of South Carolina, Columbia, SC 29208 (e-mail: walsh{at}med.sc.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Browe DM and Baumgarten CM. Stretch of {beta}1 integrin activates an outwardly rectifying chloride current via FAK and Src in rabbit ventricular myocytes. J Gen Physiol 122: 689–702, 2003.[Abstract/Free Full Text]
  2. Calalb MB, Polte TR, and Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol 15: 954–963, 1995.[Abstract]
  3. Cheng Q, Ross RS, and Walsh KB. Overexpression of the integrin {beta}1A subunit and the {beta}1A cytoplasmic domain modifies the {beta}-adrenergic regulation of the cardiac L-type Ca2+ current. J Mol Cell Cardiol 36:809–819, 2004.[CrossRef][ISI][Medline]
  4. Du X, Gao Z, Lau C, Chiu S, Tse H, Baumgarten CM, and Li G. Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases. J Gen Physiol 123: 427–439, 2004.[Abstract/Free Full Text]
  5. Eble DM, Strait JB, Govindarajan G, Lou J, Byron KL, and Samarel AM. Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase. Am J Physiol Heart Circ Physiol 278: H1695–H1707, 2000.[Abstract/Free Full Text]
  6. Hagiwara N, Masuda H, Shoda M, and Irisawa H. Stretch-activated anion currents of rabbit cardiac myocytes. J Physiol (Lond) 456: 285–302, 1992.[Abstract/Free Full Text]
  7. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth J. Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[CrossRef][ISI][Medline]
  8. Hume JR, Duan D, Collier ML, Yamazaki J, and Horowitz B. Anion transport in heart. Physiol Rev 80: 31–81, 2000.[Abstract/Free Full Text]
  9. Nilius B and Droogmans G. Amazing chloride channels: an overview. Acta Physiol Scand 177: 119–147, 2003.[CrossRef][ISI][Medline]
  10. Parsons JT. Focal adhesion kinase: the first ten years. J Cell Sci 116: 1409–1416, 2003.[Abstract/Free Full Text]
  11. Rathi SS, Saini HK, Xu YJ, and Dhalla NS. Mechanisms of low sodium-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes. Mol Cell Biochem 263: 151–162, 2004.[CrossRef][ISI][Medline]
  12. Reisin IL, Prat AG, Abraham EH, Amara JF, Gregory RJ, Ausiello DA, and Cantiello HF. The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J Biol Chem 269: 20584–20591, 1994.[Abstract/Free Full Text]
  13. Ren Z and Baumgarten CM. Antagonistic regulation of swelling-activated Cl– current in rabbit ventricle by Src and EGFR protein tyrosine kinases. Am J Physiol Heart Circ Physiol 288: H2628–H2636, 2005.[Abstract/Free Full Text]
  14. Riordan JR. Assembly of functional CFTR chloride channels. Ann Rev Physiol 67: 701–718, 2005.[CrossRef][ISI][Medline]
  15. Roos K. Length, width, and volume changes in osmotically stressed myocytes. Am J Physiol Heart Circ Physiol 251: H1373–H1378, 1986.[Abstract/Free Full Text]
  16. Sadoshima J and Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J 12: 1681–1692, 1993.[ISI][Medline]
  17. Sadoshima J, Qiu Z, Morgan JP, and Izumo S Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes. EMBO J 15: 5535–5546, 1996.[ISI][Medline]
  18. Sakai R, Hagiwara N, Kasanuki H, and Hosoda S. Chloride conductance in human atrial cells. J Mol Cell Cardiol 27: 2403–2408, 1995.[CrossRef][ISI][Medline]
  19. Shi C, Barnes S, Coca-Prados M, and Kelly ME. Protein tyrosine kinase and protein phosphatase signaling pathways regulate volume-sensitive chloride currents in a nonpigmented ciliary epithelial cell line. Invest Ophthalmol Vis Sci 43: 1525–1532, 2002.[Abstract/Free Full Text]
  20. Shuba YM, Prevarskaya N, Lemonnier L, Van Coppenolle F, Kostyuk P, Mauroy B, and Skryma R. Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line. Am J Physiol Cell Physiol 279: C1144–C1154, 2000.[Abstract/Free Full Text]
  21. Simpson DC, Terracio L, Terracio M, Price RL, Turner DC, and Borg TK. Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix. J Cell Physiol 161: 89–105, 1994.[CrossRef][ISI][Medline]
  22. Sorota S. Swelling-induced chloride-sensitive current in canine atrial cells revealed by whole-cell patch-clamp method. Circ Res 70: 679–687, 1992.[Abstract/Free Full Text]
  23. Sorota S. Pharmacologic properties of the swelling-induced chloride current of dog atrial myocytes. J Cardiovasc Electrophysiol 5: 1006–1016, 1994.[ISI][Medline]
  24. Sorota S. Insights into the structure, distribution and function of the cardiac chloride channels. Cardiovasc Res 42: 361–376, 1999.[Abstract/Free Full Text]
  25. Sorota S. Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current. Pflügers Arch 431: 178–185, 2005.
  26. Szucs G, Heinke S, De Greef C, Raeymackers L, Eggermont J, Droogmans G, and Nilius B. The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation. Pflügers Arch 431: 540–548, 1996.[ISI][Medline]
  27. Taylor JM, Rovin JD, and Parsons JT. A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes. J Biol Chem 275: 19250–19257, 2000.[Abstract/Free Full Text]
  28. Thomas SM and Brugge JS. Cellular functions regulated by Src family kinases. Ann Rev Cell Dev Biol 13: 513–609, 1997.[CrossRef][ISI][Medline]
  29. Tilly BC, Bezstarosti K, Boomaars WE, Marino CR, Lamers JMJ, and de Jonge HR. Expression and regulation of chloride channels in neonatal rat cardiomyocytes. Mol Cell Biochem 157: 129–135, 1996.[ISI][Medline]
  30. Tilly BC, van den Berghe N, Tertoolen LGJ, Edixhoven MJ, and de Jonge HR. Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances. J Biol Chem 268: 19919–19922, 1993.[Abstract/Free Full Text]
  31. Trouet D, Carton I, Hermans D, Droogmans G, Nilius B, and Eggermont J. Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains. Am J Physiol Cell Physiol 281: C248–C256, 2001.[Abstract/Free Full Text]
  32. Tseng GN. Cell swelling increases membrane conductance of canine cardiac cells: evidence for a volume-sensitive Cl– channel. Am J Physiol Cell Physiol 262: C1056–C1068, 1992.[Abstract/Free Full Text]
  33. Turner CE. Paxillin and focal adhesion signalling. Nature Cell Biol 2: E231–E236, 2000.[CrossRef][ISI][Medline]
  34. Vandenberg JI, Yoshida A, Kirk K, and Powell T. Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology. J Gen Physiol 104: 997–1017, 1994.[Abstract/Free Full Text]
  35. Voets T, Manlolpoulos V, Eggermont J, Ellory C, Droogmans G, and Nilius B. Regulation of a swelling-activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins. J Physiol (Lond) 506: 341–352, 1998.[Abstract/Free Full Text]
  36. Walsh KB and Parks GE. Changes in cardiac myocyte morphology alter the properties of voltage-gated ion channels. Cardiovasc Res 55: 64–75, 2002.[Abstract/Free Full Text]
  37. Wei H, Mei YA, Sun JT, Zhou HQ, and Zhang ZH. Regulation of swelling-activated chloride channels in embryonic chick heart cells. Cell Res 13: 21–28, 2003.[CrossRef][ISI][Medline]
  38. Zhang J, Rasmusson RL, Hall SK, and Lieberman M. A chloride current associated with swelling of cultured chick heart cells. J Physiol (Lond) 472: 801–820, 1993.[Abstract/Free Full Text]



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