Swelling-Activated Chloride Currents In The Heart: Regulation And Function

IntroductionA volume -sensitive Cl- current,ICl, swell, is expressed throughout the heart and in many other tissues. It is stimulated by cell swelling and/or membrane deformation, exhibits outwardly rectifying, partially inactivates at positive potentials, reverses between the plateau and resting potentials (Em) ,is time - independent over the physiologic voltage range and is blocked by tamoxifen, biophysical and pharmacologic characteristics that distinguish ICl, swell from other Cl~ currents. Under isosmotic conditions, ICl, swell contributes to the background Cl-current and is activated in models of cardiac disease. Functionally, the activation of ICl,swell influences both cardiac electrical activity and cell volume.The signaling underlying the activation of ICl,swell is complex, and evidence implicates protein kinase C, protein kinase A, and protein tyrosine kinase (PTK) in its regulation in heart and other tissues. PTK is activated by osmotic swelling of myocytes within 5 s and, therefore, is well - positioned to be an early step in the signaling process. Although substantial evidence indicates that phosphorylation and dephosphorylation of tyrosine residues are involved in the control of ICl,swell, the details remain obscure.The FTP inhibitor orthovanadate reduced ICl,swell and precluded its activation by Src inhibition. Src activity is not, however, the primary factor that controls the response of ventricular ICl, swell to osmotic stress. It has been revealed that specific inhibition of EGF receptor kinase causes a suppression of the ICl, Swell in human atrial myocytes and Src stimulating the regualtion of ICl,swell might be due to a receptor - mediated PTK such as epidermal growth factor receptor (EGFR) tyrosine kinase. The EGFR has been implicated in the development of myocyte hypertrophy and load - dependent cardiac hypertrophy in vivo. However, theprecise role of EGFR and Src regulating the activation of ICl,swell ventricular myocytes remains undefined.The renin -angiotensin (RAS) plays an important role in the regulation of cardiovascular functions. Enhanced RAS activity has been reported in several cardiovascular diseases, such as hypertension, diabetes, coronary insufficiency, congestive heart failure. The effects of angiotensin(Ang) Ⅱ are mediated primarily through its type I receptor which is a G protein - couple receptor that mediates most of the known biologic effects of Ang Ⅱ. Ang Ⅱ stimulates ·O2- production through NAD(P)H oxidase (NOX) activation. Stimulation of AT, receptor involves the generation of oxygen - derived free radicals, which has detrimental effects. The expression of AT, is elevated in cardiovascular tissue from hypertension and LV hypertrophy. Ang II is known to directly stimulate tyrosine phosphorylation and NAD(P)H oxidase activation. PKC and losartan regulated the Ang Ⅱ - stimulated Cl- current in rabbit sino - atrial node. All these data bring up the possibility that AT1 receptors might be involved in the activation ofICl,swell.Recently we identified another signaling cascade that participates in the activation of ICl,swell in response to stretching β1 integrins. Integrins stretch elicits ICl,swell in rabbit ventricular myocytes via Src and the angiotensin Ⅱ ( Angll) AT, receptor signaling cascade, which involves transactivation of EGFR kinase, phosphatidylinositol -3 - kinase( PI -3K) ,and generation of superoxide anion · O2- , by sarcolemmal NAD(P) H oxidase,and ultimately H2O2. It is long -established that Angll is released by mechanical stretch of cultured myocytes and induces hypertrophy. The downstream components of this pathway and the critical role of reactive oxygen species ( ROS) has been extensively studied in vascular smooth muscle and endothelium and cardiac myocytes. NADPH oxidase is a protein that transfers electron from NAD(P)H to an electron acceptor leading to the formation of ROS. NAD(P) H oxidase is also a membrane - bound enzyme that catalyzes the electron reduction of oxygen,with NADH or NAD(P)H as el-cetron donors. We can block the activity of the NAD(P)H oxidase with diphe-nylene iodonium ( DPI) which inhibits ROS formation and apocynin which blocks NOX assembly. NOX and its production of ROS are inhibited by applica-tion of DPI,an specific inhibitor of NAD(P)H oxidase activity which prevents ROS accumulation and blocks NOX electron transport. It has been revealed that Angiotensin Ⅱ -induced cardiac hypertrophy was regulated by NAD(P)H oxidase - derived superoxide anion.A volume -sensitive Cl current,ICl,swell ,is activated in heart failure and ischemia under isosmotic (1T) conditions and by osmotic swelling. In fact the modulation of ICl,swell signaling cascades is incompletely understood and may be species and tissue dependent. Although ICl,swell is evoked both by stretching β1 in-tegrins and by osmotic swelling, these stimuli are distinct. The goal of the present study was to determine whether activation of ICl,swell by osmotic swelling depends upon the same Angll - ROS signaling pathway that is engaged upon stretch of β1 integrins. We found that activation of ICl,swell by osmotic swelling was abrogated by inhibition of AT1. receptors, EGFR kinase, PI -3K, or NAD (P)H oxidase and by scavenging H2O2 with catalase. Moreover, exogenous EGF elicited ICl,swell, as was previously demonstrated with exogenous Angll and H2O2. Taken together,these data argue that the Angll - ROS signaling cascade participates in response to osmotic swelling. These findings also are likely to have implications for cardiac pathophysiology.MATERIALS AND METHODSRabbits and Ventricular Myocyte Isolation Left ventricular myocytes were freshly isolated from New Zealand white rabbits (3 kg). Hearts were excised using methods approved by the Institutional Animal Care and Use Committee, mounted on a Langendorff apparatus, and initially perfused with 37 ℃ oxygenated Tyrode solution containing (in mM) : 130 NaCl, 5 KCl, 3 MgCl2, 1. 8 CaCl2, 0.4 KH2PO4, 5 HEPES, 15 taurine, 5 creatine, 10 glucose, pH7.25. Then, after a 5 — min perfusion with Ca 2+ -free Tyrode solution containing 0.1 mM Na2EGTA, the perfusate was switched to Ca 2+ - free Tyrode solution containing 0.4-0.5 mg · mL-1 collagenase (type Ⅱ; Worthington) , 0.05mg · mL-1 pronase (type XIV; Sigma - Aldrich) , and 1. 5 mg · mL-1 BSA (Sigma -Aldrich ). At selected intervals (10 ~ 20 min) , portions of the left ventricle wereexcised, cut into strips, placed in test tubes, and gently agitated. Following filtration through nylon mesh to remove debris, myocytes were washed twice and stored in modified KB solution containing (in mM) : 120 K glutamate, 10 KCl, 10 KH2PO4, 1.8 MgSO4, 0.5 K2EGTA, 10 taurine, 20 glucose, 10 mannitol, and 10 HEPES (pH 7. 2; 295 mOsmol · L-1). Myocytes were used within 8 h of isolation, and only rod - shaped quiescent cells with well - defined regular striations and no evidence of membrane blebbing were selected for study.Experimental solutions and drugs Cells were placed in a poly -1 - lysine - coated glass - bottomed chamber (0. 3 ml) mounted on an inverted microscope (Diaphot; Nikon) and were supervised with bathing solution at 2 -3 ml · min-1; solution changes were complete within 10s. Anion currents were i-solated by replacing Na + and K + in the bathing media with equimolar amounts of N - methyl - d - glucamine ( NMDG) and adding Cs + to the bath and pipette solutions. Standard bathing solution contained (in mM) : 90 NMDG - Cl, 3 MgCl2, 4.63 CaCl2, 5 Cs2EGTA, 10 HEPES, 10 glucose, and 0 to 100 mannitol (pH 7.4). This gives a free - Ca2+ of 1. 5 μM (WinMaxC 2.4; www. Stanford, edu/ ~ cpatton/maxc. html). In some experiments ,0.2 mM CdCl2 or both 1 mM BaCl2 and 0.2 mM CdCl2 were added to the bath solution. This also necessitated omission of Cs2EGTA (replaced by 5 mM CsCl) , and bath CaCl2 was reduced to 0.1 mM. Experimental solutions were designed to allow adjustment of osmolarity with mannitol at a constant ionic strength. Isosmotic (IT) solution was set as 300 mOsmol · L-1. An osmometer ( Osmette S; Precision Systems) was used to routinely verify solution composition.Tamoxifen (10 mM) , genistein (100mM), the pyrazolopyrimidines PP2 (10mM) and PP3 (10mM), and PD153035 (250 μM),Diphenyleneiodonium chloride(DPI,30mM) , Acetovanillone( Apocynin100mM) , 4 - (3 - Chloroani-lino) -6,7 -dimethoxyquinazoline (AG1478,10mM) were dissolved in 0. 1% dimethyl sulfoxide (DMSO) at the indicated concentrations and kept frozen ( -20℃ in aliquots until use. Cs3VO4(orthovanadate) was added directly to bath solution. Losarten -K (Merck,Rahway,NJ,U. S. A) was prepared as stock solution in deionized water and kept frozen ( -20 ℃). Catalase was dissolved directly in bath solution according to 1000 units per ml bath solution. Tamoxifen,orthovanadate, DPI and DMSO were from Sigma - Aldrich, and the remaining a-gents were from Calbiochem.The inhibitor, gp91ds - tat, is a fusion peptide made from a 9 - mer, CSTRIRRQL, that inhibits assembly of NAD(P) H oxidase by mimicking the gp91phox(Nox2) docking site for the cytoplasmic p47phox subunit and a 9 - mer from the tat HIV coat - protein that confers membrane permeability. The gp91phox docking site sequence is identical in a number of experimental species including rabbit, but experimental evidence and homology with other NAD(P)H isoforms suggest that gp91 - TAT may not distinguish between NOX isoform. The inactive fusion peptide, scamb - TAT, was constructed by scrambling the 9 - mer docking sequence so as to minimize matches in the GenBank database. Stock solutions of the peptides (1.2 mg ·mL-1) were made in 150 mM NaCl plus 10 mM acetic acid and kept frozen ( - 20 ℃ ) in small aliquots until use.Electrophysiological recordings Patch electrodes were made from thin -walled 7740 borosilicate glass (Sutter) and fire polished (initial resistance,2 ~ 3MΩ). Standard electrode filling solution contained (mM) : 110 Cs aspartate, 20 CsCl, 2.5 MgATP, 8 Cs2EGTA, 0.15 CaCl2, 10 HEPES, pH 7.1 (liquid junction potential ,11.5 ±0.7 mV; n = 9 ). For some experiments,a high Cl-pipette solution was used; it contained (mM) : 31.7 Cs aspartate, 98.3 CsCl, 2.5 MgATP, 8 Cs2EGTA, 0. 15 CaCl2, 10 HEPES, pH 7. 1 (liquid junction potential,6.5 ±0.5 mV; n=5). This gave a free -Ca2+ of 60 nM for both pipette solutions. A 3M KCl agar bridge was used as ground. Seal resistances of 5 ~ 30 GΩ were typically achieved, and the measured junction potential was subtracted before seal formation.Whole - cell currents recorded with an Axoclamp 200A or 200B amplifier (Axon) were low - pass filtered at 2 kHz (Bessel) and digitized at 5 kHz. Myocytes were dialyzed for 10 min before the recordings commenced. Voltage -clamp protocols and data acquisition were governed by a Digidata 1321A and pClamp 8.0 ( Axon). Successive 500 - ms voltage steps were made from a holding potential of - 60 mV to test potentials ranging from -100 to + 60 or +100 mV in +10 mV increments. Current - voltage (I - V) relationships were plotted from the quasi steady - state current, except for Figs. 1 and 2,which showcurrents at 25 ms. Capacitance was calculated in pClamp using a 5mV step.Preliminary studies established that ICl,swell fully activated in < 5 min and remained stable for at least 45 min. All interventions were applied for a time sufficient for currents to reach a steady state as judged from Ⅰ - Ⅴ curves taken at selected intervals.Statistics Data are expressed as mean ± SEM, and n refers to the number of cells. Mean currents are presented as current density ( pA ? pF ) to account for differences in myocyte surface membrane area. Statistical analyses were done using SigmaStat 2. 3 or 3. 0 (Systat). For multiple comparisons, a repeated measures ANOVA was used, and the Student - Newman - Keuls test was performed to compare groups. In one case, a Friedman Repeated measures ANOVA on ranks was applied instead because the data set was not normally distributed. Fractional block, reported as a percentage, was calculated using each myocyte as its own control. Statistical significance was taken as P < 0. 05.ResultsIn solutions designed to isolate anion currents, osmotic swelling activates an outwardly -rectifying, tamoxifen -sensitive Cl- current,ICl,swell, , in isolated rabbit ventricular myocytes under whole - cell patch clamp conditions that isolated Cl- currents. Regulation of the swelling - activated chloride current (ICl,swell) is complex and multiple signaling cascades have been implicated. Stretch of myocytes has long been known to release angiotensin Ⅱ, which binds AT1 receptors and acts on myocytes in an autocrine - paracrine loop and signals via Src and EGFR kinase in a multistep pathway ultimately leading to the formation of reactive oxygen species (ROS). To determine whether angiotensin Ⅱ AT1 receptors, EGFR kinase, Src, and ultimately sarcolemmal NADPH oxidase and reactive oxygen species modulates ICl,swell, we assessed the role of this signaling cascades in ICl,swell activation by hypoosmotic swelling (0.7T) after isolating Cl- current. In order to know the effect of AT1 receptors and their downstream signaling cascades participating in mechanotransduction, we use Losartan, the AT1 receptor competitive antagonist. ICl,swell evoked in 0.7T was inhibited 110 ± 13% (n =4, p < 0.002) at +60 mV by 30 μM Losartan. AT1 stimulation causes EGF receptor transactivation. AG1478 (1μM) and PD153035 (20 nM) , specific EG-FR kinase inhibitors,blocked 101 ± 6%(n = 5,p<0.001) and 97 ± 2% (n = 4,p <0. 002) of ICl, swell, respectively. PI -3K is downstream of EGFR kinase, two selective PI -3 kinase inhibitors, worbnannin and LY 294002, inhibited ICl,swell by 60 ± 2% (n = 6, p < 0.001) and 99 ± 7% (n=4, p<0. 001) seperately. EGFR kinase activates NOX, and NOX inhibitors blocked ICl,swell. The NOX electron transport blocker diphenyliodonium (DPI; 60 μM) completely inhibited ICl,swell 116 ± 16% (n = 5, p <0.004) ,and the NOX assembly blocker apocynin (500 μM) inhibited 84 ± 12 % (n =5 ,p <0.001). NOX produces · O2- that is converted to H2O2- Catalase (1000U · mL-1) ,an H2O2 scavenger, blocked 90 ± 11% of ICl,swell. Mechanotransduction involved protein tyrosine kinase. PTK potentially has multiple roles in signaling. ICl,swell was enhanced 181 ± 17% (n =5, p<0.001) by genistein (100 μM), a broad — spectrum PTK blocker, and the swelling - and genistein - stimulated current was fully inhibited by 10 μM tamoxifen, a selective potent blocker of ICl,swell that is able to distinguish this current from the other major cardiac Cl-currents. Src is the principal upstream protein tyrosine kinase, we tested whether this specific tyrosine kinase mediated the response to osmotic swelling. 10 μM PP2, a selective block of Src family PTK augmented outwardly rectifying ICl,swell by 234 ± 27% (n = 6, p< 0.001) in physiological and 217 ± 13% (n = 6, p <0.001) in symmetrical high Cl~ solutions. In contrast,whereas its inactive analogue PP3 had no effect (n = 6,ns).DiscussionThe present study provides the first evidence that ICl,swell in osmotically swollen ventricular myocytes is regulated in an opposing fashion by the Src and EGFR kinase families of PTK and by the Angiotensin II signaling cascade and NADPH oxidase. PTK are well placed to be sensors of cell volume and mechanical stretch. These signaling molecules interact with cytoskeleton, integral membrane proteins,and the sarcolemma,and tyrosine phosphorylation is among theearliest responses to osmotic swelling in cardiac myocytes and other cells.Antagonistic regulation of ICl,swell by Src and EGFR kinase ICl,swell was enhanced by the selective Src family inhibitor PP2. Blocking PTP and thereby tyrosine dephosphorylation with orthovanadate inhibited ICl,swell 0.7T, an effect opposite to that obtained by blocking Src - dependent tyrosine phosphorylation. Ultimately, stimulation of ICl,swell upon block of Src must result from accumulation of critical tyrosine residues in the dephosphorylated state. Consistent with this i-dea, suppressing the rate of dephosphorylation by PTP with orthovanadate also precluded ICl,swell stimulation by PP2.Regulation of ICl,swell critically depended on EGFR kinase, a second distinct family of PTK. PD 153035, an extremely potent and selective inhibitor of EGFR kinase,completely suppressed ICl,swell in 0.7T. AG1478,the less potent chloro -derivative of PD153035, also fully inhibited ICl,swell in rabbit ventricular myocytes. The antagonistic effect of inhibiting Src and EGFR kinase PTK families suggests that at least two distinct tyrosine residues that are phosphorylated by Src and EGFR kinase, respectively, must be involved in the regulation of ICl,swell in rabbit ventricle. Orthovanadate inhibited IC1,swell, which is expected if the PTP inhibitor primarily opposed the action of Src rather than EGFR kinase, however. This suggests that the Src family PTK site may be dominant or that orthovanadate differentially modulates the dephosphorylation of the targets of these two PTK families.Regulation of ICl,swell by the Angiotensin II signaling cascade and NAD-PH oxidase Blocking AngII AT1 receptors with losartan, a competitive antagonist, inhibits the activation of ICl,swell upon osmotic swelling. EGFR kinase is downstream from ATt receptors in the Angll - ROS signaling cascade. This member of the receptor protein tyrosine kinase family undergoes transactivation upon AT1 receptor occupancy. Transactivation of EGFR kinase is a required step in the cascade leading to ICl,swell activation, exogenous EGF elicits a volume -sensitive Cl- current under isosmotic conditions. PI -3K is downstream of EGFR kinase in the Angll - ROS signaling cascade and required for NAD( P) H oxidase activation. Activation of AT1 receptors ultimately generates ROS by initiating assembly of sarcolemmal NAD(P) H oxidase from cytoplasmic and mem-...