The ICl,swell inhibitor DCPIB blocks Kir channels that possess weak affinity for PIP2.

Inwardly rectifying K(+) (Kir) channels are important contributors to the resting membrane potential and regulate cellular excitability. The activity of Kir channels depends critically on the phospholipid PIP2. Several modulators of the activity of Kir channels alter the apparent affinity of the channel to PIP2. Channels with high apparent affinity to PIP2 may not respond to a given modulator, but mutations that decrease such affinity can render the channel susceptible to modulation. Here, we identify a known inhibitor of the swelling-activated Cl(-) current, DCPIB, as an effective inhibitor of a number of Kir channels both in native cardiac cells and in heterologous expression systems. We show that the apparent affinity to PIP2 determines whether DCPIB will serve as an efficient blocker of Kir channels. These effects are consistent with a model in which DCPIB competes with PIP2 for a common binding site.

Transient complex I inhibition at the onset of reperfusion by extracellular acidification decreases cardiac injury.

A reversible inhibition of mitochondrial respiration by complex I inhibition at the onset of reperfusion decreases injury in buffer-perfused hearts. Administration of acidic reperfusate for a brief period at reperfusion decreases cardiac injury. We asked if acidification treatment decreased cardiac injury during reperfusion by inhibiting complex I. Exposure of isolated mouse heart mitochondria to acidic buffer decreased the complex I substrate-stimulated respiration, whereas respiration with complex II substrates was unaltered. Evidence of the rapid and reversible inhibition of complex I by an acidic environment was obtained at the level of isolated complex, intact mitochondria and in situ mitochondria in digitonin-permeabilized cardiac myocytes. Moreover, ischemia-damaged complex I was also reversibly inhibited by an acidic environment. In the buffer-perfused mouse heart, reperfusion with pH 6.6 buffer for the initial 5 min decreased infarction. Compared with untreated hearts, acidification treatment markedly decreased the mitochondrial generation of reactive oxygen species and improved mitochondrial calcium retention capacity and inner mitochondrial membrane integrity. The decrease in infarct size achieved by acidic reperfusion approximates the reduction obtained by a reversible, partial blockade of complex I at reperfusion. Extracellular acidification decreases cardiac injury during reperfusion in part via the transient and reversible inhibition of complex I, leading to a reduction of oxyradical generation accompanied by a decreased susceptibility to mitochondrial permeability transition during early reperfusion.

Hypercholesterolemia induces up-regulation of KACh cardiac currents via a mechanism independent of phosphatidylinositol 4,5-bisphosphate and Gßγ.

Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of K(ACh) mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on K(ACh) is unknown. Here we show that cholesterol plays a critical role in modulating K(ACh) currents (I(K,ACh)) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed I(K,ACh). Moreover, a high-cholesterol diet resulted in up to 3-fold increase in I(K,ACh) in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies I(K,ACh). Furthermore, our data suggest that cholesterol affects I(K,ACh) via a mechanism which is independent of both PI(4,5)P(2) and Gßγ. Interestingly, the effect of cholesterol on I(K,ACh) is opposite to its effect on I(K1) in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating I(K,ACh) in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.

HIV protease inhibitors elicit volume-sensitive Cl- current in cardiac myocytes via mitochondrial ROS.

HIV protease inhibitors (HIV PI) reduce morbidity and mortality of HIV infection but cause multiple untoward effects. Because certain HIV PI evoke production of reactive oxygen species (ROS) and volume-sensitive Cl(-) current (I(Cl,swell)) is activated by ROS, we tested whether HIV PI stimulate I(Cl,swell) in ventricular myocytes. Ritonavir and lopinavir elicited outwardly rectifying Cl(-) currents under isosmotic conditions that were abolished by the selective I(Cl,swell)-blocker DCPIB. In contrast, amprenavir, nelfinavir, and raltegravir, an integrase inhibitor, did not modulate I(Cl,swell) acutely. Ritonavir also reduced action potential duration, but amprenavir did not. I(Cl,swell) activation was attributed to ROS because ebselen, an H(2)O(2) scavenger, suppressed ritonavir- and lopinavir-induced I(Cl,swell). Major ROS sources in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin failed to block ritonavir- or lopinavir-induced currents, although it blocks I(Cl,swell) elicited by osmotic swelling or stretch. In contrast, rotenone, a mitochondrial e(-) transport inhibitor, suppressed both ritonavir- and lopinavir-induced I(Cl,swell). ROS production was measured in HL-1 cardiomyocytes with C-H(2)DCFDA-AM and mitochondrial membrane potential (ΔΨ(m)) with JC-1. Flow cytometry confirmed that ritonavir and lopinavir but not amprenavir, nelfinavir, or raltegravir augmented ROS production, and HIV PI-induced ROS production was suppressed by rotenone but not NADPH oxidase blockade. Moreover, ritonavir, but not amprenavir, depolarized ΔΨ(m). These data suggest ritonavir and lopinavir activated I(Cl,swell) via mitochondrial ROS production that was independent of NADPH oxidase. ROS-dependent modulation of I(Cl,swell) and other ion channels by HIV PI may contribute to some of their actions in heart and perhaps other tissues.

Regulation of swelling-activated Cl(-) current by angiotensin II signalling and NADPH oxidase in rabbit ventricle.

AIMS: We assessed whether hypoosmotic swelling of cardiac myocytes activates volume-sensitive Cl(-) current (I Cl,swell) via the angiotensin II (AngII)-reactive oxygen species (ROS) signalling cascade. The AngII-ROS pathway previously was shown to elicit I(Cl,swell) upon mechanical stretch of beta(1D) integrin. Integrin stretch and osmotic swelling are, however, distinct stimuli. For example, blocking Src kinases stimulates swelling-induced but inhibits stretch-induced I Cl,swell. METHODS AND RESULTS: I Cl,swell was measured in rabbit ventricular myocytes by whole-cell voltage clamp. Swelling-induced I Cl,swell was completely blocked by losartan and eprosartan, AngII type I receptor (AT1) antagonists. AT1 stimulation transactivates epidermal growth factor receptor (EGFR) kinase. Blockade of EGFR kinase with AG1478 abolished both I Cl,swell and AngII-induced Cl(-) current, whereas exogenous EGF evoked a Cl(-) current that was suppressed by osmotic shrinkage. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase, and PI-3K inhibitors LY294002 and wortmannin blocked I Cl,swell. Ultimately, AngII signals via NADPH oxidase (NOX) and superoxide anion, O2*. NOX inhibitors, diphenyleneiodonium, apocynin and gp91ds-tat, eliminated I Cl,swell, whereas scramb-tat, an inactive gp91ds-tat analogue, was ineffective. O2* rapidly dismutates to H2O2. Consistent with H2O2 being a downstream effector, catalase inhibited I Cl,swell, and exogenous H2O2 overcame suppression of I Cl,swell by AT1 receptor, EGFR kinase, and PI-3K blockers. H2O2-induced current was not blocked by osmotic shrinkage, however. CONCLUSION: Activation of I Cl,swell by osmotic swelling is controlled by the AngII-ROS cascade, the same pathway previously implicated in I Cl,swell activation by integrin stretch. This in part explains why I Cl,swell is persistently activated in several models of cardiac disease.

Noradrenaline up-regulates volume-regulated chloride current by PKA-independent cAMP/exchange protein activated by cAMP pathway in human atrial myocytes.

BACKGROUND AND PURPOSE: Adrenergic regulation of cell volume-regulated chloride current (ICl.vol ) is species-dependent. The present study investigates the mechanism underlying adrenergic regulation of ICl.vol in human atrial myocytes. EXPERIMENTAL APPROACH: Conventional whole-cell patch voltage-clamp techniques were used to record membrane current in human atrial myocytes. ICl.vol was evoked by hyposmotic bath solution (0.6 times isosmotic, 0.6 T). KEY RESULTS: ICl.vol was augmented by noradrenaline (1 µM) during cell swelling in 0.6 T but not under isosmotic (1 T) conditions. Up-regulation of ICl.vol in 0.6 T was blocked by the ß-adrenoceptor antagonist propranolol (2 µM), but not by the α1 -adrenoceptor antagonist prazosin (2 µM). This ß-adrenergic response involved cAMP but was independent of PKA; the protein kinase inhibitor H-89 (2 µM) or PKI (10 µM in pipette solution) failed to prevent ICl.vol up-regulation by noradrenaline. Moreover, the PI3K/PKB inhibitor LY294002 (50 µM) and the PKG inhibitor KT5823 (10 µM) did not affect noradrenaline-induced increases in ICl.vol . Interestingly, the exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2'-O-Me-cAMP (50 µM) also up-regulated ICl.vol , and the noradrenaline-induced increase of ICl.vol in 0.6 T was reversed or prevented by the Epac inhibitor ESI-09 (10 µM). CONCLUSION AND IMPLICATIONS: These data show that ICl.vol evoked by cell swelling of human atrial myocytes is up-regulated by noradrenaline via a PKA-independent cAMP/Epac pathway in human atrial myocytes. cAMP/Epac-induced ICl.vol is expected to shorten action potential duration during human atrial myocytes swelling and may be involved in abnormal cardiac electrical activity during cardiac pathologies that evoke ß-adrenoceptor signalling.

EGFR kinase regulates volume-sensitive chloride current elicited by integrin stretch via PI-3K and NADPH oxidase in ventricular myocytes.

Stretch of beta1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl(-) SAC resembles the volume-sensitive Cl(-) current (I(Cl,swell)). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl(-) SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for beta1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl(-) SAC that was 1.13 +/- 0.10 pA/pF at +40 mV. AG1478 (10 muM), an EGFR kinase blocker, inhibited 93 +/- 13% of Cl(-) SAC, and intracellular pretreatment with 1 muM AG1478 markedly suppressed Cl(-) SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl(-) current (0.81 +/- 0.05 pA/pF at +40 mV) that was fully blocked by 10 muM tamoxifen, an I(Cl,swell) blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 microM), blockers of PI-3K, inhibited Cl(-) SAC by 67 +/- 6% and 91 +/- 25% respectively, and the EGF-induced Cl(-) current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl(-) current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl(-) SAC and EGF-induced Cl(-) current by 88 +/- 9% and 127 +/- 11%, respectively. These results suggest that beta1 integrin stretch activates Cl(-) SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl(-) SAC and the EGF-induced Cl(-) currents are likely to be the volume-sensitive Cl(-) current, I(Cl,swell).

Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes.

Direct stretch of beta1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl(-) SAC resemble those of the volume-sensitive Cl(-) current, I(Cl,swell). Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for beta1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 microM), an AT1R competitive antagonist, blocked Cl(-) SAC but did not significantly alter the background Cl(-) current in the absence of integrin stretch. AT1R signaling is mediated largely by H(2)O(2) produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 microM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl(-) SAC elicited by stretch and the background Cl(-) current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl(-) SAC as well as a large fraction of the background Cl(-) current. With continuing integrin stretch, Cl(-) SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl(-) current that was rapidly and completely blocked by DPI (60 microM). Moreover, exogenous H(2)O(2) (10, 100, and 500 microM), the eventual product of NADPH oxidase activity, also activated Cl(-) SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H(2)O(2) scavenger, attenuated the response to stretch. Application of H(2)O(2) during NADPH oxidase inhibition by either DPI (60 microM) or AEBSF (0.5 mM) did not fully reactivate Cl(-) SAC, however. These results suggest that stretch of beta1-integrin in cardiac myocytes elicits Cl(-) SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl(-) SAC, providing a second regulatory pathway.

Stretch of beta 1 integrin activates an outwardly rectifying chloride current via FAK and Src in rabbit ventricular myocytes.

Osmotic swelling of cardiac myocytes and other types of cells activates an outwardly rectifying, tamoxifen-sensitive Cl- current, ICl,swell, but it is unclear whether Cl- currents also are activated by direct mechanical stretch. We tested whether specific stretch of beta1-integrin activates a Cl- current in rabbit left ventricular myocytes. Paramagnetic beads (4.5-microm diameter) coated with mAb to beta1-integrin were applied to the surface of myocytes and pulled upward with an electromagnet while recording whole-cell current. In solutions designed to isolate anion currents, beta1-integrin stretch elicited an outwardly rectifying Cl- current with biophysical and pharmacological properties similar to those of ICl,swell. Stretch-activated Cl- current activated slowly (t1/2 = 3.5 +/- 0.1 min), partially inactivated at positive voltages, reversed near ECl, and was blocked by 10 microM tamoxifen. When stretch was terminated, 64 +/- 8% of the stretch-induced current reversed within 10 min. Mechanotransduction involved protein tyrosine kinase. Genistein (100 microM), a protein tyrosine kinase inhibitor previously shown to suppress ICl,swell in myocytes, inhibited stretch-activated Cl- current by 62 +/- 6% during continued stretch. Because focal adhesion kinase and Src are known to be activated by cell swelling, mechanical stretch, and clustering of integrins, we tested whether these tyrosine kinases mediated the response to beta1-integrin stretch. PP2 (10 microM), a selective blocker of focal adhesion kinase and Src, fully inhibited the stretch-activated Cl- current as well as part of the background Cl- current, whereas its inactive analogue PP3 (10 microM) had no significant effect. In addition to activating Cl- current, stretch of beta1-integrin also appeared to activate a nonselective cation current and to suppress IK1. Integrins are the primary mechanical link between the extracellular matrix and cytoskeleton. The present results suggest that integrin stretch may contribute to mechano-electric feedback in heart, modulate electrical activity, and influence the propensity for arrhythmogenesis.

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases.

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.

Swelling-activated chloride channels in cardiac physiology and pathophysiology.

Characteristics and functions of the cardiac swelling-activated Cl current (I(Cl,swell)) are considered in physiologic and pathophysiologic settings. I(Cl,swell) is broadly distributed throughout the heart and is stimulated not only by osmotic and hydrostatic increases in cell volume, but also by agents that alter membrane tension and direct mechanical stretch. The current is outwardly rectifying, reverses between the plateau and resting potentials (E(m)), and is time-independent over the physiologic voltage range. Consequently, I(Cl,swell) shortens action potential duration, depolarizes E(m), and acts to decrease cell volume. Because it is activated by stimuli that also activate cation stretch-activated channels, I(Cl,swell) should be considered as a potential effector of mechanoelectrical feedback. I(Cl,swell) is activated in ischemic and non-ischemic dilated cardiomyopathies and perhaps during ischemia and reperfusion. I(Cl,swell) plays a role in arrhythmogenesis, myocardial injury, preconditioning, and apoptosis of myocytes. As a result, I(Cl,swell) potentially is a novel therapeutic target.

Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine.

Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-ß-erythroidine, and CP-601932 (a partial agonist of the α3ß4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

Real-time monitoring of changes in brain extracellular sodium and potassium concentrations and intracranial pressure after selective vasopressin-1a receptor inhibition following focal traumatic brain injury in rats.

Brain swelling and increased intracranial pressure (ICP) following traumatic brain injury (TBI) contribute to poor outcome. Vasopressin-1a receptors (V1aR) and aquaporin-4 (AQP4) regulate water transport and brain edema formation, perhaps in part by modulating cation fluxes. After focal TBI, V1aR inhibitors diminish V1aR and AQP4, reduce astrocytic swelling and brain edema. We determined whether V1aR inhibition with SR49059 after lateral controlled-cortical-impact (CCI) injury affects extracellular Na(+) and K(+) concentrations ([Na(+)]e; [K(+)]e). Ion-selective Na(+) and K(+) electrodes (ISE) and an ICP probe were implanted in rat parietal cortex, and [Na(+)]e, [K(+)]e, and physiological parameters were monitored for 5 h post-CCI. Sham-vehicle-ISE, CCI-vehicle-ISE and CCI-SR49059-ISE groups were studied, and SR49059 was administered 5 min to 5 h post-injury. We found a significant injury-induced decrease in [Na(+)]e to 80.1 ± 15 and 87.9 ± 7.9 mM and increase in [K(+)]e to 20.9 ± 3.8 and 13.4 ± 3.4 mM at 5 min post-CCI in CCI-vehicle-ISE and CCI-SR49059-ISE groups, respectively (p<0.001 vs. baseline; ns between groups). Importantly, [Na(+)]e in CCI-SR49059-ISE was reduced 5-20 min post-injury and increased to baseline at 25 min, whereas recovery in CCI-vehicle-ISE required more than 1 hr, suggesting SR49059 accelerated [Na(+)]e recovery. In contrast, [K(+)]e recovery took 45 min in both groups. Further, ICP was lower in the CCI-SR49059-ISE group. Thus, selective V1aR inhibition allowed faster [Na(+)]e recovery and reduced ICP. By augmenting the [Na(+)]e recovery rate, SR49059 may reduce trauma-induced ionic imbalance, blunting cellular water influx and edema after TBI. These findings suggest SR49059 and V1aR inhibitors are potential tools for treating cellular edema post-TBI.

Selective vasopressin-1a receptor antagonist prevents brain edema, reduces astrocytic cell swelling and GFAP, V1aR and AQP4 expression after focal traumatic brain injury.

A secondary and often lethal consequence of traumatic brain injury is cellular edema that we posit is due to astrocytic swelling caused by transmembrane water fluxes augmented by vasopressin-regulated aquaporin-4 (AQP4). We therefore tested whether vasopressin 1a receptor (V1aR) inhibition would suppress astrocyte AQP4, reduce astrocytic edema, and thereby diminish TBI-induced edematous changes. V1aR inhibition by SR49059 significantly reduced brain edema after cortical contusion injury (CCI) in rat 5h post-injury. Injured-hemisphere brain water content (n=6 animals/group) and astrocytic area (n=3/group) were significantly higher in CCI-vehicle (80.5±0.3%; 18.0±1.4 µm(2)) versus sham groups (78.3±0.1%; 9.5±0.9 µm(2)), and SR49059 blunted CCI-induced increases in brain edema (79.0±0.2%; 9.4±0.8µm(2)). CCI significantly up-regulated GFAP, V1aR and AQP4 protein levels and SR49059 suppressed injury induced up regulation (n=6/group). In CCI-vehicle, sham and CCI-SR49059 groups, GFAP was 1.58±0.04, 0.47±0.02, and 0.81±0.03, respectively; V1aR was 1.00±0.06, 0.45±0.05, and 0.46±0.09; and AQP4 was 2.03±0.34, 0.49±0.04, and 0.92±0.22. Confocal immunohistochemistry gave analogous results. In CCI-vehicle, sham and CCI-SR49059 groups, fluorescence intensity of GFAP was 349±38, 56±5, and 244±30, respectively, V1aR was 601±71, 117.8±14, and 390±76, and AQP4 was 818±117, 158±5, and 458±55 (n=3/group). The results support that edema was predominantly cellular following CCI and documented that V1aR inhibition with SR49059 suppressed injury-induced up regulation of GFAP, V1A and AQP4, blunting edematous changes. Our findings suggest V1aR inhibitors may be potential therapeutic tools to prevent cellular swelling and provide treatment for post-traumatic brain edema.

Endothelin signalling regulates volume-sensitive Cl- current via NADPH oxidase and mitochondrial reactive oxygen species.

AIMS: We assessed regulation of volume-sensitive Cl(-) current (I(Cl,swell)) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes. METHODS AND RESULTS: ET-1 elicited I(Cl,swell) under isosmotic conditions. Outwardly rectified Cl(-) current was blocked by the I(Cl,swell)-selective inhibitor DCPIB or osmotic shrinkage and involved ET(A) but not ET(B) receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of I(Cl,swell) by osmotic swelling or angiotensin II (AngII) was suppressed by ET(A) blockade, whereas AngII AT(1) receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I(Cl,swell). As expected, blockade of NOX suppressed ET-1-induced I(Cl,swell), but blockade of mitochondrial ROS production with rotenone also suppressed I(Cl,swell). I(Cl,swell) was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I(Cl,swell) was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade. CONCLUSION: ET-1-ET(A) signalling activated I(Cl,swell) via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ET(A) receptors were downstream effectors when I(Cl,swell) was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I(Cl,swell) is likely to participate in multiple physiological and pathophysiological processes.

Exogenous and endogenous ceramides elicit volume-sensitive chloride current in ventricular myocytes.

AIMS: Because ceramide accumulates in several forms of cardiovascular disease and ceramide-induced apoptosis may involve the volume-sensitive Cl(-) current, I(Cl,swell), we assessed whether ceramide activates I(Cl,swell). METHODS AND RESULTS: I(Cl,swell) was measured in rabbit ventricular myocytes by whole-cell patch clamp after isolating anion currents. Exogenous C(2)-ceramide (C(2)-Cer), a membrane-permeant short-chain ceramide, elicited an outwardly rectifying Cl(-) current in both physiological and symmetrical Cl(-) solutions that was fully inhibited by DCPIB, a specific I(Cl,swell) blocker. In contrast, the metabolically inactive C(2)-Cer analogue C(2)-dihydroceramide (C(2)-H(2)Cer) failed to activate Cl(-) current. Bacterial sphingomyelinase (SMase), which generates endogenous long-chain ceramides as was confirmed by tandem mass spectrometry, also elicited an outwardly rectifying Cl(-) current that was inhibited by DCPIB and tamoxifen, another I(Cl,swell) blocker. Bacterial SMase-induced current was partially reversed by osmotic shrinkage and fully suppressed by ebselen, a scavenger of reactive oxygen species. Outward rectification with physiological and symmetrical Cl(-) gradients, block by DCPIB and tamoxifen, and volume sensitivity are characteristics that identify I(Cl,swell). Insensitivity to C(2)-H(2)Cer and block by ebselen suggest involvement of ceramide signalling rather than direct lipid-channel interaction. CONCLUSION: Exogenous and endogenous ceramide elicited I(Cl,swell) in ventricular myocytes. This may contribute to persistent activation of I(Cl,swell) and aspects of altered myocyte function in cardiovascular diseases associated with by ceramide accumulation.

Long-term fish oil supplementation induces cardiac electrical remodeling by changing channel protein expression in the rabbit model.

Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography-mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (I(CaL)) and a decrease in the transient outward current (I(to)) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in I(CaL) and I(to) were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes.

Antagonistic regulation of swelling-activated Cl- current in rabbit ventricle by Src and EGFR protein tyrosine kinases.

Regulation of swelling-activated Cl(-) current (I(Cl,swell)) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates I(Cl,swell) and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). I(Cl,swell) evoked by hyposmotic swelling was increased 181 +/- 17% by 100 microM genistein, and the genistein-induced current was blocked by the selective I(Cl,swell) blocker tamoxifen (10 microM). Block of Src with PP2 (10 microM) stimulated tamoxifen-sensitive I(Cl,swell) by 234 +/- 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 microM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited I(Cl,swell) and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited I(Cl,swell). Several lines of evidence argue that the PP2-stimulated current was I(Cl,swell): 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl(-) gradients, and 4) the current reversed near the Cl(-) equilibrium potential. To rule out contributions of other currents, Cd(2+) (0.2 mM) and Ba(2+) (1 mM) were added to the bath. Surprisingly, Cd(2+) suppressed the decay of I(Cl,swell), and Cd(2+) plus Ba(2+) eliminated time-dependent currents between -100 and +100 mV. Nevertheless, these divalent ions did not eliminate I(Cl,swell) or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls I(Cl,swell), and regulation of I(Cl,swell) by the Src and EGFR kinase families of PTK is antagonistic.

Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes.

We studied the effects of osmotic swelling on the components of excitation-contraction coupling in ventricular myocytes. Myocyte volume rapidly increased 30% in hyposmotic (0.6T) solution and was constant thereafter. Cell shortening transiently increased 31% after 4 min in 0.6T but then decreased to 68% of control after 20 min. In parallel, the L-type Ca(2+) current (I(Ca-L)) transiently increased 10% and then declined to 70% of control. Similar biphasic effects on shortening were observed under current clamp. In contrast, action potential duration was unchanged at 4 min but decreased to 72% of control after 20 min. Ca(2+) transients were measured with fura 2-AM. The emission ratio with excitation at 340 and 380 nm (f(340)/f(380)) decreased by 12% after 3 min in 0.6T, whereas shortening and I(Ca-L) increased at the same time. After 8 min, shortening, I(Ca-L), and the f(340)/f(380) ratio decreased 28, 25, and 59%, respectively. The results suggest that osmotic swelling causes biphasic changes in I(Ca-L) that contribute to its biphasic effects on contraction. In addition, swelling initially appears to reduce the Ca(2+) transient initiated by a given I(Ca-L), and later, both I(Ca-L) and the Ca(2+) transient are inhibited.