James J. Matsuda

Enhancement of rabbit cardiac sodium channels by beta-adrenergic stimulation.

Voltage-dependent sodium channels from a variety of tissues are known to be phosphorylated by the cAMP-dependent protein kinase, protein kinase A. However, the functional significance of sodium channel phosphorylation is not clearly understood. Using whole-cell voltage-clamp techniques, we show that sodium currents (INas) in rabbit cardiac myocytes are enhanced by isoproterenol (ISO). This enhancement of INa by ISO 1) is holding potential dependent, 2) can be mimicked by forskolin and dibutyryl cAMP, and 3) is accompanied by an increase in the rate of Na+ channel inactivation. In single-channel, inside-out patch experiments, the catalytic subunit of protein kinase A also enhances INa and increases the rate of inactivation, suggesting that cardiac Na+ channel phosphorylation may be physiologically important. Addition of the protein kinase A inhibitor to the pipette solution in whole-cell experiments blocks the stimulatory effect of forskolin without blocking the effect of ISO, suggesting that ISO also enhances INa through a cAMP-independent pathway. To determine if ISO may stimulate INa through a direct G protein pathway, single channels were recorded in the presence of the Gs-activating GTP analogue, GTP gamma S, and the stimulatory G protein subunit, Gs alpha. Both of these agents enhanced INa without affecting the rate of Na+ channel inactivation. These results suggest that ISO enhances rabbit cardiac INa through a dual (direct and indirect) G protein regulatory pathway.

A voltage-dependent potassium current in rabbit coronary artery smooth muscle cells.

1. Voltage‐ and time‐dependent outward currents were recorded from relaxed enzymatically isolated smooth muscle cells from the rabbit left descending coronary artery using a single pipette voltage clamp technique. The calcium‐activated potassium current was blocked by inclusion of EGTA in the pipette solution and CdCl2 in the extracellular bath. 2. Outward currents were elicited with depolarizing voltage steps to potentials positive to ‐20 mV. Long (5 s) voltage steps revealed slow inactivation of the current with a time constant of nearly 3 s at +60 mV. Potassium was identified as the predominant charge carrier by reversal potential measurements in potassium substitution experiments. 3. The results of kinetic analyses compared favourably with the Hodgkin‐Huxley model for a delayed rectifier with some deviations. The sigmoid current onset was best fitted by raising the activation variable (n) to the second power. Deactivation tail currents were consistently found to be comprised of two exponential components. The kinetics of activation and deactivation were strongly voltage‐dependent from ‐80 to +60 mV. 4. Envelope of tails experiments showed that the scaled tail current amplitudes followed the kinetic behaviour of current activation. The contribution of each of the two exponential tail components was also measured in these experiments. They did not reveal kinetically separable currents, nor were they differentially altered by 4‐aminopyridine (4‐AP), tetraethylammonium (TEA), or elevated [K+]o. 5. The steady‐state voltage‐dependence curves for both activation and inactivation were well fitted by a Boltzmann distribution with V1/2 = ‐5.60 mV and k = ‐8.66 mV for n infinity act and V1/2 = ‐24.20 mV and k = 5.16 mV for n infinity act. Super‐imposition of the two curves revealed a ‘window’ of voltage where channels are available for activation without completely inactivating. 6. Neither of the commonly used potassium channel blockers, TEA or 4‐AP, were particularly effective blockers of IK, reducing current by only 50‐70% at an extracellular concentration of 10 mM. TEA block was mildly voltage‐dependent and was more effective in reducing current towards the end of a 500 ms depolarization. 4‐AP, on the other hand, demonstrated considerable voltage‐dependence and preferentially reduced early currents. 7. Outward currents recorded from guinea‐pig and human coronary artery myocytes under the same conditions as in the rabbit cell experiments displayed similar characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium currents in isolated rabbit coronary arterial smooth muscle myocytes.

1. Calcium inward currents were recorded from relaxed enzymatically isolated smooth muscle cells from the rabbit epicardial left descending coronary artery using a single‐pipette voltage‐clamp technique. Outward K+ currents were blocked with CsCl‐tetraethylammonium‐filled pipette solutions. 2. Relaxed coronary smooth muscle cells had a maximum diameter of 8.6 +/‐ 0.6 microns and a cell length of 96.7 +/‐ 3.3 microns when bathed in 2.5 mM [Ca2+]o. The average resting membrane potential at room temperature was ‐32 +/‐ 10 mV. The mean cell capacitance was 18.5 +/‐ 1.7 pF and the input resistance was 3.79 +/‐ 0.58 G omega. 3. Depolarizing voltage‐clamp steps from a holding potential of ‐80 mV elicited a single time‐ and voltage‐dependent inward current which was dependent upon extracellular [Ca2+]. In 2.5 mM [Ca2+]o, the inward current was activated at a potential of ‐40 mV and peaked at +10 mV. This current was inhibited by 0.5 mM‐CdCl2 and 1 microM‐nifedipine and was enhanced with 1 microM‐Bay K 8644. No detectable low‐threshold, rapidly inactivating T‐type calcium current was observed. 4. The apparent reversal potential of this inward current in 2.5 mM [Ca2+]o was +70 mV and shifted by 33.0 mV per tenfold increase in [Ca2+]o. This channel was also more permeable to barium and strontium ions than to calcium ions. 5. Single calcium channel recordings with 110 mM‐Ba2+ as the charge carrier revealed a mean slope conductance of 20.7 +/‐ 0.8 pS. 6. This calcium current (ICa) exhibited a strong voltage‐dependent inactivation process. However, the steady‐state inactivation curve (f infinity) displayed a slight nonmonotonic, U‐shaped dependence upon membrane potential. The potential at which half of the channels were inactivated was ‐27.9 mV with a slope factor of 6.9 mV. The steady‐state activation curve (d infinity) was also well‐described by a Boltzmann distribution with a half‐activation potential at ‐4.4 mV and a slope factor of ‐63 mV. ICa was fully activated at approximately +20 mV. 7. The rate of inactivation was dependent upon the species of ion carrying the current. Both Sr2+ and Ba2+ decreased the rate as well as the degree of inactivation. The tau f (fitted time constant of inactivation) curve displayed a U‐shaped relationship in 2.5 mM [Ca2+]o. The reactivation process was voltage dependent and could be described by a single exponential. 8. The current amplitude and the inactivation kinetics were temperature dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

The ClC-3 Cl-/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl−/H+ antiport current (IClC-3) in HEK293 cells. The outward rectification and time dependence of IClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl− current (IClacid) seen at extracellular pH ≤ 5.5. IClacid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. IClacid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (Erev) of IClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl− and H+ transport become uncoupled at low pH. At pH 4.0 Erev was completely Cl− dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native IClacid; 1) RNA interference directed at ClC-3 message reduced native IClacid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type IClC-3 and IClacid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type IClC-3 and from native IClacid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native IClacid. Low pH uncouples Cl−/H+ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl−/H+ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.

Activation of Swelling-activated Chloride Current by Tumor Necrosis Factor-α Requires ClC-3-dependent Endosomal Reactive Oxygen Production

ClC-3 is a Cl−/H+ antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (IClswell), but overexpression of ClC-3 can activate currents that resemble IClswell. Because H2O2 activates IClswell directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates IClswell. Whole-cell perforated patch clamp methods were used to record Cl− currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-α (TNF-α) (10 ng/ml) activated outwardly rectifying Cl− currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 μm) and by hypertonicity (340 mosm) identified them as IClswell. IClswell was also activated by H2O2 (500 μm), and the effect of TNF-α was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced IClswell in ClC-3 null cells in the absence of swelling or TNF-α, and this effect was also blocked by catalase. IClswell activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-α-mediated activation of IClswell. Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H2O2-dependent activation of IClswell, but this effect was not ClC-3- or Rab5-dependent. Thus, activation of IClswell by TNF-α requires ClC-3-dependent endosomal H2O2 production. This demonstrates a functional link between two distinct anion currents, ClC-3 and IClswell.

Endotoxin Priming of Neutrophils Requires NADPH Oxidase-generated Oxidants and Is Regulated by the Anion Transporter ClC-3

Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes.

Reversal of lidocaine effects on sodium currents by isoproterenol in rabbit hearts and heart cells.

We demonstrated recently that isoproterenol enhanced the cardiac voltage-dependent sodium currents (INa) in rabbit ventricular myocytes through dual G-protein regulatory pathways. In this study, we tested the hypothesis that isoproterenol reverses the sodium channel blocking effects of class I antiarrhythmic drugs through modulation of INa. The experiments were performed in rabbit ventricular myocytes using whole-cell patch-clamp techniques. Reversal of lidocaine suppression of INa by isoproterenol (1 microM) was significant at various concentrations of lidocaine (20, 65, and 100 microM, P < 0.05). The effects of isoproterenol were voltage dependent, showing reversal of INa suppression by lidocaine at normal and hyperpolarized potentials (negative to -80 mV) but not at depolarized potentials. Isoproterenol enhanced sodium channel availability but did not alter the steady state activation or inactivation of INa nor did it improve sodium channel recovery in the presence of lidocaine. The physiological significance of the single cell INa findings were corroborated by measurements of conduction velocities using an epicardial mapping system in isolated rabbit hearts. Lidocaine (10 microM) significantly suppressed epicardial impulse conduction in both longitudinal (theta L, 0.430 +/- 0.024 vs. 0.585 +/- 0.001 m/s at baseline, n = 7, P < 0.001) and transverse (theta T, 0.206 +/- 0.012 vs. 0.257 +/- 0.014 m/s at baseline, n = 8, P < 0.001) directions. Isoproterenol (0.05 microM) significantly reversed the lidocaine effects with theta L of 0.503 +/- 0.027 m/s and theta T of 0.234 +/- 0.015 m/s (P = 0.014 and 0.004 compared with the respective lidocaine measurements). These results suggest that enhancement of INa is an important mechanism by which isoproterenol reverses the effects of class I antiarrhythmic drugs.

Acetylcholine reversal of isoproterenol-stimulated sodium currents in rabbit ventricular myocytes.

We have recently shown that beta-adrenergic agonists enhance the cardiac sodium current (INa) in rabbits through dual G-protein regulatory pathways. To determine if muscarinic cholinergic receptor stimulation can also modulate INa, we studied the effects of acetylcholine (ACh) and carbachol on INa in enzymatically dispersed rabbit ventricular myocytes. Whole-cell patch-clamp experiments done at room temperature using 20 mM [Na+]o showed that 100 nM isoproterenol increased INa and accelerated current decay as previously described. ACh (1 microM) or carbachol (1 microM) significantly reversed the stimulatory isoproterenol effects at test potentials throughout the INa activation range and at holding potentials negative to -80 mV. This effect was completely inhibited by atropine (1 microM) and was confirmed by studying single-channel INa from cell-attached patches. When INa was stimulated by forskolin (1 microM), carbachol (1 microM) significantly reversed the effect. The muscarinic-mediated inhibition of INa was inhibited by pertussis toxin (0.1 or 1.0 microgram/ml) incubation (12-15 hours), suggesting that the effect was inhibitory G-protein dependent. Further investigation of the ACh inhibitory mechanism revealed that ACh alone had no effect on INa and that when cells were dialyzed with cAMP (5 microM), ACh failed to inhibit INa. Furthermore, cGMP failed to inhibit the effect of isoproterenol on INa. These data suggest that ACh acts at or proximal to adenylate cyclase stimulation. Thus, rabbit cardiac Na+ channels are regulated by muscarinic agonists in a fashion similar to cardiac Ca2+ channels.

51 CLC-3 IS REQUIRED FOR NADPH OXIDASE-DEPENDENT NUCLEAR FACTOR κB ACTIVATION BY SIGNALING ENDOSOMES.

Reactive oxygen species (ROS) are mediators of intracellular signals for a myriad of normal and pathologic cellular events, including differentiation, hypertrophy, proliferation, and apoptosis. NADPH oxidases are important sources of ROS that are present in diverse tissues throughout the body and activate many redox-sensitive signal transduction and gene expression pathways. To avoid toxicity and provide specificity of signaling, ROS production and metabolism necessitate tight regulation that likely includes subcellular compartmentalization. However, the constituent elements of NADPH oxidase-dependent cell signaling are not known. Here we show that activation of NADPH oxidase by inflammatory cytokines generates ROS within early endosomes and requires ClC-3, a member of the chloride channel (ClC) family. Nox1, one of multiple membrane-bound catalytic subunits of NADPH oxidase, colocalizes with ClC-3 in early endosomes. Both Nox1 and ClC-3 are necessary for tumor necrosis factor α and interleukin-1β generation of ROS and subsequent activation of the transcription factor NF-κB. We propose that ClC-3 functions as a chloride-proton exchanger and thereby influences ROS production via charge neutralization of the electron flow generated by Nox1 in the endosome. These findings identify ClC-3 as a critical component of the signaling endosome and a novel intermediate in redox-dependent control of gene expression.