Fred S. Lamb

Cytokine Activation of Nuclear Factor κB in Vascular Smooth Muscle Cells Requires Signaling Endosomes Containing Nox1 and ClC-3

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. To address this issue, we examined cytokine generation of ROS and subsequent activation of the transcription factor nuclear factor &kgr;B in vascular smooth muscle cells (SMCs). Tumor necrosis factor-&agr; and interleukin (IL)-1&bgr; stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22phox, integral membrane subunits of NADPH oxidase, coimmunoprecipitated with early endosomal markers in SMCs. ClC-3, an anion transporter that is primarily found in intracellular vesicles, also colocalized with Nox1 in early endosomes and was necessary for tumor necrosis factor-&agr; and interleukin-1&bgr; generation of ROS. Cytokine activation of nuclear factor &kgr;B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-&agr; and interleukin-1&bgr;, NADPH oxidase generates ROS within early endosomes and that Nox1 cannot produce sufficient ROS for cell signaling in the absence of ClC-3. These data best support a model whereby ClC-3 is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signaling endosomes.

Anion Channels, Including ClC-3, Are Required for Normal Neutrophil Oxidative Function, Phagocytosis, and Transendothelial Migration

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(–/–)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(–/–) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(–/–) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(–/–) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.

Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl− channel gene

Salivary gland acinar cells shrink when Cl− currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium‐mobilizing agonists. The molecular identity of the Cl− channel(s) in salivary cells involved in these processes is unknown, although ClC‐3 has been implicated in several tissues as a cell‐volume‐sensitive Cl− channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild‐type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell‐activated Cl− currents in cells from ClC‐3‐deficient mice were equivalent to those from wild‐type mice. It has also been suggested that ClC‐3 is activated by Ca2+‐calmodulin‐dependent protein kinase II; however, the magnitude of the Ca2+‐dependent Cl− current was unchanged in the Clcn3−/‐ animals. In addition, we observed that ClC‐3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild‐type and null mutant animals were comparable. Finally, in some cells ClC‐3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3−/‐ mice. Our results demonstrate that the ClC‐3 Cl− channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva.

Altered properties of volume‐sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3‐/‐ mice

ClC‐3, a member of the large superfamily of ClC voltage‐dependent Cl– channels, has been proposed as a molecular candidate responsible for volume‐sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC‐3 disrupted (Clcn3−/−) mice casts considerable doubt on this proposed role for ClC‐3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3+/+ and Clcn3−/− mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3−/− mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock‐out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3+/+ and Clcn3−/− mice are remarkably similar in terms of activation and inactivation kinetics, steady‐state current densities, rectification, anion selectivity (I− > Cl−≫ Asp−) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3−/− mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti‐ClC‐3 antibodies, and differential sensitivities to [ATP]i and free [Mg2+]i. These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3−/− mice compared to Clcn3+/+ mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC‐1, ClC‐2, and ClC‐3, and protein expression profiles obtained using two‐dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3−/− mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.

Chloride ion currents contribute functionally to norepinephrine-induced vascular contraction

Norepinephrine (NE) increases Cl- efflux from vascular smooth muscle (VSM) cells. An increase in Cl- conductance produces membrane depolarization. We hypothesized that if Cl- currents are important for agonist-induced depolarization, then interfering with cellular Cl- handling should alter contractility. Isometric contraction of rat aortic rings was studied in a bicarbonate buffer. Substitution of extracellular Cl- with 130 mM methanesulfonate (MS; 8 mM Cl-) did not cause contraction. NE- and serotonin-induced contractions were potentiated in this low-Cl- buffer, whereas responses to K+, BAY K 8644, or NE in the absence of Ca2+ were unaltered. Substitution of Cl- with I- or Br- suppressed responses to NE. Inhibition of Cl-transport with bumetanide (10-5 M) or bicarbonate-free conditions (10 mM HEPES) inhibited NE- but not KCl-induced contraction. The Cl--channel blockers DIDS (10-3 M), anthracene-9-carboxylic acid (10-3 M), and niflumic acid (10-5 M) all inhibited NE-induced contraction, whereas tamoxifen (10-5 M) did not. Finally, disruption of sarcoplasmic reticular function with cyclopiazonic acid (10-7 M) or ryanodine (10-5 M) prevented the increase in the peak response to NE produced by low-Cl- buffer. We conclude that a Cl- current with a permeability sequence of I-> Br- > Cl- > MS is critical to agonist-induced contraction of VSM.

Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels

Mice lacking ClC-3 chloride channels, encoded by the Clcn3 gene, undergo neurodegeneration of the hippocampal formation and retina [Neuron, 29 (2001) 185-196; Genes Cells, 7 (2002) 597-605]. We independently created a mouse lacking the Clcn3 gene which demonstrated similar central nervous system abnormalities, including early postnatal degeneration of retinal photoreceptors. However, we observed a characteristic spatial-temporal sequence of hippocampal neurodegeneration that differs from the pattern previously reported. Anterior-to-posterior degeneration and astrogliosis of the dentate gyrus and hippocampus progressed over months. Sequential loss of hippocampal neuronal subpopulations began in the dentate gyrus and progressed to CA3, followed by CA1 neurons. Projection neurons of the entorhinal cortex degenerated, secondary to the loss of their synaptic targets within the hippocampal formation. Other characteristics of the Clcn3(-/-) mice included an abnormal gait, kyphosis, and absence of hindlimb escape extension upon tail elevation. Spontaneous seizures were observed in four adult Clcn3(-/-) mice, and one mouse died during the event. We hypothesized that neuronal injury may be related to recurrent seizures. Clcn3(-/-) mice had normal serum electrolytes and pH, and exhibited neither hyperglycemia nor rebound hypoglycemia following a glucose load. They displayed a greatly reduced susceptibility to pentylenetetrazole-induced seizures and an abnormally prolonged sedation to benzodiazepines. There was no change in vulnerability to kainic acid-induced seizures. Immunostaining revealed a progressive loss of GABA synthesizing cells in the dentate gyrus. The death of these cells was preceded by increased GABA(A) receptor immunoreactivity. These data suggest that GABA(A) inhibitory neurotransmission is altered in Clcn3(-/-) mice. The increase in GABA(A) receptor density may represent a compensatory response either to chronic excessive excitatory stimuli or reduced inhibitory input from local GABAergic interneurons within the dentate gyrus.

Calcium-activated chloride current in rabbit coronary artery myocytes.

Whole-cell patch-clamp techniques were used to study enzymatically dispersed epicardial coronary artery smooth muscle cells. Depolarizing voltage pulses of 500-millisecond duration from -60 mV (118 mmol/L CsCl, 22 mmol/L tetraethylammonium chloride, and 5 mmol/L EGTA pipette solution) elicited inward L-type calcium currents (ICa). When EGTA was omitted from the pipette solution, an outward current was superimposed on the calcium current, and repolarizing voltage steps produced an inward tail current (IT). The amplitude of these inward currents was proportional to the ICa amplitude from -30 to +50 mV. The time course of decay of the current was well fit by a single exponential equation. The time constant (tau) of this equation did not change with the size of IT but was clearly voltage dependent (shorter at more negative potentials). Changing the chloride reversal potential from -1.3 to -39.7 mV by anion substitution using methanesulfonate as the chloride replacement in the pipette solution shifted the zero current level of IT from 0.9 +/- 0.56 to -33.1 +/- 0.85 mV. The tail current was blocked by nifedipine (10(-6) mol/L) and by isosmolar calcium substitution with barium in the bath solution and was enhanced by the dihydropyridine agonist Bay K 8644 (10(-6) mol/L). IT was also blocked by the chloride channel blockers DIDS (10(-4) mol/L) and niflumic acid (10(-5) mol/L). Caffeine (10(-2) mol/L), which releases intracellular calcium stores, caused an inward current at holding potentials (-60 mV), which was inhibited by DIDS. Caffeine also inhibited subsequent attempts to elicit IT by depolarizing pulses (88% reduction in IT).(ABSTRACT TRUNCATED AT 250 WORDS)

CLC-3 Channels Modulate Excitatory Synaptic Transmission in Hippocampal Neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.

Linkage of infantile Bartter syndrome with sensorineural deafness to chromosome 1p.

Bartter syndrome (BS) is a family of disorders manifested by hypokalemic hypochloremic metabolic alkalosis with normotensive hyperreninemic hyperaldosteronism. We evaluated a unique, inbred Bedouin kindred in which sensorineural deafness (SND) cosegregates with an infantile variant of the BS phenotype. Using a DNA-pooling strategy, we screened the human genome and successfully demonstrated linkage of this unique syndrome to chromosome 1p31. The genes for two kidney-specific chloride channels and a sodium/hydrogen antiporter, located near this region, were excluded as candidate genes. Although the search for the disease-causing gene in this family continues, this linkage further demonstrates the genetic heterogeneity of BS. In addition, the cosegregation of these phenotypes allows us to postulate that a single genetic alteration may be responsible for the SND and the BS phenotype. The identification and characterization of this gene would lead to a better understanding of the normal physiology of the kidney and the inner ear.

Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current

CLC‐3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium–calmodulin‐dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC‐3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII‐dependent regulation of CLC‐3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole‐cell voltage clamp. In CLC‐3‐expressing cells, we observed the activation of a Cl− conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage‐clamped cell via the patch pipette. The CaMKII‐dependent Cl− conductance was not observed following exposure of the cells to 1 μm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII‐activated Cl− conductance; however, CLC‐3−/− cells did not. The N‐terminus of CLC‐3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII‐dependent Cl− conductance, indicating that this residue is important in the gating of CLC‐3 at the plasma membrane.