Swelling-activated or volume-sensitive Cl- currents are found in numerous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Recently, the molecular entity responsible for swelling-activated Cl- current in cardiac myocytes has been identified as ClC-3. The goal of our study was to determine whether such a channel exists in smooth muscle cells of the canine colon using both molecular biological and electrophysiological techniques and, if present, to characterize its functional and molecular properties. We hypothesized that ClC-3 is present in colonic smooth muscle and is regulated in a manner similar to the molecular entity cloned from heart. Indeed, the ClC-3 gene was expressed in colonic myocytes, as demonstrated by reverse transcriptase polymerase chain reaction performed on isolated cells. The current activated by decreasing extracellular osmolarity from 300 to 250 mosM was outwardly rectifying and dependent on the Cl- gradient. Current magnitude increased and reversed at more negative potentials when Cl- was replaced by I- or Br-. Tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl-1-butene; 10 microM) and DIDS (100 microM) inhibited the current, whereas 25 microM niflumic acid, 10 microM nicardipine, and Ca2+ removal had no effect. Current was inhibited by 1 mM extracellular ATP in a voltage-dependent manner. Cl- current was also regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM) decreased Cl- current magnitude, while chelerythrine chloride (30 microM) activated it under isotonic conditions. Our findings indicate that a current activated by hypotonic solution is present in colonic myocytes and is likely mediated by ClC-3. Furthermore, we suggest that the ClC-3 may be an important mechanism controlling depolarization and contraction of colonic smooth muscle under conditions that impose physical stress on the cells.

1. Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. 2. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. 3. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. 4. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(-) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. 5. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel.

The voltage-dependent chloride channel ClC-1 stabilizes resting membrane potential in skeletal muscle. Mutations in the ClC-1 gene are responsible for both human autosomal recessive generalized myotonia and autosomal dominant myotonia congenita. To understand the tissue distribution and subcellular localization of ClC-1 and to evaluate its role in an animal model of myotonia, antibodies were raised against the carboxyl terminus of this protein. Expression of the 130-kDa ClC-1 protein is unique to skeletal muscle, consistent with its mRNA tissue distribution. Immunolocalization shows prominent ClC-1 antigen in the sarcolemma of both type I and II muscle fibers. Sarcolemma localization is confirmed by Western analysis of skeletal muscle subcellular fractions. The ADR myotonic mouse (phenotype ADR, genotype adr/adr), in which defective ClC-1 mRNA has been identified, is shown here to be absent in ClC-1 protein expression, whereas other skeletal muscle sarcolemma protein expression appears normal. Immunohistochemistry of skeletal muscle from ADR and other mouse models of human muscle disease demonstrate that the absence of ClC-1 chloride channel is a defect specific to ADR mice.

The muscular chloride channel (ClC-1) is essential for a normal excitability of mature mammalian muscle fibers; inactivation of the corresponding gene by mutations leads to hyperexcitability of muscle, the hallmark of the disease myotonia. In the mouse, there is very little ClC-1 mRNA in myotubes, and its concentration increases steeply during postnatal development, suggesting a role of the motor nerve in ClC-1 expression. We investigated the response of the expression of the corresponding gene Clc-1 to different patterns of muscle activity as controlled by sarcolemmal excitability and by innervation. In rat and mouse, the level of ClC-1 mRNA was higher in fast (extensor digitorum longus) than in slow (soleus) muscle. Myotonia in the ADR mouse is caused by an insertional mutation leading to the adr allele of the Clc-1 gene and to grossly abnormal ClC-1 mRNAs. Nevertheless, in +/adr heterozygous, phenotypically wild type (WT) animals, the expression levels of both alleles correspond to the gene dosage. However, in the myotonic ADR mouse in which both Clc-1 genes are defective, ClC-1 mRNA levels in slow muscle were nearly as high as in WT fast muscle. In WT muscle, denervation within 2 days caused a drastic reduction of the ClC-1 mRNA level and at the same time an increase of myogenin and MyoD mRNAs. Neither effect of denervation was observed in myotonic mice (homozygous for the alleles adr or adrK), suggesting that spontaneous electrical activity of the hyperexcitable sarcolemma may substitute for nerve activity. Furthermore, potential MyoD/myogenin-binding sequence motifs were identified in the 5' regulatory region of the Clc-1 gene. These findings suggest that the activity-dependent regulation of the muscular chloride channel 1 gene is mediated by myogenic factors.

X-ray structures permit theoretical study of Cl(-) permeation along bacterial ClC Cl(-) pores. We determined the lowest energy curvilinear pathway, identified anion-coordinating amino acids, and calculated the electrostatic potential energy profiles. We find that all four bacterial ClC Cl(-) crystal structures correspond to closed states. E148 and S107 side chains form steric barriers on both sides of the crystal binding site in the StClC wild-type and EcClC wild-type crystals; both the EcClC(E148A) and EcClC(E148Q) mutants are blocked at the S107 site. We studied the effect that mutating the charge of some strongly conserved pore-lining amino acids has on the electrostatic potential energy profiles. When E148 is neutralized, it creates an electrostatic trap, binding the ion near midmembrane. This suggests a possible electrostatic mechanism for controlling anion flow: neutralize E148, displace the side chain of E148 from the pore pathway to relieve the steric barrier, then trap the anion at midmembrane, and finally either deprotonate E148 and block the pore (pore closure) or bring a second Cl(-) into the pore to promote anion flow (pore conductance). Side-chain displacement may arise by competition for the binding site between the oxygens of E148 and the anion moving down the electrostatic energy gradient. We also find that the charge state of E111 and E113 may electrostatically control anion conductance and occupancy of the binding site within the cytoplasmic pore.

BACKGROUND

Dent's disease is a proximal tubule (PT) disorder characterized by low-molecular-weight proteinuria (LWMP) that may be associated with hypercalciuria, nephrocalcinosis, and renal failure. It is caused by inactivating mutations of the renal chloride channel ClC-5, which colocalizes with the vacuolar H+-ATPase in PT cells and alpha-type intercalated cells. Examinations of knockout mice have established the role of ClC-5 in PT endocytosis, but the consequences of ClC-5 mutations on the polarity of H+-ATPase and other plasma membrane proteins remain unknown.

METHODS

We have studied renal biopsies from eight patients with Dent's disease, due to inactivating ClC-5 mutations, by light and electron microscopy, and by immunohistochemical staining. All patients exhibited LMWP, and renal function ranged from normal to end-stage renal failure.

RESULTS

Light microscopy revealed either normal renal architecture or glomerulosclerosis, tubular dedifferentiation and atrophy, and mild interstitial fibrosis. Focal, hyaline casts, sometimes calcified, were identified at all stages. Electron microscopy did not reveal any ultrastructural abnormalities in PT cells, and the endocytic apparatus was apparently normal. However, immunohistochemical studies demonstrated a consistent inversion of H+-ATPase polarity in PT cells to a basolateral distribution contrasting with its apical location in the normal kidney. This inversion of polarity was specific for H+-ATPase and did not affect distribution of aminopeptidase, megalin, and Na+/K+-ATPase. Furthermore, apical H+-ATPase expression was absent in alpha-type intercalated cells.

CONCLUSIONS

ClC-5 mutations are associated with modifications in the polarity and expression of H+-ATPase, but not ultrastructural alterations in PT cells. These findings help further understanding of the role of ClC-5 and the pathophysiology of Dent's disease.

The ClC family of anion channels mediates the efficient, selective permeation of Cl(-) across the biological membranes of living cells under the driving force of an electrochemical gradient. In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl(-) anion. We infer details of this gating mechanism by studying the free energetics of Cl(-) occupancy in the pore of a prokaryotic ClC homolog. These free energetics were gleaned from 30 ns of molecular dynamics simulation on an approximately 133,000-atom system consisting of a hydrated membrane embedded StClC transporter. The binding sites for Cl(-) in the transporter were determined for the cases where the putative gating residue, Glu(148), was protonated and unprotonated. When the glutamate gate is protonated, Cl(-) favorably occupies an exterior site, S(ext), to form a queue of anions in the pore. However, when the glutamate gate is unprotonated, Cl(-) cannot occupy this site nor, consequently, pass through the pore. An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu(148). Although this suggests that, for the prokaryotic homolog, protonation of Glu(148) may be the first step in transporting Cl(-) at the expense of H(+) transport in the opposite direction, an evolutionary argument might suggest that Cl(-) opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation. During an additional 20 ns free dynamics simulation, the newly discovered outermost site, S(out), and the innermost site, S(int), were seen to allow spontaneous exchange of Cl(-) ions with the bulk electrolyte while under depolarization conditions.

Human peripheral blood LGL that mediated NK and small T cells were isolated in high purity (98% by morphology) by density sedimentation on discontinuous Percoll gradients. The proliferative frequency of these subpopulations in the presence of lectin-free conditioned media containing IL 2 was determined by limiting dilution analysis. LGL showed a 20-fold greater frequency of proliferative cell precursors than small T cells (1/200 and 1/4970, respectively). The NK-like nature of cells expanded from LGL preparations in IL 2 was confirmed by parallel testing of the cytotoxicity against K562. Whereas T cell microcultures showed no lytic activity against K562 (cytotoxic precursor frequency less than 1/10,000), LGL cultures showed frequencies of cytotoxic precursors (1/170) comparable to those of proliferative precursors. Neither responder cell type gave rise to detectable lytic activity against NK-insusceptible mouse lymphoma RL male 1 or alloblasts. LGL proliferation was only minimally affected by the presence of PHA at the onset of culture (rise to 1/74 with 2 micrograms/ml PHA). By contrast, small T cells showed a dose-dependent increase of proliferative frequency, to reach 1/11 with 2 micrograms/ml PHA, provided accessory cells in the form of PBMC, monocytes, or LGL but not T cells were present. The cytotoxic activity of LGL and small T cells expanded in IL 2 was confirmed in bulk cultures. LGL-CLC showed high lytic activity against NK-susceptible cell lines and a majority of freshly isolated allogeneic human tumor targets. T cell-CLC showed little activity against cell line targets (K562, Raji, L1210, RL male 1) but were lytic for some fresh tumor cells. These data establish optimal conditions for the growth of human LGL in IL 2-dependent culture and suggest that a major contributor to lysis of allogeneic human tumors by CLC is likely to be NK cells. The data indicate that large numbers of activated T cells cannot be detected in vivo and that in vitro induction of IL 2 receptors by lectin/antigen is necessary for the establishment of antigen-reactive T cell lines. In contrast, a proportion of LGL appear to be spontaneously activated and susceptible to IL 2-dependent growth. Thus, in the absence of stimulation, culture of unfractionated lymphoid cells in the presence of IL 2 is likely to select for the growth of LGL with NK activity.

BACKGROUND

Human lung mast cells (HLMCs) and the human mast cell line HMC-1 express a strongly outwardly rectifying Cl- current characteristic of that carried by the voltage-dependent Cl- channel ClC-5. A similar but distinct current has been implicated in the control of cell proliferation in astrocytes.

OBJECTIVE

In this study, we have examined the effects of the Cl- channel blocker tamoxifen on ion channel activity and cell proliferation in both HMC-1 and HLMCs.

METHODS

We used the whole-cell patch-clamp technique to characterize macroscopic ion currents in mast cells before and after addition of tamoxifen. HMC-1 proliferation was assessed by incorporation of tritiated thymidine, HLMC proliferation was determined by counting cells in long-term culture, and cell viability was assessed by annexin V binding and propidium iodide uptake.

RESULTS

In HMC-1, tamoxifen reduced the outward Cl- current at +130 mV by 73% +/- 9% at a concentration of 3 micromol/L and simultaneously opened a novel inwardly rectifying nonselective cation current with a mean inward current of 153 +/- 18 pA at -130 mV. Tamoxifen produced a dose-dependent inhibition of HMC-1 proliferation (90.3% +/- 4.0% inhibition at 30 micromol/L) without altering cell viability. Tamoxifen inhibited the outward ClC-5-like current in HLMCs, did not open an inward current, and produced a dose-dependent inhibition of HLMC proliferation in long-term culture.

CONCLUSIONS

Tamoxifen inhibits HMC proliferation, possibly through ion channel modulation. This suggests that tamoxifen might be useful in the treatment of mast-cell-mediated diseases, including mastocytosis, asthma, and pulmonary fibrosis.

BACKGROUND

Previous meta-analyses that compared the outcome of SILC and CLC have not presented consistent conclusions. This meta-analysis was performed after adding many recent RCTs, to clarify this issue.

METHODS

Relevant articles published in English were identified by searching PubMed, Embase, Web of Knowledge, and the Cochrane Controlled Trial Register from January 1997 to February 2013. Reference lists of the retrieved articles were reviewed to identify additional articles. Primary outcomes (postoperative pain scores, cosmetic score, and length of incision) and secondary outcomes (operating time, blood loss, conversion rates, postoperative complications, postoperative hospital stay, time to initial oral intake, and time to resume work) were pooled. Quantitative variables were calculated using the weighted mean difference (WMD), and qualitative variables were pooled using odds ratios (OR).

RESULTS

25 appropriate RCTs were identified from 2128 published articles. 1841 patients were treated, 944 with SILC and 897 with CLC. SILC was superior to CLC in cosmetic score (WMD = 1.155, P<0.001), shorter length of incision (WMD = -3.285, P = 0.029), and postoperative pain within 12 h (VAS in 3-4 h, WMD = -0.704, P = 0.026; VAS in 6-8 h, WMD = -0.613, P = 0.010). CLC was superior to SILC in operating time (OT) (WMD = 13.613, P<0.001) and need of additional instruments (OR = 7.448, P<0.001). Other secondary outcomes were similar.

CONCLUSIONS

SILC offered a better cosmetic result and less postoperative pain for patients with uncomplicated cholelithiasis or polypoid lesions of the gallbladder. However, SILC was associated with a longer OT and required additional instruments.

Hypercalciuria is the major risk factor promoting stone formation in Dent's disease, also known as X-linked recessive nephrolithiasis, but the effects of diuretics on calcium excretion and other stone risk factors in this disease are unknown. This study examined urine composition in eight male patients with Dent's disease, ages 6 to 49 yr, all of whom were hypercalciuric and had inactivating mutations of CLCN5. Eight males, ages 7 to 34 yr, with idiopathic hypercalciuria (IH) served as controls. Patients were instructed to maintain a consistent intake of sodium, potassium, calcium, and protein. Two consecutive 24-h urine collections were obtained after a baseline period and after 2 wk of chlorthalidone (25 mg), amiloride (5 mg), and the two diuretics in combination, with a week off drug separating the treatment periods in a randomized crossover design. Doses were reduced by half in boys under age 12 yr. Chlorthalidone alone (P < 0.002) and the combination of chlorthalidone and amiloride (P < 0.003) reduced calcium excretion significantly in either patient group. With chlorthalidone, calcium excretion fell to normal (<4.0 mg/kg per d) in all but one patient in each group. Amiloride alone had no significant effect on urinary calcium excretion, in either patient group. In patients with Dent's disease during chlorthalidone therapy, the supersaturation ratios for calcium oxalate and calcium phosphate fell by 25% and 35%, respectively. Mean citrate excretion was reduced by chlorthalidone (P <.04) and by chlorthalidone in combination with amiloride (P <.02). There were no significant differences in the responses to these diuretics between the patient groups in any of the urinary parameters. The intact hypocalciuric response to a thiazide diuretic indicates that inactivation of the ClC-5 chloride channel does not impair calcium transport in the distal convoluted tubule and indicates that thiazides should be useful in reducing the risk of kidney stone recurrence in patients with Dent's disease.

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.

Electrophysiologic and volumetric evidence link the swelling-activated Cl- channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl(-)-channel/Cl(-)-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl- channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl- channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 microM) which blocks>> 90% of the MDR1-associated Cl- currents, forskolin had no effect on the volume-activated Cl- channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mM) and niflumic acid (IC50 approximately 200 microM) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl(-)-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2(+)-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrink-age. These results support the concept that the volume-sensitive protein pICln regulates the Cl- channels, and that the same conduits subserve volume- and staurosporine-activated Cl- release. Of the cloned and sequenced Cl- channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl- channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl- channels of human nonpigmented ciliary epithelial cells.

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.

Using the patch-clamp technique, we investigated Cl- channels on the basolateral membrane of the connecting tubule (CNT) and cortical collecting duct (CCD). We found a approximately 10-pS channel in CNT cell-attached patches. Substitution of sodium gluconate for NaCl in the pipette shifted the reversal potential by +25 mV, whereas N-methyl-D-gluconate chloride had no effect, indicating anion selectivity. On inside-out patches, we determined a selectivity sequence of Cl->> Br- approximately NO3(-)>> F-, which is compatible with that of ClC-K2, a Cl- channel in the distal nephron. In addition, the number of open channels (NP(o)) measured in cell-attached patches was significantly increased when Ca2+ concentration or pH in the pipette was increased, which is another characteristic of ClC-K. These findings suggest that the basis for this channel is ClC-K2. A similar Cl- channel was found in CCD patches. Because CNT and CCD are heterogeneous tissues, we studied the cellular distribution of the Cl- channel using recording conditions (KCl-rich solution in the pipette) that allowed us to detect simultaneously Cl- channels and inwardly rectifying K+ channels. We detected Cl- channels alone in 45% and 42% and K+ channels alone in 51% and 58% of CNT and CCD patches, respectively. Cl- and K+ channels were recorded simultaneously from two patches (4% of patches) in the CNT and from none of the patches in the CCD. This indicates that Cl- and K+ channels are located in different cell types, which we suggest may be the intercalated cells and principal cells, respectively.

Cl- channels have been implicated in essential cellular functions including volume regulation, progression of cell cycle, cell proliferation and contraction, but the physiological functions of the ClC-3 channel are controversial. We tested the hypothesis that the ClC-3 gene (ClCn-3) is upregulated in hypertensive pulmonary arteries of monocrotaline-treated rats, and upregulated ClC-3 channel aids viability of pulmonary artery smooth muscle cells (PASMCs). Experimental pulmonary hypertension was induced in rats by a single subcutaneous administration of monocrotaline (60 mg kg(-1)). Injected animals developed characteristic features of pulmonary hypertension including medial hypertrophy of pulmonary arteries and right ventricular hypertrophy. Reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry and Western immunoblot analysis indicated that histopathological alterations were associated with upregulation of the ClC-3 mRNA and protein expression in both smooth muscle cells of hypertensive pulmonary arteries and in cardiac myocytes. RT-PCR analysis of mRNA, extracted from canine cultured PASMCs, indicated that incubation with the inflammatory mediators endothelin-1 (ET-1), platelet-derived growth factor (PDGF), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF alpha), but not transforming growth factor beta (TGFbeta), upregulated ClC-3 mRNA. Adenovirus-mediated delivery and overexpression of ClC-3 in canine PASMCs improved cell viability against increasing concentrations of hydrogen peroxide (H2O2, range 50-250 microM). In conclusion, upregulation of ClC-3 in rat hypertensive lung and heart is a novel observation. Our functional data suggest that upregulation of ClC-3 is an adaptive response of inflamed pulmonary artery, which enhances the viability of PASMCs against reactive oxygen species.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.

Dent's disease is a nephrolithiasis disorder associated with hypercalciuria and low molecular weight proteinuria that is caused by mutations in the voltage-gated chloride channel ClC-5. Because the exact cause of hypercalciuria in this disease is unknown and could come from a renal, intestinal, or bone origin, we have investigated overall calcium handling in the ClC-5 knockout mouse (ClC-5 KO). On a high calcium diet, ClC-5 KO mice had elevated serum 1alpha,25-dihydroxyvitamin D3 (1alpha,25D3), alkaline phosphatase (AP), osteocalcin (OC), and urinary deoxypyridinoline (DPD), but serum parathyroid hormone (PTH), calcium, and intestinal calcium uptake was similar to that of wild-type (WT) mice. A 30-fold decrease in dietary calcium intake caused elevation of serum PTH and urinary cyclic adenosine monophosphate in ClC-5 KO mice and decreased the renal calcium excretion, which still remained 2-fold above that of WT mice. On this low calcium diet, both groups of mice had the same serum 1alpha,25D3, with similar increments in intestinal calcium absorption, serum AP, OC, and urinary DPD. These data indicate that the hypercalciuria in the ClC-5 KO mice on low and high calcium diets is of bone and renal origin and is not caused by increased intestinal calcium absorption, despite an elevated serum 1alpha,25D3. These mice data suggest that young patients with this disease may have a propensity for altered bone homeostasis that should be monitored clinically.

Previously, we have demonstrated that the chloride channel ClC-2 modulates intestinal mucosal barrier function. In the present study, we investigated the role of ClC-2 in epithelial barrier development and maintenance in Caco-2 cells. During early monolayer formation, silencing of ClC-2 with small interfering (si)RNA led to a significant delay in the development of transepithelial resistance (TER) and disruption of occludin localization. Proteomic analysis employing liquid chromatography-mass spectrometry /mass spectrometry revealed association of ClC-2 with key proteins involved in intracellular trafficking, including caveolin-1 and Rab5. In ClC-2 siRNA-treated cells, occludin colocalization with caveolin-1 was diffuse and in the subapical region. Subapically distributed occludin in ClC-2 siRNA-treated cells showed marked colocalization with Rab5. To study the link between ClC-2 and trafficking of occludin in confluent epithelial monolayers, a Caco-2 cell clone expressing ClC-2 short hairpin (sh)RNA was established. Disruption of caveolae with methyl-β-cyclodextrin (MβCD) caused a marked drop in TER and profound redistribution of caveolin-1-occludin coimmunofluorescence in ClC-2 shRNA cells. In ClC-2 shRNA cells, focal aggregations of Rab5-occludin coimmunofluorescence were present within the cytoplasm. Wortmannin caused an acute fall in TER in ClC-2 shRNA cells and subapical, diffuse redistribution of Rab5-occludin coimmunofluorescence in ClC-2 shRNA cells. An endocytosis and recycling assay for occludin revealed higher basal rate of endocytosis of occludin in ClC-2 shRNA cells. Wortmannin significantly reduced the rate of recycling of occludin in ClC-2 shRNA cells. These data clearly indicate that ClC-2 plays an important role in the modulation of tight junctions by influencing caveolar trafficking of the tight junction protein occludin.

Rhizobium tropici CIAT899 is highly tolerant to several environmental stresses and is a good competitor for nodule occupancy of common bean plants in acid soils. Random transposon mutagenesis was performed to identify novel genes of this strain involved in symbiosis and stress tolerance. Here, we present a genetic analysis of the locus disrupted by the Tn5 insertion in mutant 899-PV9, which lead to the discovery of sycA, a homolog of the ClC family of chloride channels and Cl-/H+ exchange transporters. A nonpolar deletion in this gene caused serious deficiencies in nodule development, nodulation competitiveness, and N2 fixation on Phaseolus vulgaris plants, probably due to its reduced ability to invade plant cells and to form stable symbiosomes, as judged by electron transmission microscopy. A second gene (olsC), found downstream of sycA, is homologous to aspartyl/asparaginyl beta-hydroxylases and modifies two species of ornithine-containing lipids in vivo, presumably by hydroxylation at a still-unknown position. A mutant carrying a nonpolar deletion in olsC is symbiotically defective, whereas overexpressed OlsC in the complemented strain provokes an acid-sensitive phenotype. This is the first report of a ClC homolog being essential for the establishment of a fully developed N2-fixing root nodule symbiosis and of a putative beta-hydroxylase that modifies ornithine-containing membrane lipids of R. tropici CIAT899, which, in turn, are contributing to symbiotic performance and acid tolerance.

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl- channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl- channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.

OBJECTIVE

Rheumatoid arthritis (RA) is associated with accelerated atherosclerosis. The reduction in cardiovascular risk that is induced by methotrexate (MTX) and anti-tumor necrosis factor α agents in RA is considered secondary to their anti-inflammatory action, but their effects on serum lipoprotein function and foam cell formation are unknown. The reduced capacity of high-density lipoprotein (HDL) to promote cell cholesterol efflux and the increased serum cell cholesterol-loading capacity (CLC) demonstrated in RA may contribute to foam cell development. The aim of this study was to investigate the influence of MTX and adalimumab treatment on serum cholesterol efflux capacity (CEC) and CLC in RA patients and to study the in vitro effects of the two drugs on macrophage cholesterol handling.

METHODS

Sera from RA patients treated with MTX (n = 34) or with adalimumab and MTX (n = 22) obtained before treatment, after 6 weeks of treatment, and after 6 months of treatment were analyzed for CEC and CLC by radioisotopic and fluorometric techniques, respectively. The influence of MTX and adalimumab on macrophage cholesterol efflux and uptake was evaluated in vitro using human THP-1-derived macrophages.

RESULTS

MTX treatment was associated with increases in serum HDL, low-density lipoprotein, and total cholesterol levels and with ATP-binding cassette G1-mediated and scavenger receptor class B type I (SR-BI)-mediated increases in CEC; MTX treatment was not associated with modifications in CLC. Adalimumab treatment was associated with increases in serum HDL levels, a transient increase in SR-BI-mediated CEC, a transient decrease in ATP-binding cassette A1-mediated CEC, and a significant reduction in CLC; in addition, adalimumab reduced macrophage cholesterol uptake in vitro.

CONCLUSIONS

Antiatherosclerotic activity associated with MTX and adalimumab may be mediated by beneficial and complementary effects on lipoprotein functions and on macrophage cholesterol handling. As a whole, these mechanisms may oppose foam cell formation.

Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.

ClC-5 is a chloride (Cl(-)) channel expressed in renal tubules and is critical for normal tubular function. Loss of function nonsense or missense mutations in ClC-5 are associated with Dent's disease, a condition in which patients present with low molecular weight (LMW) proteinuria (including albuminuria), hypercalciuria and nephrolithiasis. Several key studies in ClC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. ClC-5 is typically regarded as an intracellular Cl(-) channel and thus the defect in this receptor-mediated uptake pathway was initially attributed to the failure of the early endosomes to acidify correctly. ClC-5 was postulated to play a key role in transporting the Cl(-) ions required to compensate for the movement of H(+) during endosomal acidification. However, more recent studies suggest additional roles for ClC-5 in the endocytosis of albumin. ClC-5 is now known to be expressed at low levels at the cell surface and appears to be a key component in the assembly of the macromolecular complex involved in protein endocytosis. Furthermore, mutations in ClC-5 affect the trafficking of v-H(+)-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of ClC-5 at the cell surface as well as its presence in endosomes appears to be essential for normal protein uptake by the renal proximal tubule.