Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome.

BACKGROUND: The proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. Here, the role of the NLRP3 inflammasome in experimental colitis induced by dextran sodium sulfate (DSS) was examined. METHODS: IL-1beta production in response to DSS was studied in macrophages of wild-type, caspase-1(-/-), NLRP3(-/-), ASC(-/-), cathepsin B(-/-) or cathepsin L(-/-) mice. Colitis was induced in C57BL/6 and NLRP3(-/-) mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. RESULTS: Macrophages incubated with DSS in vitro secreted high levels of IL-1beta in a caspase-1-dependent manner. IL-1beta secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1beta secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3(-/-) mice developed a less severe colitis than wild-type mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection comparable with NLRP3 deficiency. CONCLUSIONS: The NLRP3 inflammasome was identified as a critical mechanism of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for patients with IBD.

TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1(-/-) mice are viable, fertile and show no altered hematopoietic compartment. In CD4(+) T cell- and dextran sodium sulfate-induced models of colitis, Trem1(-/-) mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1(-/-) mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1(-/-) mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1(-/-) mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1(+/+) controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.

STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing.

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c(+) cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3(IEC-KO) mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing.

Protective and Aggravating Effects of Nlrp3 Inflammasome Activation in IBD Models: Influence of Genetic and Environmental Factors.

Background: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation due to dysregulation of the mucosal immune system. The cytokines IL-1β and IL-18 appear early in intestinal inflammation and their pro-forms are processed via the caspase-1-activating multiprotein complex, the Nlrp3 inflammasome. Previously, we reported that the uptake of dextran sodium sulfate (DSS) by macrophages activates the Nlrp3 inflammasome and that Nlrp3(-/-) mice are protected in the acute DSS colitis model. Of note, other groups have reported opposing effects in regards to DSS susceptibility in Nlrp3(-/-) mice. Recently, mice lacking inflammasomes were found to develop a distinct intestinal microflora. Methods: To reconcile the contradicting observations, we investigated the role of Nlrp3 deficiency in two different IBD models: acute DSS colitis and TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis. In addition, we investigated the impact of the intestinal flora on disease severity by performing cohousing experiments of wild-type and Nlrp3(-/-) mice, as well as by antibiotic treatment. Results: Nlrp3(-/-) mice treated with either DSS or TNBS exhibited attenuated colitis and lower mortality. This protective effect correlated with an increased frequency of CD103+ lamina propria dendritic cells expressing a tolerogenic phenotype in Nlrp3(-/-) mice in steady state conditions. Interestingly, after cohousing, Nlrp3(-/-) mice were as susceptible as wild-type mice, indicating that transmission of endogenous bacterial flora between the two mouse strains might increase susceptibility of Nlrp3(-/-) mice towards DSS-induced colitis. Accordingly, treatment with antibiotics almost completely prevented colitis in the DSS model. Conclusions: The composition of the intestinal microflora significantly influences disease severity in IBD models comparing wild-type and Nlrp3(-/-) mice. This observation may - at least in part - explain contradictory results concerning the role of the inflammasome in different labs. Further studies are required to define the role of the Nlrp3 inflammasome in noninflamed mucosa under steady state conditions and in IBD.

In vitro and in vivo Repair Activities of Undifferentiated and Classically and Alternatively Activated Macrophages.

Objective: Macrophages play a critical role in wound repair. However, the specific role of the different macrophage subtypes in wound repair remains incompletely understood. The aim of this study was to compare the wound repair activities of undifferentiated macrophages (M0), classically activated macrophages (M1) and alternatively activated (M2) macrophages. Methods: The macrophage repair activities of intestinal wounds were evaluated using in vitro and in vivo models. Results: All three macrophage subtypes enhanced wound closure in vitro, with the M2 macrophages demonstrating greater repair activities than the M0 and M1 macrophages. Injection of M0 and M2 macrophages into mice with experimental dextran sodium sulfate-induced colitis significantly enhanced ulcer repair when compared to control mice. In contrast, injection of M1 macrophages did not affect ulcer repair. Conclusions: These results underscore the wound repair capacity of different macrophage subsets. Notably, wound repair activity is not restricted to M2 macrophages, as the current literature suggests. © 2014 S. Karger AG, Basel.

CCL17 Promotes Intestinal Inflammation in Mice and Counteracts Regulatory T Cell-Mediated Protection From Colitis.

BACKGROUND & AIMS: Priming of T cells by dendritic cells (DCs) in the intestinal mucosa and associated lymphoid tissues helps maintain mucosal tolerance but also contributes to the development of chronic intestinal inflammation. Chemokines regulate the intestinal immune response and can contribute to pathogenesis of inflammatory bowel diseases. We investigated the role of the chemokine CCL17, which is expressed by conventional DCs in the intestine and is up-regulated during colitis. METHODS: Colitis was induced by administration of dextran sodium sulfate (DSS) to mice or transfer of T cells to lymphopenic mice. Colitis activity was monitored by body weight assessment, histologic scoring, and cytokine profile analysis. The direct effects of CCL17 on DCs and the indirect effects on differentiation of T helper (Th) cells were determined in vitro and ex vivo. RESULTS: Mice that lacked CCL17 (Ccl17(E/E) mice) were protected from induction of severe colitis by DSS or T-cell transfer. Colonic mucosa and mesenteric lymph nodes from Ccl17-deficient mice produced lower levels of proinflammatory cytokines. The population of Foxp3(+) regulatory T cells (Tregs) was expanded in Ccl17(E/E) mice and required for long-term protection from colitis. CCR4 expression by transferred T cells was not required for induction of colitis, but CCR4 expression by the recipients was required. CCL17 promoted Toll-like receptor-induced secretion of interleukin-12 and interleukin-23 by DCs in an autocrine manner, promoted differentiation of Th1 and Th17 cells, and reduced induction of Foxp3(+) Treg cells. CONCLUSIONS: The chemokine CCL17 is required for induction of intestinal inflammation in mice. CCL17 has an autocrine effect on DCs that promotes production of inflammatory cytokines and activation of Th1 and Th17 cells and reduces expansion of Treg cells.

The Selective Phosphodiesterase 4 Inhibitor Roflumilast and Phosphodiesterase 3/4 Inhibitor Pumafentrine Reduce Clinical Score and TNF Expression in Experimental Colitis in Mice.

OBJECTIVE: The specific inhibition of phosphodiesterase (PDE)4 and dual inhibition of PDE3 and PDE4 has been shown to decrease inflammation by suppression of pro-inflammatory cytokine synthesis. We examined the effect of roflumilast, a selective PDE4 inhibitor marketed for severe COPD, and the investigational compound pumafentrine, a dual PDE3/PDE4 inhibitor, in the preventive dextran sodium sulfate (DSS)-induced colitis model. METHODS: The clinical score, colon length, histologic score and colon cytokine production from mice with DSS-induced colitis (3.5% DSS in drinking water for 11 days) receiving either roflumilast (1 or 5 mg/kg body weight/d p.o.) or pumafentrine (1.5 or 5 mg/kg/d p.o.) were determined and compared to vehicle treated control mice. In the pumafentrine-treated animals, splenocytes were analyzed for interferon-γ (IFNγ) production and CD69 expression. RESULTS: Roflumilast treatment resulted in dose-dependent improvements of clinical score (weight loss, stool consistency and bleeding), colon length, and local tumor necrosis factor-α (TNFα) production in the colonic tissue. These findings, however, were not associated with an improvement of the histologic score. Administration of pumafentrine at 5 mg/kg/d alleviated the clinical score, the colon length shortening, and local TNFα production. In vitro stimulated splenocytes after in vivo treatment with pumafentrine showed a significantly lower state of activation and production of IFNγ compared to no treatment in vivo. CONCLUSIONS: These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice.

Phenotypical analysis of mice deficient for the channel activating protease CAP2

Channel activating proteases (CAP) are membrane-bound serine proteases that have been identified as in vitro activators of the epithelial sodium channel (ENaC). Two of them are mainly studied in the laboratory. CAP1/Prss8 was previously shown implicated in colonic sodium homeostasis in vivo. In the first part of this thesis, we generated and characterized mice deficient for CAP2/Tmprss4. The mice are healthy and viable, and they do not show any obvious phenotype. We investigated ENaC activity and expression under regular and sodium- deficient diet, and we could demonstrate that CAP2 is not a major regulator of sodium homeostasis in vivo. We next studied whether CAP2 is implicated in potassium homeostasis. We detected a strong gender-dependency when CAP2 knock-out mice were put under a potassium-deficient diet. We showed in male mice an implication of CAP2 in the regulation of the colonic H+, K+- ATPase, and we propose an implication of membrane-associated progesterone receptors and their binding partners, as well as a possible cleavage-mediated glucocorticoid receptor signalling. We studied the possible interaction between CAPI and CAP2 by generating and characterizing two different mouse study groups, displaying different hypomorphic mutations in the CAPI gene, and deficient for CAP2. We demonstrate that balanced expression of CAPI and CAP2 is required for maintainance of skin integrity and for normal placental development. As CAPI knock-out embryos die due to a placental failure, the additional combined deletion of CAP2 resulted in survival until birth. We could evidence that CAPI and CAP2 are implicated in the same signalling pathway as proposed in cancer studies at the level of the placenta, implicating integrin a5, ERK, AKT, E- and N-cadherin. Furthermore, we investigated whether CAPI is implicated in the pathogenesis and susceptibility to experimental chronic colitis in a mutant rat model. By giving CAPI mutant rats Dextran sodium sulfate, we induced chronic inflammation of the colon, and we highlighted the protective role of CAPI at the histopathological and clinical levels. In conclusion, we showed that CAP2 is not a major regulator of ENaC-mediated sodium homeostasis in vivo, but rather a regulator of potassium homeostasis in a gender-dependent manner implicating the colonic H+, K+-ATPase, membrane progesterone receptors, and the glucocorticoid receptor. We have investigated whether CAPI and CAP2 interact at the functional level, and we show that a balanced expression of CAPI and CAP2 is required in the skin, but also in the placenta. Imbalanced expression of CAPI and CAP2 leads to impaired EMT-associated signalling. We have studied whether CAPI is implicated in the pathogenesis and susceptibility to chronic colitis, and we demonstrated the protective role of CAPI in distal colon. -- Les protéases activatrices de canal (CAP) sont des protéases à serine attachées à la membrane qui ont été identifiées comme activateurs in vitro du canal sodique épithélial (ENaC). Deux de ces protéases sont principalement étudiées dans le laboratoire. CAP1/Prss8 a été identifié préalablement comme impliqué dans l'homéostasie du sodium in vivo au niveau du côlon. Dans la première partie de cette thèse, nous avons généré et caractérisé des souris déficientes pour CAP2/Tmprss4. Les souris sont en bonne santé et viables, et elles ne présentent pas de phénotype visible. Nous avons étudié l'activité et l'expression d'ENaC sous diète normale et déficiente en sodium, et nous avons démontré que CAP2 n'est pas un régulateur essentiel de l'homéostasie sodique in vivo. Nous avons ensuite étudié si CAP2 est impliqué dans l'homéostasie du potassium. Nous avons détecté une forte dépendance du sexe lorsque les souris knock-out pour CAP2 étaient placées sous diète déficiente en potassium. Nous avons démontré dans les souris mâles une implication de CAP2 dans la régulation de la H+, K+- ATPase colonique, des récepteurs membranaires à la progestérone et de leur partenaires de liaison, ainsi que dans la possible signalisation médiée par le clivage du récepteur aux glucocorticoïdes. Nous avons étudié l'interaction possible entre CAPI et CAP2 en générant et en caractérisant deux groupes d'étude de souris différents, porteurs de différentes mutations hypomorphiques dans le gène de CAPI, et déficients pour CAP2. Nous avons pu montrer qu'une expression équilibrée de CAPI et CAP2 est requise pour le maintien de l'intégrité de la peau et pour le développement normal du placenta. Les embryons knock-out pour CAPI meurent suite à une défaillance placentaire, et la délétion additionnelle et combinée de CAP2 permet la survie jusqu'à la naissance. Nous supposons que CAPI et CAP2 sont impliqués dans la même voie de signalisation au niveau du placenta que celle proposée dans les études de cancer, impliquant l'intégrine a5, ERK, AKT, E- et N-cadhérine. De plus, nous avons étudié si CAPI est impliqué dans la pathogenèse et la susceptibilité de colite chronique expérimentale dans un modèle de rat mutant. En administrant aux rats mutants pour CAPI du Dextran sodium sulfate, nous avons induit une inflammation chronique du côlon, et nous avons pu mettre en évidence le rôle protecteur de CAPI au niveau histopathologique et au niveau clinique. En conclusion, nous avons démontré que CAP2 n'est pas un régulateur essentiel de l'homéostasie sodique médiée par ENaC in vivo, mais plutôt de l'homéostasie potassique d'une manière dépendante du sexe et impliquant la H+, K+-ATPase colonique, les récepteurs membranaires à la progestérone et le récepteur aux glucocorticoïdes. Nous avons étudié si CAPI et CAP2 interagissent au niveau fonctionnel, et nous avons montré qu'une expression équilibrée entre CAPI et CAP2 est requise dans la peau et le placenta. L'expression déséquilibrée de CAPI et CAP2 mène à une altération de la signalisation associée à l'EMT. Nous avons étudié si CAPI est impliqué dans la pathogenèse et la susceptibilité de colite chronique expérimentale, et nous avons démontré le rôle protecteur de CAPI dans le côlon distal.

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast.

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.

Renal sodium handling and nighttime blood pressure.

Blood pressure follows a circadian rhythm with a physiologic 10% to 20% decrease during the night. There is now increasing evidence that a blunted decrease or an increase in nighttime blood pressure is associated with a greater prevalence of target organ damage and a faster disease progression in patients with chronic kidney diseases. Several factors contribute to the changes in nighttime blood pressure including changes in hormonal profiles such as variations in the activity of the renin-angiotensin and the sympathetic nervous systems. Recently, it was hypothesized that the absence of a blood pressure decrease during the nighttime (nondipping) is in fact a pressure-natriuresis mechanism enabling subjects with an impaired capacity to excrete sodium to remain in sodium balance. In this article, we review the clinical and epidemiologic data that tend to support this hypothesis. Moreover, we show that most, if not all, clinical conditions associated with an impaired dipping profile are diseases associated either with a low glomerular filtration rate and/or an impaired ability to excrete sodium. These observations would suggest that renal function, and most importantly the ability to eliminate sodium during the day, is indeed a key determinant of the circadian rhythm of blood pressure.

Getting the most sulfate from soil: Regulation of sulfate uptake transporters in Arabidopsis.

Sulfur (S) is an essential macronutrient for all living organisms. Plants require large amounts of sulfate for growth and development, and this serves as a major entry point of sulfate into the food web. Plants acquire S in its ionic form from the soil; they have evolved tightly controlled mechanisms for the regulation of sulfate uptake in response to its external and internal availability. In the model plant Arabidopsis thaliana, the first key step in sulfate uptake is presumed to be carried out exclusively by only two high-affinity sulfate transporters: SULTR1;1 and SULTR1;2. A better understanding of the mode of regulation for these two transporters is crucial because they constitute the first determinative step in balancing sulfate in respect to its supply and demand. Here, we review the recent progress achieved in our comprehension of (i) mechanisms that regulate these two high-affinity sulfate transporters at the transcriptional and post-transcriptional levels, and (ii) their structure-function relationship. Such progress is important to enable biotechnological and agronomic strategies aimed at enhancing sulfate uptake and improving crop yield in S-deficient soils.

Metoprolol prevents sodium retention induced by lower body negative pressure in healthy men.

BACKGROUND: Lower body negative pressure (LBNP) has been shown to induce a progressive activation of neurohormonal systems, and a renal tubular and hemodynamic response that mimics the renal adaptation observed in congestive heart failure (CHF). As beta-blockers play an important role in the management of CHF patients, the effects of metoprolol on the renal response were examined in healthy subjects during sustained LBNP. METHODS: Twenty healthy male subjects were randomized in this double blind, placebo versus metoprolol 200 mg once daily, study. After 10 days of treatment, each subject was exposed to 3 levels of LBNP (0, -10, and -20 mbar) for 1 hour, each level of LBNP being separated by 2 days. Neurohormonal profiles, systemic and renal hemodynamics, as well as renal sodium handling were measured before, during, and after LBNP. RESULTS: Blood pressure and heart rate were significantly lower in the metoprolol group throughout the study (P < 0.01). GFR and RPF were similar in both groups at baseline, and no change in renal hemodynamic values was detected at any level of LBNP. However, a reduction in sodium excretion was observed in the placebo group at -20 mbar, whereas no change was detected in the metoprolol group. An increase in plasma renin activity was also observed at -20 mbar in the placebo group that was not observed with metoprolol. CONCLUSION: The beta-blocker metoprolol prevents the sodium retention induced by lower body negative pressure in healthy subjects despite a lower blood pressure. The prevention of sodium retention may be due to a blunting of the neurohormonal response. These effects of metoprolol on the renal response to LBNP may in part explain the beneficial effects of this agent in heart failure patients.

Metabolic and respiratory effects of infused sodium acetate in healthy human subjects.

The metabolic and respiratory effects of intravenous 0.5 M sodium acetate (at a rate of 2.5 mmol/min during 120 min) were studied in nine normal human subjects. O2 consumption (VO2) and CO2 production (VCO2) were measured continuously by open-circuit indirect calorimetry. VO2 increased from 251 +/- 9 to 281 +/- 9 ml/min (P < 0.001), energy expenditure increased from 4.95 +/- 0.17 kJ/min baseline to 5.58 +/- 0.16 kJ/min (P < 0.001), and VCO2 decreased nonsignificantly (211 +/- 7 ml/min vs. 202 +/- 7 ml/min, NS). The extrapulmonary CO2 loss (i.e., bicarbonate generation and excretion) was estimated at 48 +/- 5 ml/min. This observation is consistent with 1 mol of bicarbonate generated from 1 mol of acetate metabolized. Alveolar ventilation decreased from 3.5 +/- 0.2 l/min basal to 3.1 +/- 0.2 l/min (P < 0.001). The minute ventilation (VE) to VO2 ratio decreased from 22.9 +/- 1.3 to 17.6 +/- 0.9 l/l (P < 0.005), arterial PO2 decreased from 93.2 +/- 1.9 to 78.7 +/- 1.6 mmHg (P < 0.0001), arterial PCO2 increased from 39.2 +/- 0.7 to 42.1 +/- 1.1 mmHg (P < 0.0001), pH from 7.40 +/- 0.005 to 7.50 +/- 0.007 (P < 0.005), and arterial bicarbonate concentration from 24.2 +/- 0.7 to 32.9 +/- 1.1 (P < 0.0001). These observations indicate that sodium acetate infusion results in substantial extrapulmonary CO2 loss, which leads to a relative decrease of total and alveolar ventilation.

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis.

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.