Peripheral blood mononuclear cell response to YKL-40 and Galectin-3 in cystic fibrosis.

BACKGROUND: Elevated circulating levels of YKL-40 correlate with disease severity in Cystic Fibrosis (CF), but the role of YKL-40 in the inflammatory response in CF is still under investigation. Our main goal was to evaluate if YKL-40 can modulate the expression of major cytokines (IL-6, IL-10, IL-13) implicated in the inflammatory response in CF. A secondary goal was to explore the interactions between YKL-40 and other circulating proteins to determine the impacts on cytokine modulation. METHOD: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 83 adult CF patients in stable clinical condition. PBMCs were treated with human YKL-40 followed by the measure of IL-6, IL-10 and IL-13 gene expression. Protein arrays were used to explore the interactions between YKL-40 and circulating proteins. Interaction with Galectin-3 (GAL3) was identified, and confirmed by binding assay. Cytokine gene expressions were again monitored by RT-qPCR after PBMC treatment with GAL3, with or without YKL-40 co-stimulation. RESULTS: Following YKL-40 stimulation, PBMC gene expression of IL-6, IL-10 and IL-13 varies across patients. IL-6 and IL-13 are coexpressed, but this response was different in male and female patients. GAL3 protein was detected in the blood of CF patients, and a molecular interaction with YKL-40 was identified. GAL3 did not interfere with the YKL-40 stimulation of IL-6, IL-10 and IL-13 but may modulate the coexpression. CONCLUSION: We observed that YKL-40 stimulation had a variable impact on IL-6, IL-10, and IL-13 gene expression in CF PBMCs and uncovered an interaction between GAL3 and YKL-40 in the serum of CF patients. Our findings suggest that YKL-40 is not only a biomarker of disease severity in CF, but it might play an active role in the inflammatory pathophysiology of the disease.

Association between glucose intolerance and bacterial colonisation in an adult population with cystic fibrosis, emergence of Stenotrophomonas maltophilia.

BACKGROUND: Diabetes is common in cystic fibrosis (CF). Glucose can be detected in the airway when the blood glucose is elevated, which favours bacterial growth. We investigated the relationship between dysglycemia and lung pathogens in CF. METHODS: Cross-sectional and prospective analysis of CF patients (N=260) who underwent a 2h-oral glucose tolerance test. Clinical data was collected. RESULTS: Stenotrophomonas maltophilia (S. maltophilia) was the sole bacteria increased in dysglycemic (AGT: 20.2%, CFRD: 21.6%) patients compared to normotolerants (NGT: 8.7%). S. maltophilia positive patients with dysglycemia had more pulmonary exacerbation events compared to NGTs (1.22 vs 0.63, P=0.003). The interaction between S. maltophilia colonisation and glucose tolerance status significantly increases the risk of lower lung function (P=0.003). Its growth was not affected by the evolution of the glucose tolerance after three years follow-up. CONCLUSION: Prevalence of S. maltophilia was higher in dysglycemic patients, supporting the idea that S. maltophilia is a marker of disease severity in CF.

Ralstonia mannitolilytica in cystic fibrosis: A new predictor of worse outcomes.

BACKGROUND: Patients with Cystic Fibrosis are subject to repeated respiratory tract infections, with recent increasing isolation of unusual pathogens. Ralstonia species have lately been isolated at our institution, an organism historically frequently misidentified as Burkholderia or Pseudomonas. The prevalence of Ralstonia spp. in cystic fibrosis populations has yet to be determined, along with its clinical implications. CASE PRESENTATIONS: Seven patients out of the 301 followed at our cystic fibrosis clinic have had Ralstonia strains identified in their respiratory tract. Most strains identified were multi-drug resistant. After aquisition of Ralstonia spp., the patients' clinical course was characterized by more frequent and more severe respiratory infections along with prolonged hospitalizations, greater decline of lung function, and greater mortality. The mortality rate in this group of patients was 86%. No other factor that could explain such a dramatic evolution was identified upon review of patient data. Some of the strains involved were recognized as clones on Pulse Field Electrophoresis Gel, raising the question of person-to-person transmission. CONCLUSION: New pathogens are identified with the evolution of the microbiota in cystic fibrosis respiratory tracts. In our cohort of patients, acquisition of Ralstonia spp. was associated with dramatic outcomes in terms of disease acceleration and raised mortality rates. It is of critical importance to continue to better define the prevalence and clinical impact of Ralstonia in cystic fibrosis populations.

The association between leptin and insulin levels in adults with cystic fibrosis.

UNLABELLED: Cystic fibrosis (CF)-related diabetes is an important complication of CF caused by a decrease in insulin secretion that is associated with weight loss, poor nutritional status and increased mortality. Leptin, a hormone secreted from white adipose tissue, has an important role in energy homoeostasis by inhibiting food intake and increasing energy expenditure. Leptin secretion can be increased by nutrient signals such as insulin. AIMS: Considering that leptin plays a role in energy homoeostasis and that CF is associated to poor weight gain and decreased insulin secretion, leptin levels in CF patients with different glucose tolerances were investigated and compared with those of healthy control subjects. METHODS: Two-hour oral glucose tolerance tests (OGTT) were performed in 82 patients with CF and various glucose tolerances as well as in 17 healthy control subjects during which blood was withdrawn every 30 min to measure glucose and insulin. Fasting leptin, fibrinogen and fat mass were also measured, and body mass index (kg/m(2)) calculated for all participants. Early and late insulin secretion was separated by calculating the area under the curve from time 0 to 30 min and 30 to 120 min of the OGTT (AUC(0-30) and AUC(30-120)). RESULTS: Leptin levels were comparable between CF patients and healthy control subjects. Interestingly, correlations were observed between leptin levels and insulin (AUC(0-120) and AUC(30-120)) after adjusting for gender and fat mass (P<0.05). CONCLUSION: This study suggests a potential role of insulin in regulating leptin levels in adults with stable CF.

Normal total and high molecular weight adiponectin levels in adults with cystic fibrosis.

Cystic fibrosis related diabetes (CFRD) is an important complication of CF that increases mortality. Adiponectin, an adipokine secreted from adipose tissue, plays an important role in fatty acid and glucose metabolism. Lower total adiponectin (TA) levels have been linked to the risk of developing type 2 diabetes. However, studies show that the high molecular weight isoform (HMW), thought to be more active than TA, might be a better indicator of insulin sensitivity. Our aim was to determine the association between HMW and insulin sensitivity in CF subjects and determine if other factors might modulate its levels. Thirteen control subjects and 47 CF adults (16 with normal glucose tolerance, 16 prediabetic and 15 with CFRD) underwent an oral glucose tolerance test. Blood samples were taken at time 0, 30, 60, 90 and 120 min. Body mass index, fibrinogen, glucose and insulin, TA and HMW were measured in every subject. Regression analysis was used to determine the association between TA, HMW and glucose (fasting glucose, 2h glucose and glucose AUC) as well as insulin (fasting insulin, insulin AUC, and Stumvoll insulin sensitivity index) parameters. TA and HMW levels were similar between CF patients and controls and were not associated to insulin sensitivity. TA was negatively associated to insulin AUC (p=0.0108) and 2h glucose (p=0.0116) in controls while these relationships were either weakly negative (p=0.0208) or weakly positive (p=0.0105) in CF patients. Also, HMW was negatively associated to insulin (p=0.00301) and glucose AUC (p=0.0546) in controls whereas these associations were positive in CF patients (p=0.0388, p=0.0232 respectively). In conclusion, our exploratory study on HMW adiponectin demonstrated similar levels of TA and HMW between CF patients and controls and different relationships between forms of adiponectin to glucose metabolism and insulin in CF.

Validation of insulin secretion indices in cystic fibrosis patients.

BACKGROUND AND AIMS: Impaired insulin secretion plays a key role in the development of cystic fibrosis-related diabetes (CFDR). Numerous indices to evaluate insulin secretion have been proposed, but their validity has not been explored in cystic fibrosis (CF). The aim of the present study was to validate surrogate indices of insulin secretion in CF patients against the gold standard, the intravenous glucose tolerance test (IVGTT). METHODS: 32 subjects were enrolled: 16 controls, 10 cystic fibrosis-normal glucose tolerant (CF-NGT) and 6 CFRD patients. All subjects underwent a 2-h oral glucose tolerance test (OGTT) and an IVGTT. Insulin secretion was estimated using three indices: the HOMA-beta cell, the Stumvoll's early insulin secretion, and the insulin secretion rate (ISR). RESULTS: In control subjects, all insulin secretion indices correlated significantly with the IVGTT. In CF-NGT patients, the HOMA-beta cell correlated significantly with the IVGTT at shorter time points but not over longer time-period. On the other hand, both OGTT-derived indices (Stumvoll and IRS) correlated significantly with the IVGTT for the CF-NGT and CFRD groups. CONCLUSION: Since the OGTT is required on a regular basis in CF patients to screen for CFRD, OGTT-derived indices appear to be suitable for evaluating insulin secretion.

Dichotomy between postprandial glucose and lipid profiles in adults with cystic fibrosis: a pilot study.

BACKGROUND: Cystic fibrosis (CF) patients present a high incidence of glucose tolerance abnormalities. Altered insulin secretion combined with recommended high-fat intake could be associated with dysregulation of glucose and lipid metabolism. We examined postprandial glucose and lipid profiles during an oral glucose tolerance test (OGTT) and following a standardized high-fat test meal (TM). METHODS: Sixteen CF patients with normal glucose tolerance (NGT) or CF-related diabetes (CFRD) and 16 controls underwent a 4 h OGTT and a TM. We then measured plasma glucose, insulin, free fatty acid (FFA) and triglyceride (TG) concentrations. RESULTS: CF patients presented higher glucose excursion compared to controls after the OGTT and TM. However, in CF patients, this excursion was significantly reduced in both amplitude and length after the TM. The TM provoked a comparable increase in TG levels in both groups whereas they remained stable during the OGTT. FFAs were suppressed similarly in both groups after both challenges. CONCLUSION: CF is associated with abnormal glucose excursion in the presence of relatively normal lipid excursion. The rapid normalization of glucose values after a mixed meal should be further explored and, if confirmed, might have significant implications for CFRD diagnostic.

No relationship between mean plasma glucose and glycated haemoglobin in patients with cystic fibrosis-related diabetes.

AIM: Cystic fibrosis-related diabetes (CFRD) prevalence has increased dramatically with the improved life expectancy of patients with cystic fibrosis (CF). Glycated haemoglobin (HbA(1c)) is an important tool for monitoring blood glucose control but, unlike in type 1 and type 2 diabetes, a correlation between HbA(1c), fructosamine and mean plasma glucose has not been clearly established in CF. This study aimed to examine the relationship between mean plasma glucose and HbA(1c) or fructosamine in stable patients with CFRD. METHODS: Fifteen type 1 diabetes and 13 CFRD patients (HbA(1c)<9.0%; no anaemia), matched for age and body mass index (BMI), provided 72 capillary blood glucose profiles taken 3days/month for three months. At the end of this time, HbA(1c) and fructosamine were measured. Mean plasma glucose was estimated using the Diabetes Control and Complications Trial (DCCT) conversion formula, and linear regressions carried out to establish its relationship with HbA(1c) and fructosamine. RESULTS: In type 1 diabetes patients, mean plasma glucose correlated significantly with HbA(1c) (r=0.68; P=0.005). In CFRD patients, no correlation was found between mean plasma glucose and HbA(1c) (r=0.24; P=0.460). Also, no association was found between mean plasma glucose, representing the month before blood sampling, and fructosamine in either group. CONCLUSION: Unlike in type 1 diabetes, HbA(1c) did not correlate with mean plasma glucose in CFRD subjects. Thus, having a normal HbA(1c) may not be sufficient to indicate a low risk of diabetes complications in CFRD. Further studies are required to explain such a discrepancy.

Normal adiponectin levels despite abnormal glucose tolerance (or diabetes) and inflammation in adult patients with cystic fibrosis.

RATIONALE: Circulating adiponectin levels are negatively associated with glucose intolerance, inflammation and central adiposity. Since these conditions are common in cystic fibrosis (CF), we examined whether adiponectin values are altered in these patients. AIM: To determine if CF patients have altered adiponectin levels and if these levels correlate with glucose tolerance categories (normal, impaired glucose tolerance (IGT) and cystic fibrosis-related diabetes (CFRD)), insulin resistance or inflammatory markers such as fibrinogen and C-reactive protein (CRP). METHODS: Oral glucose tolerance tests (OGTTs) were performed and adiponectin levels were measured in 90 CF patients not known to be diabetic and 15 healthy controls matched for age, sex and body mass index (BMI). Inflammatory markers, serum albumin concentrations and the clinical status of CF patients (i.e. pulmonary function) were also examined. RESULTS: CF pathology was characterized by a high prevalence (43.5%) of glucose tolerance abnormalities: 26.5% of IGT and 17.0% of newly diagnosed CFRD. CF patients also presented systemic inflammation as revealed by a significant increase of fibrinogen (P=0.029) in all patients and higher CRP levels in CFRD patients compared to the controls (P<0.05). On the other hand, CF and control subjects had similar albumin serum concentration. While CF patients and controls had similar serum adiponectin values, women had significantly higher hormone levels than men (P<0.001). Adiponectin levels did not correlate with glucose tolerance, inflammatory markers or insulin resistance. On the other hand, they correlated positively with both total and HDL-cholesterol (P<0.001). CONCLUSION: CF patients did not show any alterations in adiponectin levels despite insulin resistance, glucose intolerance and sub clinical chronic inflammation. Thus, CF appears to be one of the rare conditions in which discordance between adiponectin values and insulin resistance or inflammation is evident.

Predictors of pulmonary exacerbations in patients with cystic fibrosis infected with multi-resistant bacteria.

BACKGROUND: This study examined characteristics of adult and adolescent patients with cystic fibrosis (CF) to determine factors associated with an increased risk of pulmonary exacerbations. METHODS: 249 patients with CF infected with multidrug resistant bacteria were recruited and prospectively followed for up to 4.5 years until they experienced a pulmonary exacerbation severe enough to require intravenous antibiotics. Multivariable regression analyses were used to compare the characteristics of patients who experienced an exacerbation with those who did not. RESULTS: 124 of the 249 patients (50%) developed a pulmonary exacerbation during the first year and 154 (62%) experienced an exacerbation during the 4.5 year study period. Factors predictive of exacerbations in a multivariable survival model were younger age (OR 0.98, 95% CI 0.96 to 0.99), female sex (OR 1.45, 95% CI 1.07 to 1.95), lower forced expiratory volume in 1 second (FEV(1)) (OR 0.98, 95% CI 0.97 to 0.99), and a previous history of multiple pulmonary exacerbations (OR 3.16, 95% CI 1.93 to 5.17). Chronic use of inhaled corticosteroids was associated with an increased risk of exacerbation (OR 1.92, 95% CI 1.00 to 3.71) during the first study year. CONCLUSIONS: Patients who experience pulmonary exacerbations are more likely to be younger, female, using inhaled steroids, have a lower FEV(1), and a history of multiple previous exacerbations. It is hoped that knowledge of these risk factors will allow better identification and closer monitoring of patients who are at high risk of exacerbations.

Diabetes: a major co-morbidity of cystic fibrosis.

Cystic fibrosis-related diabetes (CFRD) is a frequent complication of cystic fibrosis, its prevalence increases with age of patient and is close to 30% at the age of 30 years. As life expectancy greatly increases, the number of cystic fibrosis patients developing diabetes will increase too. CFRD shares some features with type 1 and type 2 diabetes, initial phase is characterised by postprandial hyperglycaemia followed by a progression toward insulin deficiency. Insulin deficiency is an essential factor in the development of diabetes with an additional contribution of insulin resistance. Systematic screening with an oral glucose tolerance test is recommended from the age of 14 years because clinical signs of CFRD are often confused with signs of pulmonary infection and CFRD occurrence is associated with weight and pulmonary function deterioration. In observational studies CFRD diagnosis is associated with a significant increase in mortality, while treatment allow correction of weight and lung deterioration suggesting that CFRD has a significant impact on CF evolution. Microvascular complications are recognised, although paucity of data does not permit a clear description of their natural history. Annual screening for microvascular complication is recommended. There is no evidence by now that CF patients develop macrovascular complications. The only recommended pharmacological treatment is insulin therapy.

Purinergic-induced ion current in monolayers of C7-MDCK cells: role of basolateral and apical ion transporters.

This study examines purinergic modulation of short-circuit current (I(SC)) in monolayers of C7- and C11-MDCK cells resembling principal and intercalated cells from collecting ducts. In C7 monolayers, basolateral and apical application of ATP led to similar elevation of I(SC), consisting of a transient phase with maximal I(SC) increment of approximately 10 microA/cm2 terminating in 2-3 min, and a sustained phase with maximal I(SC) less than 2 microA/cm2 and terminating in 10 min. ATP-induced I(SC) was insensitive to the presence of Na+, Cl- and inhibitors of K+ (Ba2+, charibdotoxin (ChTX), clotrimazole (CLT), apamin) and Na + (amiloride) channels in the mucosal solution. Inhibitors of Cl- channels, DIDS and NPPB, added to apical membranes at a concentration of 100 microM, did not affect ATP-induced I(SC), whereas at 500 microM, NPPB inhibited it by 70-80%. Substitution of Cl- with NO3- in serosal medium decreased ATP-induced I(SC) by 2-3-fold and elevation of [K+]o from 6 to 60 mM changed its direction. Basolateral NPPB inhibited I(SC) by 10-fold with ED50 of approximately 30 microM, whereas ChTX (50 nM) and CLT (2 microM) diminished this parameter by 30-50%. Suppression of Na+, K+, Cl- cotransport with bumetanide did not affect the transient phase of ATP-induced I(SC) and slightly diminished its sustained phase. ATP increased ouabainand bumetanide-resistant K+ (86Rb) influx across the basolateral membrane by 7-8-fold, which was partially inhibited with ChTX and CLT. ATP-treated C11 cells exhibited negligible I(SC), and their presence did not affect I(SC) triggered by ATP in C7 cells. Thus, our results show that transcellular ion current in ATP-treated C7 cells is mainly caused by the coupled function of apical and basolateral anion transporters providing transient Cl- secretion. These transporters possess different sensitivities to anion channel blocker NPBB and are under the control of basolateral K+ channels(s) inhibited by ChTX and CLT.

Treatment of ARDS.

Improved understanding of the pathogenesis of acute lung injury (ALI)/ARDS has led to important advances in the treatment of ALI/ARDS, particularly in the area of ventilator-associated lung injury. Standard supportive care for ALI/ARDS should now include a protective ventilatory strategy with low tidal volume ventilation by the protocol developed by the National Institutes of Health ARDS Network. Further refinements of the protocol for mechanical ventilation will occur as current and future clinical trials are completed. In addition, novel modes of mechanical ventilation are being studied and may augment standard therapy in the future. Although results of anti-inflammatory strategies have been disappointing in clinical trials, further trials are underway to test the efficacy of late corticosteroids and other approaches to modulation of inflammation in ALI/ARDS.

Modulation of alpha-ENaC and alpha1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells.

cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and beta1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of alpha-ENaC and alpha1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated beta- and gamma-ENaC mRNA but not beta1-Na+-K+-ATPase mRNA. Dexamethasone augmented alpha-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated beta- and gamma-ENaC mRNA. There was a 1.6-fold increase at 8 h of beta1-Na+-K+-ATPase mRNA but no significant modulation of alpha1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of alpha-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse alpha-ENaC gene to study the impact of DBcAMP and dexamethasone on alpha-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for alpha-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of alpha-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+ transport in alveolar epithelial cells.

Phloretin inhibits Na+ and K+ uptake in cultured alveolar type II cells by reduction of cellular ATP content.

A number of Na+ and K+ transport pathways have been identified in the alveolar epithelium and multiple inhibitors have been used to uncover these mechanisms. However, the effect of phloretin, a small dipolar nonelectrolyte compound which exerts many effects on membrane transport on Na+ and K+ uptake in alveolar epithelial cells, is not known. The purpose of this study was then to determine the impact of phloretin in Na+ and K+ uptake in cultured rat alveolar type II cells. Phloretin at a dose of 250 microM decreased Na+ uptake by 80% and K+ uptake by 90%. This decrease in Na+ and K+ uptake was not associated with a cytotoxic effect of phloretin, but this treatment did decrease ATP levels in the cells to 80% of the control cells value by 5 minutes and to 95% by 10 minutes. Our study demonstrates that phloretin is a nonspecific inhibitor of ions transport in alveolar type II cells. This inhibition is probably mediated by a reduction of intracellular ATP content.

Pretreatment with cationic lipid-mediated transfer of the Na+K+-ATPase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema.

Resolution of pulmonary oedema is mediated by active absorption of liquid across the alveolar epithelium. A key component of this process is the sodium-potassium ATPase (Na+K+-ATPase) enzyme located on the basolateral surface of epithelial cells and up-regulated during oedema resolution. We hypothesised that lung liquid clearance could be further up-regulated by lipid-mediated transfer and expression of exogenous Na+K+-ATPase cDNA. We demonstrate proof of this principle in a model of high permeability pulmonary oedema induced by intraperitoneal injection of thiourea (2.5 mg/kg) in C57/BL6 mice. Pretreatment of mice (24 h before thiourea) by nasal sniffing of cationic liposome (lipid #67)-DNA complexes encoding the alpha and beta subunits of Na+K+-ATPase (160 microg per mouse), significantly (P<0.01) decreased the wet:dry weight ratios measured 2 h after thiourea injection compared with control animals, pretreated with an equivalent dose of an irrelevant gene. Whole lung Na+K+-ATPase activity was significantly (P<0.05) increased in mice pretreated with Na+K+-ATPase cDNA compared both with untreated control animals as well as animals pretreated with the irrelevant gene. Nested RT-PCR on whole lung homogenates confirmed gene transfer by detection of vector-specific mRNA in three of four mice studied 24 h after gene transfer. This demonstration of a significant reduction in pulmonary oedema following in vivo gene transfer raises the possibility of gene therapy as a novel, localised approach for pulmonary oedema in clinical settings such as ARDS and lung transplantation.

Indomethacin does not influence alveolar liquid clearance in anesthetized sheep or rats.

Active transport of sodium has been shown to be the predominant mechanism involved in alveolar liquid clearance. One regulatory mechanism involved in the modulation of this transport system is cAMP. Although it was initially thought that cAMP could directly modulate transepithelial Na+ transport, recent data suggest that this cAMP modulation could be secondary to the production of arachnidonic acid metabolites. The purpose of this study was thus to evaluate if prostaglandin products could have an indirect or direct role to play in lung liquid clearance. Addition of 10(-5) M salmeterol, known to increase intracellular cAMP, to the instilled fluid in rats stimulated lung liquid clearance. However, addition of indomethacin did not influence the stimulating effect of salmeterol. Furthermore, addition of prostaglandin E2 to the instilled fluid did not stimulate alveolar fluid clearance. In order to determine if this response could be species related, we evaluated if indomethacin could modulate alveolar liquid clearance in sheep. Presence of cAMP and aminophylline stimulated lung liquid clearance in sheep, but indomethacin did not inhibit this response. The present study demonstrates that cyclooxygenase products are not involved in the modulation of basal or stimulated alveolar or lung liquid clearance in sheep or rats.

Downregulation of epithelial sodium channel (ENaC) by CFTR co-expressed in Xenopus oocytes is independent of Cl- conductance.

Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl- into the cell and is negligible when Cl- flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (Iamil) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to -60 mV, stimulating CFTR with 1 mm IBMX reduced Iamil by up to 80%, demonstrating that ENaC is inhibited when Cl- is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of Iamil downregulation and the CFTR-mediated plasma membrane Cl- conductance, suggesting a direct correlation. However, Iamil downregulation was not affected when Cl- flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl- reversal potential (67% +/- 10% inhibition at -20 mV compared to 79% +/- 4% at -60 mV) demonstrating that Iamil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in Iamil downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit Iamil. These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitude of Cl- transport across the plasma membrane.