Mucosal morphology in Barrett's esophagus: interobserver agreement and role of narrow band imaging.

BACKGROUND AND STUDY AIMS: We have recently proposed a classification of mucosal morphology in Barrett's esophagus based on three criteria: regularity of mucosal pattern, regularity of vascular pattern, and presence of abnormal blood vessels. We aimed to evaluate the interobserver agreement with the proposed mucosal morphology classification and to assess the additional value of narrow band imaging (NBI) over high resolution white light endoscopy (HR-WLE). PATIENTS AND METHODS: Five international experts in the field of Barrett's imaging and seven community endoscopists with no expertise in this field independently evaluated magnified still images from 50 areas, obtained with HR-WLE and NBI, in Barrett's esophagus patients. Visual analogue scales (VAS) were used for scoring imaging quality. Interobserver agreement for mucosal morphology and yield for identifying early neoplasia were assessed. RESULTS: Imaging qualities of NBI were rated more highly than HR-WLE, when evaluated separately as well as in a side-by-side comparison. The interobserver agreement ranged from 0.40 to 0.56 and did not significantly differ between expert and non-expert endoscopists. The overall yield for correctly identifying images of early neoplasia was 81 % for HR-WLE, 72 % for NBI and 83 % for HR-WLE + NBI, with no significant difference between experts and non-experts. CONCLUSION: Interobserver agreement for the classification of mucosal morphology was moderate. Although NBI was rated more highly than HR-WLE for imaging quality, this did not result in improved interobserver agreement or increased yield for identifying early neoplasia in Barrett's esophagus. This applied to non-expert as well as expert endoscopists.

Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid-inducible kinase isoforms SGK1 and SGK3.

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.

Deficient membrane integration of the novel p.N14D-GJB2 mutant associated with non-syndromic hearing impairment.

Mutations in GJB2, the gene encoding for the Gap Junction protein Connexin 26 (Cx26), have been established as the major cause of hereditary, non-syndromic hearing impairment (HI). We report here the identification of a novel point mutation in GJB2, c.40A>G [p.N14D], detected in compound heterozygosity with the c.35delG mutation in two brothers with moderate non-syndromic sensorineural HI. The mother who carried one wildtype and a p.N14D allele displayed normal hearing. The mutation leads to substitution of the neutral amino acid asparagine (N) by the negatively charged aspartic acid (D) at amino acid number 14, a position that is conserved among Cx26 of different organisms and among many other connexin isoforms. To investigate the impact of this mutation on protein function, Cx26 activity was measured by depolarization activated hemichannel conductance in non-coupled Xenopus laevis oocytes. Oocytes injected with the p.N14D mutant cRNA showed strongly reduced currents compared to wildtype. Coinjection of wildtype and mutant cRNA at equimolar levels restored the conductive properties supporting the recessive character of this mutation. Total Cx26 protein expression and cell surface abundance examined by western blotting and by quantitative immunoassays revealed that the hemichannel was properly synthesized but not integrated into the plasma membrane. In this study we have shown that the GJB2 mutation p.N14D is associated with recessively inherited HI and exhibits a defective phenotype due to diminished expression at the cell surface.

Long-term survival in patients with repair of tetralogy of Fallot: 36-year follow-up of 490 survivors of the first year after surgical repair.

OBJECTIVES: We sought to analyze risk factors for long-term survival (up to 36 years) after surgical repair of tetralogy of Fallot (TOF). BACKGROUND: Survival after repair is excellent, but data >20 years are rare. METHODS: From 1958 to 1977, 658 patients underwent correction of TOF at our institution and were analyzed for survival. Of this patient group (age 12.2 +/- 8.6 years [mean +/- SD], range 2 to 67), 39.7% had a previous palliation. Operative (n = 139) and 1-year (n = 29) deaths were excluded for long-term calculations, resulting in a study group of 490 patients. RESULTS: Actuarial 10-, 20-, 30- and 36-year survival rates were 97%, 94%, 89% and 85%, respectively. Mortality increased 25 years postoperatively from 0.24%/year to 0.94%/year (p = 0.003). The most common cause of death was sudden death (n = 13), followed by congestive heart failure (n = 6). Multivariate correlates of impaired long-term survival were date of operation (before 1970, p = 0.0104), preoperative polycythemia (p = 0.0487) and use of a right ventricular (RV) outflow patch (p = 0.0079). Postoperative systolic RV/left ventricular pressure ratio and age showed no influence. Patients without preoperative polycythemia and an RV outflow patch (n = 164) had a 36-year actuarial survival rate of 96% and normal life expectancy. CONCLUSIONS: Cyanosis, operative experience of the surgeon and an RV outflow tract patch influence long-term outcome after repair of TOF in older children. Early repair by experienced surgeons to avoid polycythemia and excessive RV hypertrophy is supported by this study. However, mortality risk increases 25 years postoperatively, and thus heart monitoring should be intensified.

The epithelial Na(+) channel (ENaC) is related to the hypertonicity-induced Na(+) conductance in rat hepatocytes.

The epithelial Na(+) channel (ENaC) is composed of the subunits alpha, beta, and gamma [Canessa et al., Nature 367 (1994) 463-467] and typically exhibits a high affinity to amiloride [Canessa et al., Nature 361 (1993) 467-470]. When expressed in Xenopus oocytes, conflicting results were reported concerning the osmo-sensitivity of the channel [Ji et al., Am. J. Physiol. 275 (1998) C1182-C1190; Hawayda and Subramanyam, J. Gen. Physiol. 112 (1998) 97-111; Rossier, J. Gen. Physiol. 112 (1998) 95-96]. Rat hepatocytes were the first system in which amiloride-sensitive sodium currents in response to hypertonic stress were reported [Wehner et al., J. Gen. Physiol. 105 (1995) 507-535; Wehner et al., Physiologist 40 (1997) A-4]. Moreover, all three ENaC subunits are expressed in these cells [Böhmer et al., Cell. Physiol. Biochem. 10 (2000) 187-194]. Here, we injected specific antisense oligonucleotides directed against alpha-rENaC into single rat hepatocytes in confluent primary culture and found an inhibition of hypertonicity-induced Na(+) currents by 70%. This is the first direct evidence for a role of the ENaC in cell volume regulation.

Omeprazole: therapy of choice in intellectually disabled children.

OBJECTIVE: To study extensively the therapeutic approach of gastroesophageal reflux disease in intellectually disabled children. DESIGN: We studied the effect of omeprazole sodium on healing and symptom relief in 52 institutionalized intellectually disabled children (male-female, 21:31; mean age, 15.4 years; range, 4-19 years). INTERVENTION: Endoscopically proven esophagitis (grades I-IV, Savary-Miller classification) was treated with omeprazole sodium, 40 mg/d (20 mg/d for children weighing <20 kg) as healing dose for 3 months, and 20 mg/d (10 mg/d for children weighing <20 kg) as maintenance dose for another 3 months. After 3 and 6 months, results of treatment were evaluated using symptom scoring and/or endoscopy. For patients with relapse, the dose was increased. RESULTS: At first endoscopy, 19 patients (36%) of 52 showed grade I esophagitis; 20 (38%), grade II; 6 (12%), grade III; and 7 (13%), grade IV. In 44 (86%) of 51 patients, treatment was effective in healing esophagitis and keeping patients in remission, independent of the severity of esophagitis. In 7 patients (14%), a symptomatic relapse was observed after decreasing the dose. However, these patients became symptom free again after increasing the dose and showed healing on endoscopy at the end of the study. One child did not finish the study for reasons not related to therapy. Marked improvement of persistent vomiting, regurgitation, food refusal, iron deficiency anemia, and signs of depression was seen at the end. CONCLUSIONS: Omeprazole is highly effective for all grades of esophagitis in intellectually disabled children, without adverse effects. The dose needed to maintain the remission can be titrated according to the reflux symptoms. One disadvantage of medical therapy is that it is open ended, in contrast to operation, but surgery in this population has high mortality and complication rates.

CA 125 measurement in the follow-up of breast cancer patients.

During follow-up examinations for breast cancer the serum tumor marker CA 125 was measured routinely in the Universitätsfrauenklinik Erlangen. We found elevated CA 125 levels in 18 of 510 clinical asymptomatic patients. These patients were followed closely. A second malignant neoplasm was found in six of these patients, two at a curable early stage. We found metastases of breast cancer in nine patients and benign diseases in three patients.

The clinical relevance of lactose malabsorption in irritable bowel syndrome.

OBJECTIVE: The prevalence of lactose malabsorption (LM) in the Caucasian population of northern Europe is estimated to be low. Irritable bowel syndrome (IBS) is a very common diagnosis, and its symptoms are nearly identical to those of LM. Therefore we investigated the prevalence of LM among IBS patients in comparison with healthy volunteers. DESIGN: A double-blind clinical trial compared with healthy controls. SETTING: One out-patient gastroenterology clinic in the Netherlands. PATIENTS: 70 Caucasian IBS patients and 35 healthy volunteers (staff members). METHODS: All 105 underwent hydrogen (H2) breath and blood glucose tests, after an oral intake of 50 grams of lactose. The IBS patients were treated with a lactose-restricted diet for 6 weeks. They completed a lactose intake score before, and a symptom score scored by six separate criteria, before, during and after treatment. RESULTS: In 17 out of 70 (24.3%) IBS patients LM was detected, in comparison with 2 out of 35 (5.7%) controls (P < 0.009). There was no difference in the pre-entry mean lactose intake and symptom score between the LM positive and negative IBS patients. The mean symptom score of the LM positive group showed a marked decrease after 6 weeks of dietary therapy (P < 0.001). CONCLUSION: A substantial number of IBS patients showed a clinically unrecognized lactose malabsorption, which could not be discriminated by symptoms and dietary history, and which can be treated with a lactose-restricted diet. Therefore LM has to be excluded before the diagnosis IBS is made.

The effect of a lactose-restricted diet in patients with a positive lactose tolerance test, earlier diagnosed as irritable bowel syndrome: a 5-year follow-up study.

DESIGN AND METHODS: Prospectively, the effect of a lactose-restricted diet was evaluated among irritable bowel syndrome patients with lactose malabsorption. Lactose malabsorption was defined by a positive hydrogen breath test and a positive blood-glucose test. An analysis of symptoms was completed before, during, 6 weeks after and 5 years after starting the diet. In addition, the number of visits made by the patients to the outpatient clinic was scored during 6 years. RESULTS: In 17 out of 70 irritable bowel syndrome patients (24.3%), lactose malabsorption was detected. There was no difference in the symptom score between patients with a positive lactose tolerance test and patients with a negative lactose tolerance test. After 6 weeks of the lactose-restricted diet, symptoms were markedly improved in lactose malabsorption-positive patients (P < 0.001). After 5 years, one patient was lost for follow-up, and 14 out of the remaining 16 lactose malabsorption patients (87.5%) still had no complaints during the lactose-restricted diet. Two patients chose not to follow the diet continuously and accepted the discomfort caused by lactose intake. Only two out of 16 patients (12.5%) no longer experienced any benefit from lactose restriction. In the 5 years before their diagnosis of lactose malabsorption, these 16 patients visited the outpatient clinic a total of 192 times (mean 2.4 visits per year per person; range 1-7 visits). In the 5 years after diagnosis, they visited the outpatient clinic a total of 45 times (mean 0.6 visits per year per person; range 0-6 visits; P < 0.0001). CONCLUSIONS: In a large majority of irritable bowel syndrome patients with lactose malabsorption, which was previously clinically unrecognized, a lactose-restricted diet improved symptoms markedly both in the short term and the long term. Furthermore, visits by all patients to the outpatient clinic were reduced by 75%. We conclude that diet therapy is extremely cost- and time-saving. Therefore, it is strongly recommended that lactose malabsorption, which is easily treatable, is excluded before diagnosing irritable bowel syndrome.

The age-related incidences of oesophageal carcinoma in intellectually disabled individuals in institutes in The Netherlands.

OBJECTIVE: An increased age-related incidence of oesophageal cancer in people with intellectual disability has been suggested by studies in the Netherlands. Gastro-oesophageal reflux disease (GORD), as documented by pH testing, occurs frequently in the intellectually disabled population, being found in nearly 50% of those with an IQ less than 50, while Barrett's oesophagus is found in about 15-26%. DESIGN: We compared the age-related incidence of oesophageal cancer in institutionalized, intellectually disabled individuals in the Netherlands with the age-related incidence in the general Dutch population. METHODS: Data were provided by the Netherlands Cancer Registry. The patient's institute physician was asked to complete a questionnaire about the diagnosis, which was endoscopically and histologically confirmed. RESULTS: The incidence of oesophageal carcinoma was 20 in 168,000 person-years. The expected incidence for oesophageal cancer, based on age-related incidence in the general population, was 7.0, resulting in a standardized morbidity ratio in the population with intellectual disability of 2.9 (confidence limits, 1.8-4.1; P < 0.001). Endoscopic findings were as follows: in 18/20 intellectually disabled carcinoma patients an adenocarcinoma was found; the remaining two patients had a squamous cell carcinoma. Barrett's epithelium was observed in nine patients (45%), eight (42%) of whom showed a peptic stricture as well. In 15 (75%) cancer patients reflux oesophagitis was found, accompanied in 14 cases by a hiatal hernia. CONCLUSION: A standardized morbidity ratio for oesophageal carcinoma of 2.9 was found in the intellectually disabled population as compared to the general population. Early detection and treatment of GORD in the population with intellectual disability is of paramount importance to prevent the development of Barrett's dysplasia and carcinoma.

The prevalence of gastro-oesophageal reflux disease based on non-specific symptoms in institutionalized, intellectually disabled individuals.

OBJECTIVE: The prevalence of gastro-oesophageal reflux disease (GORD) in randomly selected, institutionalized, intellectually disabled individuals (IQ < 50) in The Netherlands is high. DESIGN: In this study we investigated the prevalence of GORD and reflux oesophagitis (RO) in intellectually disabled individuals specifically selected for symptoms such as: behavioural difficulties such as automutilation, food refusal, fear and restlessness, vomiting, regurgitation and rumination. Predisposing factors were also evaluated. METHODS: One hundred and ten individuals from one institute underwent a 24-h oesophageal pH test and were scored for predisposing factors and non-specific reflux symptoms. A pathological pH test was defined as a pH lower than 4 more than 4.5% of the measured time. Subjects with a pathological pH test (patients) were compared to those with a normal pH test (controls). RESULTS: In seven cases (6.4%) the test failed for technical reasons. In 57 (55.3%) cases a pathological pH test was found. RO was diagnosed in 33 (64.7%) patients. The use of anticonvulsant drugs and cerebral palsy appeared to be predisposing factors, while the non-specific reflux symptoms did not discriminate for GORD. CONCLUSION: The prevalence of GORD was equal in a randomly selected group of intellectually disabled individuals, and in this group, specifically selected on account of possible reflux symptoms. This study confirms that in this particular group, it is nearly impossible to discriminate for the diagnosis of GORD on non-specific reflux symptoms only. However, GORD may be suspected where there are certain predisposing factors, particularly cerebral palsy and use of anticonvulsant drugs.)

Gastro-oesophageal reflux disease in institutionalised intellectually disabled individuals.

BACKGROUND: The prevalence of reflux oesophagitis (RO) in the normally intellectual population is about 2%, while this condition in the intellectually disabled has an estimated prevalence of 10%. METHODS: We investigated the presence of RO among 1687 intellectually disabled, with an IQ < 50, from 5 different institutes in the Netherlands. All were scored for possible associated factors and reflux symptoms, and compared with the overall population (n = 1580) from the same institutes (controls). Also, the effect of treatment on symptoms was evaluated after at least one year of therapy. RESULTS: Gastro-oesophageal reflux disease (GORD) was suspected clinically in 169 patients based on the following symptoms: vomiting, haematemesis, anaemia, rumination or behaviour problems. At endoscopy RO was diagnosed in 107 of 1687 patients (6.4%): 17 (15.9%) grade I, 34 (31.8%) gr. II, 42 (39.3%) gr. III and 14 (13.1%) gr. IV RO (Savary-Miller classification). Cerebral palsy, constipation, anticonvulsant drugs, an IQ < 35, underweight and gastrostomy feeding appeared to be possible associated factors, while as reflux symptoms persistent vomiting, haematemesis, iron deficiency anaemia, rumination, and behaviour problems were found. Concerning therapy, surgery was found to be effective in 38%, H2 receptor antagonists in 60% and the proton-pump inhibitor omeprazole in 96%. CONCLUSIONS: In this group of Dutch intellectually disabled patients with IQ < 50 RO was diagnosed in about 6% (107 of 1687), mostly severe grades of oesophagitis. Several possible associated factors were significantly present. From non-specific reflux symptoms persistent vomiting was the most indicative factor. In this population the most effective treatment of RO was long-term omeprazole therapy.

Effect of a hybrid liver support system on cardiopulmonary function in healthy pigs.

The aim of this study was to evaluate the effects of a hybrid liver support system (LSS) on cardiopulmonary function in nine healthy pigs. A hybrid LSS containing primary pig hepatocytes was connected to fully alert animals. The extracorporeal blood flow was maintained between 200-240 ml/min using a roller pump. Continuous plasma flow through the hybrid LSS was 50-60 ml/min. Hemodynamic and pulmonary gas exchange parameters were compared 1 hour before and 1 hour after connection to as well as 1 hour before and 1 hour after disconnection from the hybrid LSS. The hybrid LSS did not influence significantly hemodynamics and pulmonary gas exchange in this group of healthy and awake pigs. It can be concluded that the used LSS did not cause a cardiopulmonary effect per se and should be evaluated further concerning its function as a liver support system in an animal model of acute hepatic failure.

Experimental evaluation of a cell module for hybrid liver support.

Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy. Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery. Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells. The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density. Treatment started after complete recovery from anesthesia and was performed continuously. A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group. Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.

[Gastroesophageal reflux disease in mentally retarded persons: prevalence, diagnosis and treatment]. / Gastro-oesofageale refluxziekte bij verstandelijk gehandicapten: prevalentie, diagnostiek en behandeling.

The prevalence of gastroesophageal reflux disease among institutionalised intellectually disabled individuals with an IQ < 50 is high: about 50% have an deviant 24-hour pH measurement and 70% of them have refluxoesophagitis. Intellectually disabled individuals have an increased risk of gastroesophageal reflux disease in case of cerebral palsy, IQ < 35, scoliosis, use of anticonvulsant drugs or benzodiazepines, not being ambulant, and in case of symptoms such as haematemesis, rumination or dental erosions. To establish the diagnosis is difficult because of the aspecific symptoms. Reflux disease is only diagnosed at a late stage. 24-hour pH measurement should be used in all those intellectually disabled individuals in whom gastroesophageal reflux disease is clinically suspected. For the treatment of gastro-oesophageal reflux disease in adults as well as children, proton pump inhibitors are highly effective, independent of the severity of oesophagitis. Marked improvement of symptoms and quality of life can be noticed after treatment.

Loss of function mutations of the GJB2 gene detected in patients with DFNB1-associated hearing impairment.

Mutations in GJB2, which encodes the gap junction protein connexin 26 (Cx26), are one of the major causes for inherited and sporadic nonsyndromic hearing impairment. This study aimed to functionally characterize more frequent GJB2 mutations identified in patients showing nonsyndromic hearing impairment. Following injection of wild type and mutated cRNA in Xenopus oocytes, Cx26 hemichannel activity was measured by depolarization activated conductance in noncoupled oocytes. All mutants showed a partially or completely defective phenotype, except (V27I)Cx26, a polymorphism tested as positive control. Coexpression of wild type and mutant Cx26 injected at equimolar levels revealed that p.M34T, p.V37I and p.I82M, but not p.G59V, p.L90P, p.R127H and p.R143W exert a dominant inhibitory effect. When coexpressed with Cx30, a connexin partially colocalized with Cx26 in the cochlea, all mutants had a dominant behavior. This study provides data that might be important for the improvement of genetic diagnosis and counseling for patients with hearing impairment.

Transport regulation by the serum- and glucocorticoid-inducible kinase SGK1.

The serum- and glucocorticoid-inducible kinase SGK1 is an ubiquitously expressed kinase with the ability to regulate a variety of transport systems. Recent observations point to a role of SGK1 in the regulation of diverse physiological functions such as epithelial transport and cardiac and neuronal excitability. At least partially through its effect on transport, SGK1 contributes to a number of pathophysiological conditions including metabolic syndrome and fibrosing disease.

18-beta-Glycyrrhetinic acid (BGA) as an electrical uncoupler for intracellular recordings in confluent monolayer cultures.

In the study of epithelial cell biology, primary cell cultures or cell lines grown to confluency offer considerable advantages compared with isolated cells and cell clusters. This is due mainly to the development of appropriate cell-to-cell contacts that are a prerequisite for cell polarity and thus vectorial solute transport. On the other hand, electrical coupling via gap junctions in most instances significantly hinders the use of voltage-clamp techniques for electrophysiological analysis of transport processes in single cells. In the present study we employed the gap junctional blocker 18-beta-glycyrrhetinic acid (BGA) to reduce electrical cell-to-cell coupling in confluent primary cultures of rat hepatocytes. In current-clamp experiments, 40 micromol/l BGA reversibly increased apparent cell input resistance approximately tenfold. Due to this partial electrical isolation of cells, two-channel voltage-clamp experiments became feasible and, for the first time, the hypertonicity-induced Na+ conductance of rat hepatocytes could be analysed quantitatively. In ion substitution experiments, however, it became obvious that BGA, while leaving Na+ and K+ conductances virtually unchanged, completely blocked cell membrane Cl- conductance. This additional effect of BGA necessitates independent control experiments to ensure that the transport process under consideration is itself not changed by the compound. Nevertheless, BGA may serve as a powerful tool for the quantitative electrophysiological study of epithelial cells that are in quasi physiological contact with their neighbours.

The hypertonicity-induced Na(+) conductance of rat hepatocytes: physiological significance and molecular correlate.

The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an uptake of extracellular Na(+) that is then exchanged for K(+) via stimulation of Na(+)/K(+)-ATPase. While it was generally assumed that this Na(+) uptake is mediated by the activation of Na(+)/H(+) antiport and Na(+)-K(+)-2Cl(-) symport it could be shown recently that, in addition to these transporters, hypertonic stress also stimulates conductive Na(+) entry. In a quantitative study, it was found that the relative contribution of Na(+) conductance, Na(+)/H(+) antiport, and Na(+)-K(+)-2Cl(-) symport to the initial Na(+) import as well as to the RVI process (at 300 --> 400 mosmol/l) is approximately 4 : 1 : 1. When the osmotic sensitivity of these Na(+) importers was tested (at 300 mosmol/l --> 327, 360, 400, 450 mosmol/l) it became clear that Na(+) conductance is the prominent mechanism of RVI from 360 mosmol/l upwards whereas Na(+)/H(+) antiport is the most sensitive transporter with 65 % of its maximal activation at 327 mosmol/l already. Concerning the intracellular regulation of the Na(+) importers involved in RVI it was found that Na(+) concuctance as well as Na(+)-K(+)-2Cl(-) symport - but not Na(+)/H(+) antiport - are activated via PKC. With respect to the molecular correlate of the volume activated Na(+) conductance it could be shown that it exhibits a rather low affinity to amiloride (IC(50) = 6.0 micromol/l) and an overall sensitivity profile of EIPA > amiloride > benzamil = phenamil that, at first sight, would not speak in favor of a typical epithelial type of Na(+) channel (ENaC). Western-blot analysis and RT-PCR techniques, however, revealed that alpha-, beta-, as well as gamma-ENaC are, in fact, expressed in rat hepatocytes. Moreover, by use of an antisense-DNA based approach it could be shown that at least alpha-ENaC is part of the hypertonicity induced Na(+) conductance.

Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1.

EAAC1-mediated glutamate transport concentrates glutamate across plasma membranes of brain neurons and epithelia. In brain, EAAC1 provides a presynaptic uptake mechanism to terminate the excitatory action of released glutamate and to keep its extracellular concentration below toxic levels. Here we report the effect of well known anxiolytic compounds, benzodiazepines, on glutamate transport in EAAC1-stably transfected Chinese hamster ovary (CHO) cells and in EAAC1-expressing Xenopus laevis oocytes. Functional properties of EAAC1 agreed well with already reported characteristics of the neuronal high-affinity glutamate transporter (Km D-Asp,CHO cells: 2.23+/-0.15 microM; Km D-Asp,oocytes: 17.01+/-3.42 microM). In both expression systems, low drug concentrations (10-100 microM) activated substrate uptake (up to 200% of control), whereas concentrations in the millimolar range inhibited (up to 50%). Furthermore, the activation was more pronounced at low substrate concentrations (1 microM), and the inhibition was attenuated. The activity of other sodium cotransporters such as the sodium/D-glucose cotransporter SGLT1, stably transfected in CHO cells, was not affected by benzodiazepines. In electrophysiological studies, these drugs also failed to change the membrane potential of EAAC1-expressing Xenopus laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.