The epithelial sodium channel (ENaC) is intracellularly located as a tetramer

The epithelial sodium channel (ENaC) plays an important role in Na(+) homeostasis by determining the Na(+) transport rate in so-called end-organs such as the renal collecting duct, distal colon, salivary and sweat gland ducts. ENaC is formed by heteromultimerization of three homologous subunits, termed alpha, beta, and gamma ENaC. The number of subunits and stoichiometry remain a matter of debate. In this study, sucrose gradient analysis of Xenopus laevis oocytes expressing rENaC revealed that ENaC forms heterotetramers, when the membrane fraction was solubilized in 0.1% (wt/vol) Na-deoxycholate. However, solubilization of the membrane proteins in higher concentrations of detergents dissociated the ENaC subunits of the tetramers in dimers. Co-immunoprecipitation studies with FLAG-tagged ENaC subunits suggest that during dissociation of ENaC tetramers the composition of dimers is completely random. Glycosidase digestion studies show that the ENaC subunits are retarded in the endoplasmic reticulum (ER) and pre-Golgi, whereas only a small fraction is inserted into the plasma membrane. Immunocytochemical analysis confirmed that ENaC is primarily located intracellularly. In addition, these findings are not restricted to the oocyte expression system, since identical results were found in rabbit connecting tubule and cortical collecting duct cells in primary culture and in rabbit colon.     

Dimeric architecture of the human bumetanide-sensitive Na-K-Cl Co-transporter

The primary mediator of NaCl reabsorption in the renal distal tubule is the human bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter (hNKCC2), located at the apical membrane of the thick ascending limb of Henle's loop. The physiologic importance of this transporter is emphasized by the tubular disorder Bartter syndrome type I, which arises from the functional impairment of hNKCC2 as a result of mutations in the SLC12A1 gene. The aim of the present study was to investigate the oligomeric state of hNKCC2 to understand further its operational mechanism. To this end, hNKCC2 was heterologously expressed in Xenopus laevis oocytes. Chemical cross-linking with dimethyl-3,3-dithio-bis-propionamidate indicated that hNKCC2 subunits can reversibly form high molecular weight complexes. Co-immunoprecipitation of tagged hNKCC2 subunits further substantiated a physical interaction between individual hNKCC2 subunits. The size of the hNKCC2 multimers was determined by sucrose gradient centrifugation, and a preference for dimeric complexes (approximately 320 kD) was demonstrated. Finally, concatemeric constructs consisting of two wild-type subunits or a wild-type and a functionally impaired hNKCC2 subunit (G319R) were expressed in oocytes. Subsequently, the concatemers were functionally characterized, resulting in a significant bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.2 nmol/oocyte per 30 min for the wild-type-wild-type concatemer, which was reduced to 1.3 +/- 0.1 nmol/oocyte per 30 min for the wild-type-G319R concatemer. In conclusion, this study suggests that hNKCC2 forms at least functional dimers when expressed in Xenopus laevis oocytes of which the individual subunits transport Na(+) independently.     

Functional expression of the human thiazide-sensitive NaCl cotransporter in Madin-Darby canine kidney cells

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which is expressed on the apical membrane of epithelial cells lining the distal convoluted tubule, is responsible for the reabsorption of 5% to 10% of filtered Na(+) and Cl(-). To date, functional studies on the structural and regulatory requirements for localized trafficking and ion-transporting activity of NCC have been hampered by lack of a suitable cell system expressing this cotransporter. Reported here is the functional expression of human NCC (hNCC) in a polarized mammalian cell of renal origin-that is, the high-resistance Madin-Darby canine kidney (MDCK) cell. Western blot testing revealed that the cells predominantly expressed the complex glycosylated (approximately 140 kD) form of hNCC. hNCC was present primarily in the apical part of the cell. The functionality of hNCC was demonstrated by the gain of thiazide-sensitive Na(+) uptake and transepithelial transport activity. Na(+) uptake was significantly increased after short-term (15 min) treatment with forskolin, whereas cyclic guanosine monophosphate, wortmannin, phorbol 12-myriatate 13-acetate, and staurosporine were without effect. This indicates that hNCC activity is regulated through cyclic adenosine monophosphate, rather than via cyclic guanosine monophosphate, phospho-inositide 3-kinases or protein kinase C. Aldosterone did not alter Na(+) uptake in the short term (15 min) but significantly increased the transport activity in the long term (16 h). The latter effect of aldosterone was due to an effect on the cytomegalovirus promoter/enhancer driving the expression of hNCC. hNCC-MDCK cells are a good model for the study of the regulation of apical trafficking and ion-transporting activity of hNCC.     

Effect of age and testosterone on autumnal neurogenesis in male European starlings (Sturnus vulgaris)

The male European starling (Sturnus vulgaris) is an open-ended learner that increases its repertoire throughout life. In parallel, the volume of high vocal center (HVC) is larger in older birds than in yearlings. We labeled with the thymidine analog 5'-bromodeoxyuridine (BrdU) the cells that are generated during the fall in the brain of adult males that were 2 or more years old and in yearling males that were treated with exogenous testosterone (T) or kept intact before BrdU administration. In all subjects, the singing rate was recorded and BrdU-labeled cells were quantified in HVC, in proliferative areas of the ventricular zone (VZ) and in auditory regions. BrdU-containing cells were observed in all brain regions investigated. They were significantly more numerous in the VZ of the T-treated yearlings than in any other group. In older birds, a reduced number of labeled cells was specifically observed in the VZ close to the anterior commissure. No group difference was detected in auditory processing areas or in HVC. These data show for the first time a positive influence of T on the production of new cells at the VZ level in a male songbird and a decrease of this process with age. Furthermore, in T-treated birds, a correlation was observed between the HVC volume and the number of differentiated (round) BrdU-positive cell numbers in HVC on the one hand and song rate on another hand supporting the notion that singing activity is causally related to the T-induced growth of this song control nucleus.     

Neuropathic pain and primary somatosensory cortex reorganization following spinal cord injury

Complex Regional Pain Syndrome type 1 (CRPS1) is a complication after trauma or surgery. Its pathophysiology is still a matter of debate, and psychological factors have been suggested to play a role, although their influence is unclear. The aim of this study was to investigate the evidence for the influence of psychological factors on the onset and maintenance of CRPS1 in adults. In a systematic review, articles were selected using Cochrane, Pubmed/Medline, Psychinfo, and Cinahl since 1980. Only original articles and empirical studies were included. Based on these selection criteria, 31 articles were identified. Studies were evaluated and weighted using a quality assessment instrument. The few prospective studies do not report a relationship between CRPS1 and depression, anxiety, neuroticism, or anger. The results of the retrospective/cross-sectional studies yield contradictory results regarding psychological problems in patients with CRPS1. A majority show no association, and studies with a higher methodological quality lean to a conclusion of no relationship between psychological factors and CRPS1. The majority of included studies (N = 24; 77%) had only a poor to moderate methodological quality. Although many patients with CRPS1 are stigmatized as being psychologically different, this literature review identified no relationship between CRPS1 and several psychological factors. Only life events seemed to be associated with CRPS1: patients who experienced more life events appeared to have a greater chance of developing CRPS1. More studies with greater methodological quality and more participants should be performed on the association between psychological factors and the development and course of CRPS1.     

Atrial natriuretic peptide infusion and nitric oxide inhalation in patients with acute respiratory distress syndrome

Aim  

To study the effects of infusion of atrial natriuretic peptide (ANP) versus the inhalation of nitric oxide (NO) in patients with an early acute respiratory distress syndrome (ARDS).     

Clinical outcome of immunonutrition in a heterogeneous intensive care population

Objective To study the effect of a high-protein enteral formula enriched with arginine, glutamine, and antioxidants and containing 3 fatty acids and a mixture of fibers, on the clinical outcome of a heterogeneous intensive care (ICU) population.Design and setting A randomized, prospective, double blind, controlled, two-center clinical trial in two intensive care units in The Netherlands.Patients and participants A total of 597 adult ICU patients expected to require enteral tube feeding for more than 2 days were randomized to receive immunonutrition or an isocaloric control formula. Interventions Patients received either the immunonutrition or the control feed.Measurements and results Intention-to-treat and per-protocol analyses showed no statistically significant difference in clinical outcome parameters between the two groups. Results of the intention-to-treat analysis in control vs. immunonutrition were: median ICU length of stay in days, 8.0 (IQR 5.0–16.0) vs. 7.0 (4.0–14.0); median hospital length of stay in days, 20.0 (IQR 10.0–34.0) vs. 20.0 (10.0–35.0); median days of ventilation, 6.0 (IQR 3.0–12.0) vs. 6.0 (IQR 3.0–12.0); ICU mortality, 26.8% vs. 28.2%; in-hospital mortality, 36.4% vs. 38.5%; infectious complications, 41.7% vs. 43.0%Conclusions The results of this largest randomized, controlled trial found that in the general ICU population immunonutrition has no beneficial effect on clinical outcome parameters. These results are consistent with the literature that is currently available.This revised version was published online in February 2005 with corrections to the heading of a subsection of the Results (A priori subgroup analyses).