Luminal heterodimeric amino acid transporter defective in cystinuria

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.     

Trafficking of GFP-tagged Delta F508-CFTR to the plasma membrane in a polarized epithelial cell line

The F508 mutationreduces the amount of cystic fibrosis transmembrane conductanceregulator (CFTR) expressed in the plasma membrane of epithelial cells.However, a reduced temperature, butyrate compounds, and "chemicalchaperones" allow F508-CFTR to traffic to the plasma membrane andincrease Cl permeability in heterologous and nonpolarizedcells. Because trafficking is affected by the polarized state ofepithelial cells and is cell-type dependent, our goal was to determinewhether these maneuvers induce F508-CFTR trafficking to the apicalplasma membrane in polarized epithelial cells. To this end, wegenerated and characterized a line of polarized Madin-Darby caninekidney (MDCK) cells stably expressing F508-CFTR tagged with greenfluorescent protein (GFP). A reduced temperature, glycerol, butyrate,or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP(CPT-cAMP)-stimulated transepithelial Cl secretion acrosspolarized monolayers. However, when the basolateral membrane waspermeabilized, butyrate, but not the other experimental maneuvers,increased the CPT-cAMP-stimulated Cl current across theapical plasma membrane. Thus butyrate increased the amount offunctional F508-CFTR in the apical plasma membrane. Butyrate failedto stimulate transepithelial Cl secretion because ofinhibitory effects on Cl uptake across the basolateralmembrane. These observations suggest that studies on heterologous andnonpolarized cells should be interpreted cautiously. The GFP tag onF508-CFTR will allow investigation of F508-CFTR trafficking inliving, polarized MDCK epithelial cells in real time.

     

Ecto-5′-nucleotidase is expressed by pericytes and fibroblasts in the rat heart

Ecto-5-nucleotidase is anchored at the outer surface of cell membranes and thus its reaction product adenosine is released into the extracellular space. Extracellular adenosine displays via specific receptors a wide range of physiological effects in heart. There are discrepancies in the literature concerning the distribution of ecto-5-nucleotidase in heart. Since we suspected that these may be due to technical problems, in the present study on ecto-5-nucleotidase in rat heart we attempted to circumvent some technical pitfalls. Good preservation of the tissue with open capillary lumina, providing a clear identification of endothelium, was obtained by perfusion fixation. At the light microscopic level, the distribution of ecto-5-nucleotidase studied by enzyme histochemistry and immunohistochemistry using a monoclonal and a polyclonal antibody yielded congruent results. The enzyme was rather homogeneously distributed throughout the myocardium, with a slightly higher incidence of stained cells in the outer thirds than in the inner third of the wall. Consistently high levels of ecto-5-nucleotidase were seen only in interstitial cells. The walls of large vessels and heart muscle cells were constantly negative for ecto-5-nucleotidase. The endothelia of capillaries were mostly negative but a few profiles occasionally displayed a weak immunoreaction. The interstitial cells staining positive for ecto-5-nucleotidase could be identified as pericytes and as fibroblasts according to their shapes and localizations. The immunoreactivity of fibroblasts was confirmed by electron microscopy. These data indicate that adenosine may be formed extracellularly in the interstitium of the myocardium, where it would have direct access to important targets such as myocytes, arterioles and nerve endings.     

The NH(2) terminus of the epithelial sodium channel contains an endocytic motif.

An epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. To elucidate the function of the cytoplasmic, NH(2) terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na(+) currents (I(Na)) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH(2) terminus of alpha (Delta2-109), beta (Delta2-49), or gamma-rENaC (Delta2-53) dramatically reduced I(Na). A series of progressive, NH(2)-terminal deletions of alpha-rENaC were constructed to identify motifs that regulate I(Na). Deletion of amino acids 2-46 had no effect on I(Na): however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I(Na) by approximately 4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I(Na). To evaluate the mechanism whereby Delta2-67-alpha-rENaC increased I(Na), single channels were evaluated by patch clamp. The single-channel conductance and open probability of alpha,beta,gamma-rENaC and Delta2-67-alpha,beta,gamma-rENaC were similar. However, the number of active channels in the membrane increased from 6 +/- 1 channels per patch with alpha,beta,gamma-rENaC to 11 +/- 1 channels per patch with Delta2-67-alpha,beta,gamma-rENaC. Laser scanning confocal microscopy confirmed that there were more Delta2-67-alpha,beta, gamma-rENaC channels in the plasma membrane than alpha,beta, gamma-rENaC channels. Deletion of amino acids 2-67 in alpha-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 +/- 0.2 to 3.5 +/- 1.1 h. We conclude that the cytoplasmic, NH(2) terminus of alpha-, beta-, and gamma-rENaC is required for channel activity. The cytoplasmic, NH(2) terminus of alpha-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.     

Left Preference for Sport Tasks Does Not Necessarily Indicate Left-Handedness: Sport-Specific Lateral Preferences,Relationship with Handedness and Implications for Laterality Research in Behavioural Sciences

In the elite domain of interactive sports, athletes who demonstrate a left preference (e.g., holding a weapon with the left hand in fencing or boxing in a ‘southpaw’ stance) seem overrepresented. Such excess indicates a performance advantage and was also interpreted as evidence in favour of frequency-dependent selection mechanisms to explain the maintenance of left-handedness in humans. To test for an overrepresentation, the incidence of athletes'' lateral preferences is typically compared with an expected ratio of left- to right-handedness in the normal population. However, the normal population reference values did not always relate to the sport-specific tasks of interest, which may limit the validity of reports of an excess of ‘left-oriented’ athletes. Here we sought to determine lateral preferences for various sport-specific tasks (e.g., baseball batting, boxing) in the normal population and to examine the relationship between these preferences and handedness. To this end, we asked 903 participants to indicate their lateral preferences for sport-specific and common tasks using a paper-based questionnaire. Lateral preferences varied considerably across the different sport tasks and we found high variation in the relationship between those preferences and handedness. In contrast to unimanual tasks (e.g., fencing or throwing), for bimanually controlled actions such as baseball batting, shooting in ice hockey or boxing the incidence of left preferences was considerably higher than expected from the proportion of left-handedness in the normal population and the relationship with handedness was relatively low. We conclude that (i) task-specific reference values are mandatory for reliably testing for an excess of athletes with a left preference, (ii) the term ‘handedness’ should be more cautiously used within the context of sport-related laterality research and (iii) observation of lateral preferences in sports may be of limited suitability for the verification of evolutionary theories of handedness.     

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells

Cystic fibrosis iscaused by mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) Clchannel, which mediates transepithelialCl transport in a varietyof epithelia, including airway, intestine, pancreas, and sweat duct. Insome but not all epithelial cells, cAMP stimulatesCl secretion in part byincreasing the number of CFTRCl channels in the apicalplasma membrane. Because the mechanism whereby cAMP stimulates CFTRCl secretion is cell-typespecific, our goal was to determine whether cAMP elevates CFTR-mediatedCl secretion across serousairway epithelial cells by stimulating the insertion of CFTRCl channels from anintracellular pool into the apical plasma membrane. To this end westudied Calu-3 cells, a human airway cell line with a serous cellphenotype. Serous cells in human airways, such as Calu-3 cells, expresshigh levels of CFTR, secrete antibiotic-rich fluid, and play a criticalrole in airway function. Moreover, dysregulation of CFTR-mediatedCl secretion in serouscells is thought to contribute to the pathophysiology of cysticfibrosis lung disease. We report that cAMP activation of CFTR-mediatedCl secretion across humanserous cells involves stimulation of CFTR channels present in theapical plasma membrane and does not involve the recruitment of CFTRfrom an intracellular pool to the apical plasma membrane.

     

Role of Sgk1 in salt and potassium homeostasis

Aldosterone plays a pivotal role in NaCl and K(+) homeostasis by stimulation of Na(+) reabsorption and K(+) secretion in the aldosterone-sensitive distal nephron (ASDN). Recent studies demonstrated that the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by aldosterone in the ASDN and that polymorphisms of the kinase associate with arterial blood pressure in normotensive subjects. This review discusses the role of Sgk1 in NaCl and K(+) homeostasis as evidenced by in vivo studies, including those in Sgk1-deficient mice. The studies indicate that Sgk1 is not absolutely required for Na(+) reabsorption and K(+) secretion in the ASDN. On a standard NaCl and K(+) diet, modestly enhanced plasma aldosterone concentrations appear sufficient to establish a compensated phenotype in the absence of Sgk1. The kinase is necessary, however, for upregulation of transcellular Na(+) reabsorption in the ASDN. This may involve Sgk1-mediated stimulation of basolateral Na(+)-K(+)-ATPase as well as retention of epithelial Na(+) channel, ENaC, in the apical membrane. Such an upregulation is a prerequisite for adequate adaptation of 1) renal NaCl reabsorption during restricted dietary NaCl intake, as well as 2) K(+) secretion in response to enhanced K(+) intake. Thus gain-of-function mutations of Sgk1 are expected to result in renal NaCl retention and enhanced K(+) secretion. Further studies are required to elucidate renal and nonrenal aldosterone-induced effects of Sgk1, the role of other Sgk1 activators, as well as the link of Sgk1 polymorphisms to arterial hypertension in humans.     

Distribution of ecto-5′-nucleotidase in the rat liver: effect of anaemia

In the kidney a striking parallel exists between the expression of ecto-5-nucleotidase and of erythropoietin by renal fibroblasts. It was therefore hypothesized that the expression of ecto-5-nucleotidase in fibroblasts might be controlled by oxygen tension. In order to test this hypothesis, we examined the distribution of the enzyme in a tissue which displays a defined zonation in respect to oxygen tension, namely in the liver; anaemia was used in order to exaggerate this zonation. The distribution of ecto-5-nucleotidase was investigated by light and electron microscopy using enzyme and immunohistochemical methods in the livers of healthy and of anaemic rats. Anaemia was produced by haemolysis combined with X-ray irradiation. The enzyme was detected in the bile canaliculi, in the connective tissue of the portal triads and of the central veins, and in fat-storing cells probably corresponding to a special form of fibroblasts. In healthy animals the perisinusoidal ecto-5-nucleotidase activity was slightly higher in the pericentral than in the periportal area of the acinus whereas the inverse was observed for the staining of bile canaliculi. Anaemia provoked an increase of ecto-5-nucleotidase in fat-storing cells in the pericentral zone of the acinus and in fibroblasts around the central veins, resulting in steepended gradients along the sinusoids. The intralobular gradient of ecto-5-nucleotidase in perisinusoidal cells and the effect thereon of anaemia suggest that the expression of the ecto-5-nucleotidase might be directly or indirectly controlled by local oxygen tension.