No abstract

Both arginine vasopressin (AVP) and forskolin regulate vectorial Na+ transport across high-resistance epithelia by increasing the Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. Pretreatment of A6 cells with brefeldin A partially inhibited the increase in Na+ transport in response to forskolin, suggesting recruitment of Na+ channels from an intracellular pool. The activation of Cl- secretion was not affected. Apical cell surface expression of Na+ channels was examined following activation of transepithelial Na+ transport across the epithelial cell line A6 by AVP or forskolin. Apical cell surface radioiodinated Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical plasma membrane and to determine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a potential mechanism by which AVP and forskolin increase apical membrane Na+ conductance. The activation of Na+ transport across A6 cells by AVP was accompanied by a significant increase in the biochemical pool of Na+ channels at the apical plasma membrane within 5 min after addition of hormone, which was sustained for at least 30 min. The increase in apical cell surface expression of Na+ channels was also observed 30 min after application of forskolin. No changes in the oligomeric subunit composition of the channel were noted. Brefeldin A inhibited the forskolin-stimulated increase in apical cell surface expression of Na+ channels. These results suggest that AVP and forskolin regulate Na+ transport, in part, via rapid recruitment of Na+ channels to the cell surface, perhaps from a pool of channels in the subapical cytoplasm.

The neurohypophysial peptide arginine vasopressin (AVP) increases Na+ absorption across A6 epithelia. In addition to the positive natriferic response, AVP increases net basolateral to apical Cl- flux. The time course of activation of electrogenic ion transport in A6 epithelia was examined by measuring transepithelial short-circuit current (ISC). Basolateral application of AVP (0.1 U/ml) or forskolin (10 microM) affects ISC in a biphasic manner. Shortly after addition of AVP, an early (transient) phase is observed in which ISC is rapidly stimulated, reaching a peak value at 1.4 +/- 0.1 min. A subsequent decrease in current is interrupted by a slower, late phase in which ISC reaches a peak 23 +/- 3 min after addition of AVP. The late increase in ISC is sustained over the remainder of the 40-min period of observation. The time course of ISC stimulation by forskolin is qualitatively similar. Replacement of external Cl- by aspartate lowers baseline transport nearly 40%, strongly blunts the early phase of ISC stimulation, and retains the late increase. Addition of amiloride (10 microM) to the apical bath before AVP or forskolin stimulation of ISC eliminates the late increase of ISC. Steady-state amiloride-insensitive ISC activated under these conditions was sensitive to apical application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (20 microM) and niflumic acid (100 microM). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (1 mM) was not an effective inhibitor of this current. Basolateral bumetanide (100 microM) inhibited baseline ISC and reduced both the peak transient and steady-state amiloride-insensitive ISC.(ABSTRACT TRUNCATED AT 250 WORDS)

We have identified monoclonal anti-DNA antibodies derived from lupus prone MRL-lpr/lpr mice that produce glomerular immune deposits and nephritis after passive transfer to normal mice. Particularly noteworthy is that the location of immune deposition varied among nephritogenic Ig, and this was associated with distinctive histologies and clinical disease profiles. Although their autoantigen binding properties differed, they were highly cross-reactive, in a manner similar to Ig deposited in glomeruli of lupus mice. This antigen binding profile was also typical of other previously described nephritogenic autoantibodies that bound directly to glomerular antigens to initiate immune deposit formation. In this study, we questioned whether ligation of different glomerular antigens by individual autoantibodies could contribute to the observed differences in the location of immune deposits. To examine this possibility, monoclonal anti-DNA antibodies (IgG2a) that produced glomerular immune deposits in different locations were evaluated. H221 produced mesangial, intracapillary (that is, intraluminal or within the capillary lumen) and subendothelial deposits associated with heavy proteinuria, whereas H147 produced mesangial, subendothelial and linear basement membrane deposits associated with proliferative glomerulonephritis. Initially, the capacity of H221 and H147 to bind directly to glomerular and vascular cell surfaces was evaluated. As demonstrated by FACS, H221 bound preferentially to mesangial cells whereas H147 bound preferentially to endothelial cells. To identify possible target cell surface antigens, Western blots, immunoprecipitation of surface labeled cells, and 2D gel electrophoresis were employed. H221 reacted with a 108 kDa protein on mesangial cells not identified by H147, whereas H147 reacted with a 45 kDa protein on endothelial cells not identified by H221. These results support the hypothesis that some nephritogenic lupus autoantibodies initiate immune deposit formation through direct interaction with glomerular antigens. Furthermore, they suggest that the site of immune deposition is determined by both antigen binding properties of the relevant antibody and the location of its target ligand within the glomerulus. In a given individual, therefore, the predominant autoantibody-glomerular antigen interaction may influence the morphologic and clinical phenotype expressed. Variation in the predominant interaction may also contribute to variations in disease expression among individuals with lupus nephritis.

The Na+/H+ exchanger isoform NHE1 is localized to the basolateral membrane of renal and intestinal epithelia. We examined the plasma membrane distribution, biosynthesis, and cell surface delivery of NHE1 in A6 epithelia. NHE1 was localized to the basolateral membrane. Studies of NHE1 biosynthesis with a pulse-chase protocol demonstrated that a core glycosylated, endoglycosidase H-sensitive, 90-kDa NHE1 was present 0-5 h into the chase period and that mature 110-kDa NHE1 was present 1-24 h into the chase period. Studies of plasma membrane delivery of newly synthesized NHE1 demonstrated that the 90-kDa NHE1 was detected at both apical and basolateral membranes 2-5 h into the chase period. The 110-kDa NHE1 was observed at the basolateral membrane 5-24 h into the chase period. These results suggest that NHE1 is expressed primarily at the basolateral membrane of A6 cells, that core glycosylated NHE1 is delivered to the plasma membrane in a nonpolarized manner, and that nature 110-kDa NHE1 is delivered to the basolateral membrane.