S-adenosyl-L-homocysteine hydrolase is necessary for aldosterone-induced activity of epithelial Na(+) channels.

The A6 cell line was used to study the role of S-adenosyl-L-homocysteine hydrolase (SAHHase) in the aldosterone-induced activation of the epithelial Na(+) channel (ENaC). Because aldosterone increases methylation of several different molecules, and because this methylation is associated with increased Na(+) reabsorption, we tested the hypothesis that aldosterone increases the expression and activity of SAHHase protein. The rationale for this work is that general methylation may be promoted by activation of SAHHase, the only enzyme known to metabolize SAH, a potent end-product inhibitor of methylation. Although aldosterone increased SAHHase activity, steroid did not affect SAHHase expression. Antisense SAHHase oligonucleotide decreased SAHHase expression and activity. Moreover, this oligonucleotide, as well as a pharmacological inhibitor of SAHHase, decreased aldosterone-induced activity of ENaC via a decrease in ENaC open probability. The kinetics of ENaC in cells treated with antisense plus aldosterone were similar to those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate from open states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gating mode with short open and closed times to one with longer open and closed times.

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway.

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.

Application of the method of multiple thresholding to white blood cell classification.

A new method of image segmentation based on the principle of multiple grey level thresholding has been applied to a data set consisting of 1149 white blood cells of 13 different, clinically important types, randomly chosen on 20 blood smears from leukemia patients. Classification of these cells on the basis of quantitative measurements in the segmented images yields an accuracy of 82.6%. Some of the erroneous classifications must be attributed to intrinsic problems in the assignment of a priori labels. Correcting for such cases, the performance of the method, as measured on the present data set, increases to 89.8%. This illustrates the practical applicability of the segmentation method in automated white blood cell and possibly other cytological and histological analysis systems.

Automated white blood cell classification revisited.

A novel approach to the problem of automated white blood cell classification is described. Whereas in most earlier attempts the segmentation of the cells has been recognized as the most difficult and most critical step in the sequence of operations resulting in the classification, the method described here eliminates the necessity of the detection of the contour of the nucleus and of the cytoplasm, and is therefore less sensitive to such disturbing factors as the presence of granules, of other cells touching the cell of interest, etc. The multiple sequential threshold method to be described here in two slightly different variants yields a correct classification rate of 94.7% for a 4 class problem (90 cells in the test set), and 91.8% for an 8 class problem (279 cells in the test set). Both experiments include immature cell types.