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The saccular membranes of trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus) were examined to characterize specialized epithelial cells that might be responsible for ion exchange. The approach for localizing cell types was new for this tissue, as observations were made with a stereomicroscope and a light microscope in order to have a general view of the epithelium. No important differences between the two species were seen. The saccular tissue is a monolayer epithelium (except for the macula neural zone) surrounded by a layer of connective tissue invaded by many blood vessels. The use of the fluorescent probe DAPSMI and zinc iodide/osmium fixation-coloration defined two areas in which ionocytes were present. In the first, large ionocytes were grouped into a nearly complete, crowned meshwork around, but separated from, the macula. In the second area, opposite the macula, the ionocytes were smaller, cubical, and grouped in patches. Cells rich in Na+, K+-ATPase and carbonic anhydrase II were present in both areas. Contrary to previous studies in mammals and fish, ionocytes were also found in the epithelium of the saccule.

Cl− conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl− conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl−currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl− currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl− currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl− current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl− pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.

1. Protein constituents were determined in eight amyloid deposits from eight patients (five male and three female), 53 +/- 4 years of age, treated by haemodialysis for 9-20 years using only cuprophane membranes and operated for carpal tunnel syndrome. 2. Soluble proteins were removed by solubilization in phosphate-buffered saline after osmotic lysis. The proteins of the insoluble fibrils were characterized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two-dimensional gel electrophoresis, and immunologically identified by Western blotting. 3. In addition to beta 2-microglobulin, alpha 2-macroglobulin was identified in the fibrillar material. The presence of these two proteins in amyloid deposits was confirmed by immunofluorescent microscopic studies. 4. Our data confirm the presence of beta 2-microglobulin in haemodialysis-associated amyloidosis, and also suggest a possible role for alpha 2-microglobulin: it may protect beta 2-microglobulin from proteolytic digestion, leading to its accumulation in intact form and to amyloid fibril formation.

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Interaction of plasma membrane with the cytoskeleton involves a large number of proteins, among them a 36 kDa protein that was found to be involved in the interaction with actin filaments. We have isolated a 36 kDa protein from bovine aorta as a monomer and in a complex with a 10 kDa protein. Partial amino acid sequence determinations show that the 36 kDa and 10 kDa proteins isolated from bovine aorta are analogous to or identical with corresponding proteins purified from bovine intestine already described by Kristensen, Saris, Hunter, Hicks, Noonan, Glenney & Tack [(1986) Biochemistry 25, 4497-4503]. We report here that the association of the 10 kDa protein with the 36 kDa protein confers specific calmodulin-binding and actin-severing properties on the complex that are not possessed by the 36 kDa monomer alone. These findings suggest that the protein complex could be involved in thin-filament-related structures or could modulate some Ca2+-regulated events mediated by calmodulin.

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Ionic (Na+, K+, Cl-, PO4(3-), pH), total CO2, total calcium and protein concentrations in the plasma and endolymph of the inner ear were compared in trout Oncorhynchus mykiss and turbot Scophthalmus maximus. In both species, saccular endolymph was characterized by high levels of K+ and total CO2 and in trout by an alkaline pH. The kinetic characteristics of proton secretion across the saccular epithelium of trout were investigation using a titration technique in which isolated saccules were mounted as closed sacs. The rate of proton secretion depends strongly on the pH of the Ringer's solution and secretion stops at a pH below 7.2. Proton secretion is driven by an energy-dependent mechanism involving basolateral ouabain-sensitive Na+/K+ exchangers. Proton secretion was partially inhibited by acetazolamide and completely inhibited in Na(+)-free Ringer or in the presence of 1 mmol l-1 amiloride. A cellular model stressing the importance of proton exchange through the saccular epithelium is proposed to explain the regulation of endolymph pH, a crucial factor for the deposition of otolith calcium.