ISSN:1094-9313    

DOI:10.1159/000154788

出版商:S. Karger AG    

年代:1996

数据来源: Karger

ISSN:1094-9313    

DOI:10.1159/000154789

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

The effect of bromosulfophthalein (BSP) on potential-stimulated p-aminohippurate (PAH) uptake as well as on Na+-D-glucose and Na+-L D L-alanine. Half-maximal inhibition of initial uptakes was observed at approximately 10,75 and 250 µM BSP, respectively. The inhibition of initial Na+-D-glucose uptake could not be attributed to increased cation permeability, since equilibrium exchange was equally affected by BSP. The nonspecific short-term inhibition of unrelated transport systems by BSP suggests a more general effect, such as a change in membrane fluidity. In addition, long-term incubation with 0.5 mM BSP resulted in a loss of vesicle integrity

ISSN:1094-9313    

DOI:10.1159/000154790

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Binding proteins for weak organic acids, in particular bile acids and loop diuretics, were separated by two-dimensional gel electrophoresis after photoaffinity labelling of rat liver basolateral plasma membranes. After staining with Coomassie Blue the protein spots were isolated, electroeluted and concentrated in an SDS gel. The proteins were digested in the gel by endoproteinase Lys-C. Appropriate fragments were separated by HPLC and the sequence was determined. A 57-kD protein with an apparent pi of 5.3, which was marked by photoaffinity labelling with 7,7-azotaurocholate and stained with an antibody against recombinant protein disulfide isomerase (PDI), showed complete identity with PDI in three fragments. [3H]bumetanide, a loop diuretic, marked two 54-kD proteins. One 54-kD protein with an apparent pi of 6.35 showed homology to the tyrosine kinase inhibitor of the insulin receptor. The amino acid sequence of three fragments belonging to the other 54-kD protein with an apparent pi of 6.2 was identical with cytokeratin 8. Two further proteins that were present in large amounts were also sequenced. A 35-kD protein was identified to be arginase. The internal sequences of a 58-kD protein showed 100% homology to endoproteinase ER 60. The results show that the proposed method of isolation of proteins from 2D gels (a) provides sequence data, even if there are only minute amounts of protein obtainable, and (b) allowed identification of yet unknown binding proteins of organic anions in the liver.

ISSN:1094-9313    

DOI:10.1159/000154791

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Organic anions (OA) (e.g., p-aminohippurate and fluorescein) undergo net secretion, apparently by a similar process in renal proximal tubules of most vertebrates. In isolated tubules from fish, amphibians, reptiles, birds, and mammals, net secretion involves transport into the cells against an electrochemical gradient at the basolateral membrane and movement from the cells to the lumen down an electrochemical gradient. In all vertebrate classes, basolateral uptake involves a tertiary active process, the final step in which is OA/α-ketoglutarate (αKG) countertransport. An outwardly directed αKG gradient is maintained by metabolism and basolateral and luminal Na-dicarboxylate cotransport into the cells, the importance of each apparently varying with the species and the metabolic state of the tubules. Little is known about transport into the lumen, but it may involve carrier-mediated diffusion or anion countertransport, the process apparently differing between vertebrate classes and between species within a vertebrate class. Intracellular compartmental accumulation may occur during transepithelial transport in fish, reptiles, birds, and mammals, but it may be significant in mammals only with reduced metaboli

ISSN:1094-9313    

DOI:10.1159/000154792

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

A mouse fibroblast L cell line defective in Na+/H+ antiport activity (LAP1) was used to develop a screening assay for the Na+-dependent Cl-/HCO–3 exchanger (NCBE). Regulation of intracellular pH (pHi) via NCBE was studied in suspended LAP1 cells using the pH-sensitive fluorescent dye 2’, 7’-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Besides the well-known irreversible NCBE blocker 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS), different compounds were tested. None of these had any higher effect than DIDS. Nevertheless, 3’,5-dichlorodiphenylamine-2-carboxylic acid and the corresponding 3’-trifluoromethyl derivative were found to be approximately as potent as DIDS with IC50 values of 29.2 ± 2.5 and

ISSN:1094-9313    

DOI:10.1159/000154793

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Although plasma membrane events mediating organic anion and cation secretion have been intensively investigated, intracellular events associated with transcellular fluxes of secreted solutes have received much less attention. Recent data obtained through epifluorescence and confocal microscopy in renal tissue of mammals, teleosts, and crustaceans indicates that sequestration within intracellular organelles is intimately involved in secretion of both organic anions (OA) and cations (OC). For OA, one compartment is mitochondrial and a second is smaller and more mobile. OA-loaded vesicles were shown to move in a basolateral-to-apical direction in crustacean urinary bladder. This movement was reversibly blocked by nocodazole, a microtubule-disrupting agent. Furthermore, secretion into the lumen of teleost proximal tubule was also reversibly inhibited by nocodazole. For OA, the small mobile vesicles have yet to be identified or isolated. For OC, a population of acidifying endosomes was identified which accumulated tetraethylammonium (TEA) by TEA/proton exchange. Finally, in cultured monolayers of choroid plexus, an epithelium which transports both OA and OC from cerebrospinal fluid to blood, fusion of vesicles with the basolateral membrane and concomitant release of OC was seen during transepithelial transport. This process was also blocked by nocodazole. The roles of sequestration in transepithelial transport are as yet uncertain, but these data argue that such roles are likely.

ISSN:1094-9313    

DOI:10.1159/000154794

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Earlier studies demonstrated that an apical anion exchanger which accepts urate, chloride, aliphatic monocarboxylates, and the aromatic anions, pyrazinoate (PZA) and nicotinate, plays a major role in urate reabsorption in human proximal tubules. In substrate competition studies for transport, we investigated the effect of PZA on 50 µM [14C]-urate uptake in exchange of lactate, human brush-border membrane vesicles (BBMV) being loaded with 5 mM lactate. 1 mM PZA cis-inhibited, whereas 0.1 mM PZA cis-stimulated [14C]-urate uptake in exchange for lactate. Such paradoxical effects suggest the cooperation of two anion exchangers, PZA having more affinity for a ‘lactate/anion exchanger’ than for a ‘urate/ anion exchanger’. Apparent Km of [14C]-PZA uptake were measured in lactate- or urate-loaded BBMV, and were 0.33 ± 0.06 and 0.98 ± 0.44 mM, respectively. Unlike [14C]-PZA, [14C]-orotate and [14C]-nicotinate uptakes were stimulated only to a minor extent in BBMV loaded with lactate. In contrast, cis -inhibition studies in urate-loaded BBMV demonstrated that nicotinate and orotate had similar affinity as PZA for the ‘urate/anion exchanger’. Oxalate was shown to have affinity for the ‘lactate/anion exchanger’, but not for the ‘ur

ISSN:1094-9313    

DOI:10.1159/000154795

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

The permanent cationic fluorescent probe 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+) has recently been reported to be transported by the organic cation transporters in the proximal tubule of the kidney [Kidney Int 1995;47:1647-1657]. A new application for the fluorescence microscopy technique using this dye as a substrate for organic cation transport systems is described, which can be used for dynamic monitoring of organic cation transport in cultured renal cells of proximal origin (LLC-PK1 cells). The uptake of ASP+ was concentration dependent and linear over the first 10 min at a concentration of 1 µmol/l. The results are compatible with a carrier-mediated transport process presenting characteristics of organic cation transport systems

ISSN:1094-9313    

DOI:10.1159/000154796

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

In this study the influence of probenecid as inhibitor of the renal excretory system for organic anions was tested on the release of cortisol from primary cultures of bovine adrenocortical cells. ACTH-stimulated cortisol release (30 min to 24 h) was inhibited by probenecid (Ki = 0.093 mM). Analysis of steroid synthesis with 3H-pregnenolone as precursor indicated that part of the inhibition resulted from an attenuation of steroid synthesis by probenecid. The increased intracellular cortisol levels in the presence of probenecid however indicate an inhibitory effect on an efflux mechanism. Cortisol efflux was trans-stimulated by p-aminohippurate (PAH) and tetrafluorosuccinate, substrates of the renal PAH/anion exchanger. Further evidence for the PAH exchanger provided the expression of a probenecid-sensitive PAH transporter after injection of mRNA from adrenal cells into Xenopus laevis oocytes.

ISSN:1094-9313    

DOI:10.1159/000154797

出版商:S. Karger AG    

年代:1996

数据来源: Karger