Voltage-driven p-aminohippurate, chloride, and urate transport in porcine renal brush-border membrane vesicles.

p-Aminohippurate (PAH) and urate are secreted into the proximal tubule lumen across the brush-border membrane. Here we used brush-border membrane vesicles from pig kidney to study PAH and urate transport. Efflux and influx of [3H]PAH were influenced by K+-diffusion potentials indicating electrogenic PAH transport. An outside>inside PAH concentration difference accelerated voltage-sensitive, Na+-coupled D-glucose uptake as efficiently as did an outside>inside Cl- concentration difference, suggesting comparable conductances for PAH and Cl- in brush-border membrane vesicles. Up to 1 mM of the uricosurics indacrinone, tienilic acid, losartan and probenecid, as well as of the stilbenes, DIDS and SITS, and of the loop diuretics furosemide and bumetanide inhibited voltage-driven PAH uptake, but not, or only slightly, voltage-driven Cl- uptake. Voltage-driven [14C]urate uptake, however, was inhibited by 0.1 mM DIDS, 0.2 mM losartan and 0.5 mM probenecid to a similar extent as [3H]PAH uptake. One millimolar pyrazinoic acid, oxonate, xanthine and adenosine inhibited neither [3H]PAH nor [14C]urate uptake. These results suggest that PAH and urate share an anion conductance which is distinct from the Cl- conductance and is probably not the same as a recently identified urate channel (Leal-Pinto E et a]. J Biol Chem 272:617-625, 1997).

Chloride conductance in HT29 cells: investigations with apical membrane vesicles and RT-PCR.

Vesicles enriched in a marker enzyme for apical membranes were isolated from HT29 cells. These vesicles contain an anion conductance with the selectivity gluconate approximately sulphate4, 4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS)>glibenclamide. The Cl- conductance was insensitive to Ca2+ and to extravesicular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inosine 5'-triphosphate (ITP). Using the reverse transcription polymerase chain reaction (RT-PCR) technique and sequencing of the amplified products we detected messenger ribonucleic acid (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR), the putative Cl- channel or Cl- channel regulator pICln, and the Cl- channels ClC-2, ClC-3, ClC-5 and ClC-6 in HT29 cells. The properties of the vesicles' Cl- conductance resemble those of the intermediate conductance outwardly rectifying Cl- channel and tentatively exclude contributions of CFTR, pICln and ClC-2. Whether ClC-3, ClC-5, ClC-6 are involved in Cl- conductance remains to be determined.

S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types.

Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Endosomes: another extra-mitochondrial location of type-1 porin/voltage-dependent anion-selective channels.

Endocytotic vesicles (EV) isolated from rat renal cortex were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. A monoclonal antibody against human type-1 porin (31 kDa) detected a strong band of 31 kDa. The same antibody has been used as the primary antibody in indirect immunocytochemistry. Light microscopy of cryostat sections of rat renal cortex showed a heavy staining of EV underneath the brush-border membrane. Electron microscopy was performed by "preembedding immunogold staining" of rat renal cortex, the sections of which showed an extensive labelling of EV with gold particles. These results demonstrate that the expression of type-1 porin is not restricted to outer mitochondrial membranes. The biological function of endosomal type-1 porin has as yet to be ascertained.

Characterization of the chloride conductance in porcine renal brush-border membrane vesicles.

The chloride conductance in brush-border membrane vesicles prepared from pig kidney cortex was investigated using a light-scattering assay, anion-diffusion-potential-dependent Na+-D-glucose cotransport and 36Cl- influx. K+-diffusion-potential-driven salt exit from, or entry into, the vesicles was slow in the presence of gluconate, SO42- and F-, intermediate with Cl- and Br-, and fast with I-, NO3-, and SCN-. Stimulation of Na+-D-glucose uptake followed a similar anion sequence. Conductive Cl- flux had a low activation energy and was inhibited by suphhydryl reagents, the stilbene disulphonates 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) and 4, 4'-diisothiocyanato-2,2'-disulphonate (DIDS), and the arylaminobenzoates diphenylamine-2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Intravesicular Ca2+ and extravesicular nucleotides were without effect on conductive Cl- flux. These characteristics tentatively exclude some known Cl- channels and leave members of the ClC family as possible candidates responsible for the Cl- conductance in brush-border membranes.

Evidence for a cytosolic inhibitor of epithelial chloride channels.

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.