Membrane translocation of glutaric acid and its derivatives.

The neurodegenerative disorder glutaric aciduria type I (GA I) is characterized by increased levels of cytotoxic metabolites such as glutaric acid (GA) and 3-hydroxyglutaric (3OHGA). The present report summarizes recent investigations providing insights into mechanisms of intra- and intercellular translocation of these metabolites. Initiated by microarray analyses in a mouse model of GA I, the sodium-dependent dicarboxylate cotransporter 3 (NaC3) was the first molecule identified to mediate the translocation of GA and 3OHGA with high and low affinity, respectively. More recently, organic anion transporters (OAT) 1 and 4 have been reported to be high-affinity transporters for GA and 3OHGA as well as D-2- and L-2-hydroxyglutaric acid (D2OHGA, L2OHGA). The concerted action of NaC3 and OATs may be important for the directed uptake and excretion of GA, 3OHGA, D2OHGA and L2OHGA in kidney proximal tubule cells. In addition, experimental data on cultured neuronal and glial cells isolated from mouse brain demonstrated that GA rather than 3OHGA may competitively inhibit the anaplerotic supply of tricarboxylic acid cycle intermediates from astrocytes to neurons. The identification of GA and GA derivative transporters may represent targets for new approaches to treat patients with GA I and related disorders.

Molecular evidence of organic ion transporters in the rat adrenal cortex with adrenocorticotropin-regulated zonal expression.

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.

Interaction of the metal chelator 2,3-dimercapto-1-propanesulfonate with the rabbit multispecific organic anion transporter 1 (rbOAT1).

The metal chelator DMPS (2,3-dimercapto-1-propanesulfonate) is used to treat heavy metal intoxication because it increases renal excretion of these toxins, which are accumulated in proximal tubule cells. To evaluate the involvement of the organic anion transporter 1 (OAT1) in the renal flux of DMPS, we examined the effect of DMPS on transport mediated by the rabbit ortholog of OAT1 and compared these characteristics with those observed in intact isolated rabbit proximal tubules. The rabbit OAT1 (rbOAT1) cDNA consisted of 2124 base pairs encoding a protein of 551 amino acids. Heterologous expression in COS-7 cells revealed rbOAT1-mediated transport of p-aminohippurate (PAH; K(t) = 16 microM). A 1 mM concentration of unlabeled PAH, alpha-ketoglutarate, urate, or probenecid inhibited [(3)H]PAH uptake by 70 to 90%. cis-Inhibition and trans-stimulation experiments using several Krebs cycle intermediates implicated alpha-ketoglutarate as the main intracellular exchange anion. Reduced DMPS inhibited rbOAT1-mediated fluorescein transport with an apparent K(i) of 102 microM. These characteristics paralleled those observed in isolated rabbit proximal tubules. PAH was transported into nonperfused single proximal tubule S(2) segments with a K(t) of 76 microM. DMPS inhibited FL uptake into single tubule segments with a K(i-app) of 71 microM. Fluorescein efflux from preloaded tubules was trans-stimulated by 1 mM PAH and 1 mM DMPS, consistent with DMPS entry into tubule cells by rbOAT1. In summary, rbOAT1 mediates basolateral uptake of DMPS into proximal tubule cells, implicating this process in the detoxification process of heavy metals in the kidneys.

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.

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.

Liquid-phase hybridization and capture of hepatitis B virus DNA with magnetic beads and fluorescence detection of PCR product.

The polymerase chain reaction (PCR) exceeds all hitherto known detection limits. This sensitivity could lead to false positive results. Every manipulation increases the risk of contamination via, for example, aerosols. Most protocols for the extraction of template nucleic acids are complicated and possible centrifugation steps do not reduce the risk of aerosols. In addition, most of the methods for analysis are time-consuming and cannot be applied to different template materials. An alternative extraction method has been developed. The fast chemical denaturation of template by guanidine thiocyanate was followed by liquid hybridization to biotinylated oligonucleotides. The template nucleic acid could be washed after binding to streptavidin-coated paramagnetic beads to reduce influence on the enzymatic amplification steps. PCR of hepatitis B virus deoxyribonucleic acid was used to demonstrate how easy, versatile, and time-saving this method is without centrifugation. The level of extracted nucleic acids was quantitated and the properties for sensitive extraction were evaluated. After PCR an additional step was developed which used fluorescent staining to detect positive amplifications. This is useful to identify positive results in predominantly negative samples.