Organic anion transporting polypeptides (OATP/SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 (Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 (SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C (SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.
N-Acetylcysteine (NAC) and dimercaptopropanesulfonate (DMPS) are sulfhydryl-containing compounds that produce a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned animals, but the molecular mechanism for this effect is unknown. NAC and DMPS are themselves excreted in urine in high concentrations. The present study tested the hypothesis that the complexes formed between MeHg and these anionic chelating agents are transported from blood into proximal tubule cells by the basolateral membrane organic anion transporters (Oat) 1 and Oat3. Xenopus laevis oocytes expressing rat Oat1 showed increased uptake of [ 14 C]MeHg when complexed with either NAC or DMPS but not when complexed with L-cysteine, glutathione, dimercaptosuccinate, penicillamine, or ␥-glutamylcysteine. In contrast, none of these MeHg complexes were transported by Oat3-expressing oocytes. The apparent K m values for Oat1-mediated transport were 31 Ϯ 2 M for MeHg-NAC and 9 Ϯ 2 M for MeHg-DMPS, indicating that these are relatively high-affinity substrates. Oat1-mediated uptake of [ 14 C]MeHg-NAC and [ 14 C]MeHg-DMPS was inhibited by prototypical substrates for Oat1, including p-aminohippurate (PAH), and was trans-stimulated when oocytes were preloaded with 2 mM glutarate but not glutamate. Conversely, efflux of [ 3 H]PAH from Oat1-expressing oocytes was trans-stimulated by glutarate, PAH, NAC, DMPS, MeHg-NAC, MeHg-DMPS, and a mercapturic acid, indicating that these are transported solutes. [3 H]PAH uptake was competitively inhibited by NAC (K i of 2.0 Ϯ 0.3 mM) and DMPS (K i of 0.10 Ϯ 0.02 mM), providing further evidence that these chelating agents are substrates for Oat1. These results indicate that the MeHg antidotes NAC and DMPS and their mercaptide complexes are transported by Oat1 but are comparatively poor substrates for Oat3. This is the first molecular identification of a transport mechanism by which these antidotes may enhance urinary excretion of toxic metals.
Pathways for transport of choline by human placental epithelia were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of choline transport mechanisms mediating Na(+)-choline cotransport, choline/H+ exchange and facilitated diffusion were assessed from [3H]choline tracer flux measurements. The rate and magnitude of intravesicular choline accumulation was unaffected by the imposition of an inwardly directed Na+ gradient suggesting an absence of a mechanism mediating brush border membrane Na(+)-choline cotransport. The imposition of inside-acid or inside-alkaline pH gradients was observed to have no significant effect on choline uptake suggesting choline is not a substrate for placental epithelial organic cation/H+ exchange. Conditions favoring the development of an inside-negative K+ diffusion potential was observed to induce a concentrative accumulation of choline to levels exceeding equilibrium suggesting the presence of a conductive uptake pathway for choline in placental brush border membrane. Evidence to suggest conductive choline uptake resulted from a mediated transport process includes a demonstration of the counterflow phenomena, the concentration-dependent inhibition by hemicholinium-3 (IC50 approximately equal to 100 microM) and the saturable rate of conductive choline uptake (Km approximately equal to 300 microM, Vmax approximately equal to 30 nmol/mg per min). Substrate specificity studies of the mechanism mediating conductive choline uptake suggest the interaction of choline with the transport protein occurs at a minimum of two sites: a site of negativity with the positively charged nitrogen group and a site of hydrogen bonding to the primary alcohol. Several commonly prescribed pharmaceuticals known to cross the placental barrier including imipramine, verapamil, propranolol, quinine, flurazepam, amiloride and ritodrin were observed to inhibit conductive choline uptake suggesting an interaction with the mechanism mediating conductive choline transport. Conductive choline uptake was unaffected by the presence of the basic amino acids lysine, arginine and histidine; the neurotransmitters serotonin, dopamine and histamine and the vitamins thiamine and carnitine which suggests the mechanism mediating conductive choline transport is not a pathway for placental uptake of these compounds.
Exercise is associated with an increase in plasma renin activity (PRA). The purpose of this study was to determine the role of the prostaglandin (PG) and adrenergic pathways in the renin release with exercise in the dog. One group of animals (n = 4) was exercised under control untreated and indomethacin- and meclofenamate- (2 mg/kg) treated conditions. A 155% increase in PRA was not influenced by PG inhibition. In a second group (n = 7) PRA was 1.22 +/- 0.32, 3.29 +/- 1.59, 6.28 +/- 2.85, and 5.30 +/- 2.00 ng ANG I X ml-1 X h-1 at rest and during light, moderate, and heavy exercise, respectively. Guanethidine treatment (15 mg/kg) decreased mean PRA by 41, 50, 70, and 73% at rest and during the three levels of exercise, respectively. In a third group (n = 5) control exercise runs were repeated after metoprolol treatment. Selective beta 1-blockade completely abolished the increment in PRA observed with exercise. These data demonstrate that the elevation of PRA during exercise in the dog is mediated by increased sympathetic nerve activity involving beta 1-receptors and that it is not dependent on alterations in PG synthesis.
Pathways for transport of thiamine by the human placental epithelium were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of thiamine transport mechanisms mediating Na+-thiamine cotransport, proton/thiamine exchange and facilitated diffusion was assessed from [3H]-thiamine tracer flux measurements. The magnitude of intravesicular thiamine accumulation was unaffected by the imposition of an inwardly directed sodium gradient suggesting an absence of a mechanism mediating brush border membrane Na+-thiamine cotransport. Intravesicular thiamine accumulation was indistinguishable when measured in the presence and absence of conditions favoring the development of an inside-negative, potassium diffusion potential. The observed absence of conductive thiamine uptake suggests the absence of a mechanism mediating facilitated diffusion of thiamine in placental brush border membrane. The imposition of an inside-acid pH gradient was observed to induce concentrative accumulation of thiamine to levels exceeding equilibrium, suggesting the presence of a placental brush border membrane proton/thiamine exchange mechanism. Protonophore- induced dissipation of an imposed inside-acid pH gradient in the absence of membrane potential was observed to abolish concentrative accumulation of thiamine, suggesting a direct chemical coupling of protons and thiamine via a mediated exchange mechanism. Consistent with the functional properties expected for a mechanism mediating thiamine transport by organic cation exchange, the rate and magnitude of intravesicular [3H]-thiamine accumulation was increased when measured in the presence compared to the absence of an outwardly directed thiamine concentration gradient. Substrate specificity studies of the proton/thiamine exchange mechanism suggest that the amine at position four of the pyrimidine ring, but not the hydroxyethyl side chain or an unmodified thiazolium ring, is an important chemical determinant for interaction with the transporter substrate binding site(s). Substrate specificity studies further suggest the possible presence of three separate organic cation exchange mechanisms mediating transport of thiamine, guanidine and MIA across placental brush border membrane.
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