Richard Béliveau

Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols.

Many beneficial proprieties have been associated with polyphenols from green tea, such as chemopreventive, anticarcinogenic, antiatherogenic and antioxidant actions. In this study, we investigated the effects of green tea polyphenols (GTPs) and their principal catechins on the function of P-glycoprotein (P-gp), which is involved in the multidrug resistance phenotype of cancer cells. GTPs (30 microg/ml) inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 (R-123) 3-fold in the multidrug-resistant cell line CH(R)C5, indicating that GTPs interact with P-gp and inhibit its transport activity. Moreover, the modulation of P-gp transport by GTPs was a reversible process. Among the catechins present in GTPs, EGCG, ECG and CG are responsible for inhibiting P-gp. In addition, EGCG potentiates the cytotoxicity of vinblastine (VBL) in CH(R)C5 cells. The inhibitory effect of EGCG on P-gp was also observed in human Caco-2 cells, which form an intestinal epithelial-like monolayer. Our results indicate that, in addition to their anti-cancer properties, GTPs and more particularly EGCG inhibit the binding and efflux of drugs by P-gp. Thus, GTPs or EGCG might be potential agents for modulating the bioavailability of P-gp substrates at the intestine and the multidrug resistance phenotype associated with expression of this transporter in cancer cells.

P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries

A significant proportion of P‐glycoprotein (P‐gp) and caveolin was co‐localized in caveolae isolated from resistant (CHRC5) cells overexpressing P‐gp and from drug‐sensitive Chinese hamster ovary cells (AuxB1). The proportion of P‐gp and caveolin associated with caveolar microdomains was higher in CHRC5 cells grown in the presence of P‐gp substrates (cyclosporin A or colchicine) than in untreated CHRC5 cells. Co‐immunoprecipitation of P‐gp and caveolin from CHRC5 lysates suggests that there is a physical interaction between them. Furthermore, co‐localization of P‐gp and caveolin was found in caveolae from brain capillaries, indicating that this association also takes place in vivo.

Phosphate uptake by superficial and deep nephron brush border membranes

Dietary phosphate restriction and acute parathyroidectomy in rat are known to be associated with a selective increase in phosphate uptake by renal cortical brush border membranes (BBM). Conversely, phosphate loading and parathyroid hormone (PTH) administration result in a decrease of this uptake.In the present study, we investigated whether the response of the membrane to these various stimuli implies similar or different modifications of the kinetic properties of this membrane, whether these modifications affect one or both of the two systems of phosphate transport previously described, whether both superficial and deep nephron populations are involved, and whether the two stimuli: dietary phosphate, and parathyroid activity, are additive or not.Kinetic studies of phosphate (PO4) uptake by BBM vesicles were performed in seven groups of rats: control (N), acutely thyroparathyroidectomized (TPTX), PTH loaded (PTH), phosphate loaded (P+), phosphate depleted (P−) phosphate depleted with acute thyroparathyroidectomy (P−TPTX), and phosphate depleted-PTH loaded (P−PTH). In each of these experimental conditions, superficial and deep nephrons were investigated.Results indicate that 1. BBM from deep nephrons present a greater capacity for PO4 transport than those from superficial nephrons; 2. Whereas a dual system of PO4 uptake is observed in superficial BBM, deep BBM present only one single system; 3. Phosphate in the diet influences PO4 uptake by BBM to a greater extent in the deep than in the superficial nephrons; 4. PTH status on the contrary, equally influences both populations; 5. TPTX does not significantly enhance PO4 uptake in phosphate depleted rats; however, PTH loading curtails this uptake; 6. In the deep BBM neither the PTH status nor the phosphate content of the diet modify the apparentKm. In the superficial BBM, the apparentKm of the high affinity system (low substrate concentrations) varies with the PTH activity.

Molecular size of the Na+-H+ antiport in renal brush border membranes, as estimated by radiation inactivation

The radiation inactivation method was applied to brush border membrane vesicles from rat kidney, in order to estimate the molecular size of the Na+-H+ antiporter. Sodium influx (1mM) driven by an acid intravesicular pH was unaffected by the high osmolarity of the cryoprotective solution. Initial rate of influx was estimated by linear regression performed on the first 10 seconds of transport: 0.512 pmol/micrograms protein/s. There was no binding component involved. Incubation performed in the presence of 1 mM amiloride, an inhibitor of the Na+-H+ antiport gave an initial rate of only 0.071 pmol/microgram/s, an 82% inhibition. Membrane vesicles were irradiated at -78 degrees C in a Gammacel Model 220. Sodium influx was reduced, as the dose of radiation increased, but the influx remained linear for the period of time (10s) during which the initial rate was estimated, indicating no alteration of the proton driving force during this time period. Amiloride-insensitive flux remained totally unaffected by the radiation dose, indicating that the passive permeability of the membrane towards sodium was unaffected. The amiloride-sensitive pathway presented a monoexponential profile of inactivation, allowing the molecular size to be estimated at 321 kDa. Based on DCCD-binding studies suggesting the molecular size of the monomer to be around 65 kDa for rat kidney, our results suggest that the functional transporter in the membrane to be a multimer.

Normal molecular size of the Na+-phosphate cotransporter and normal Na+-dependent binding of phosphonoformic acid in renal brush border membranes of X-linked Hyp mice

X-linked Hyp mice have a specific defect in Na(+)-dependent phosphate (Pi) transport at the renal brush border membrane (BBM). In the present study we examined the effect of the Hyp mutation on the molecular size of the Pi transporting unit and on Na(+)-dependent 14C-phosphonoformic (PFA) binding in renal BBM vesicles. By radiation inactivation analysis, we demonstrated that the molecular size of the Na(+)-Pi cotransporter is similar in normal (242 +/- 16 kDa) and Hyp mice (227 +/- 39 kDa). Moreover, while BBM Na(+)-dependent Pi transport is significantly reduced in Hyp mice (249 +/- 54 vs 465 +/- 82 pmol/mg protein/6s), genotype differences in (1) Na(+)-dependent PFA binding (1020 +/- 115 vs 1009 +/- 97 pmol/mg protein/30 min), (2) Pi-displaceable Na(+)-dependent PFA binding (605 +/- 82 vs 624 +/- 65 pmol/mg protein/6s), and (3) phosphate uptake at Na(+)-equilibrium (67 +/- 10 vs 54 +/- 7 pmol/mg protein/6s) are not apparent. The present data demonstrate that the molecular size of the renal BBM Na(+)-Pi cotransporter is normal in Hyp mice and suggest that the number of Na(+)-Pi cotransporters may not be reduced in the mutant strain.

Membrane topography of the renal phosphate carrier NaPi-2: limited proteolysis studies.

The rat sodium/phosphate cotransporter NaPi-2 is a 70 kDa polypeptide (p70) for which eight transmembrane segments have been predicted. We have shown that p70 exists predominantly as p45 and p40 fragments which are linked by disulfide bonds. In this work, the p40 fragment, corresponding to the C-terminus of NaPi-2, was purified from renal brush-border membranes using non-reducing and then reducing column electrophoresis followed by enzymatic deglycosylation and SDS-PAGE. The N-terminal sequence obtained for this fragment, VEAIG, indicates that the formation of p45 and p40 arises from the cleavage of p70 between arginine-319 and valine-320. In order to determine the membrane topography of NaPi-2, brush-border membrane vesicles were digested with various proteases and the transporter-derived proteolytic peptides were subsequently identified by Western blotting using N- and C-terminal-directed antibodies. Our results lead us to propose an alternative topographical model in which p45 and p40 possess three transmembrane domains each and indicate that the processing site of p70 for the generation of p45 and p40 is localized in a large protein core facing the extracellular milieu. This localization of the cleavage site indicated that NaPi-2 could either be processed intracellularly by vesicular proteases or extracellularly by secretory proteases or by brush-border membrane ectoenzymes.

Characterization of essential arginine residues implicated in the renal transport of phosphate and glucose

We have characterized the reaction of arginine-specific reagents with the phosphate and glucose carriers of the kidney brush-border membrane. The inhibition of phosphate and glucose transport by phenylglyoxal follows pseudo-first-order kinetics. The rate of inactivation of phosphate transport by 50 mM phenylglyoxal was about 3-fold higher than that for glucose transport (kapp was 0.052 s-1 for the uptake of phosphate and 0.019 s-1 for the uptake of glucose). The order of the reaction, n, with respect to phenylglyoxal was 1.25 and 1.31 for the inactivation of phosphate and glucose transport, respectively. The inactivation of phosphate flux by p-hydroxyphenylglyoxal also follows pseudo-first-order kinetics, but the inhibition rate (kapp = 0.0012 s-1) was slower than with phenylglyoxal. The inactivation increased with the alkalinity of the preincubation medium for both phosphate and glucose fluxes and was maximal at pH 9.0. The inactivation of phosphate flux by phenylglyoxal depends upon the presence of an alkaline intravesicular pH. Extravesicular pH does not affect the reaction. Phenylglyoxal does not interfere with the recycling of the protonated carrier since phosphate uptake is inhibited independently of the pH used for transport measurements. Moreover, phenylglyoxal completely abolished trans stimulation by phosphate. Trans sodium inhibited phosphate uptake and abolished the pH profile of phosphate uptake.

Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransport

Polyclonal antibodies raised against the 14-amino acid C-terminal portion of the rabbit renal brush-border membrane Na+/Pi cotransporter, as deduced from the nucleotide sequence of the cloned NaPi-1 gene, were used for Western blot analysis of renal brush-border membrane proteins from rat, rabbit and beef. Proteins of 65 kDa from the rat, 64 kDa from the rabbit, and 38, 66, 77, 92, 110, 176 and 222 kDa from the beef were specifically labelled. The affinity of the antibodies was much greater, however, for the proteins of the rat and rabbit than for those of the beef. The rat 65-kDa antigen was readily detected in brush-border membranes isolated from kidney cortex, but was absent from the basolateral membrane and the cytosolic and microsomal fractions of this tissue, in agreement with the subcellular localization of the Na+/Pi cotransporter. This antigen was however several-fold more abundant in the juxtamedullary portion of the cortex than in the outer portion. Despite a strong stimulation in phosphate transport, a low-phosphate diet had little influence on the amount of antigen detected. An additional peptide-displaceable band corresponding to a protein of 250 kDa appeared when beta-mercaptoethanol was omitted during electrophoresis, in agreement with the possibility that disulfide bonds may be involved in the regulation of renal phosphate transport activity.

Radiation-inactivation analysis of the oligomeric structure of the renal sodium/D-glucose symporter.

The radiation-inactivation size (RIS) of the rat renal brush-border membrane sodium/D-glucose cotransporter was estimated from the loss of transport activity in irradiated membrane vesicles. The RIS depended on the electrochemical conditions present when measuring transport activity. A RIS of 294 +/- 40 kDa was obtained when transport was measured in the presence of a sodium electrochemical gradient. Under sodium equilibrium conditions, the RIS was 84 +/- 25 kDa in the presence of a glucose gradient, and 92 +/- 20 kDa in its absence. In the absence of a sodium gradient, but in the presence of an electrical potential gradient, the RIS increased to 225 +/- 49 kDa. The 294 kDa result supports earlier suggestions that the Na+ gradient-dependent glucose transport activity is mediated by a tetramer. Individual monomers appear, however, to carry out glucose transport under equilibrium exchange conditions or when a glucose gradient serves as the only driving force. The electrical potential gradient-driven glucose transport RIS appears to involve three functional subunits.

The Renal Brush Border Membrane in Man

Brush border membranes (BBM) have been prepared from fresh samples of normal human kidney cortex and compared to that from mouse, rat, and rabbit. Human BBM presents a sodium dodecyl sulfate gel elect