Biomedical subjects

X Bossuyt

Publications and source records attributed to X Bossuyt.

44 records · Page 3Linked to original sources

PubMed1996

Multispecific amphipathic substrate transport by an organic anion transporter of human liver.

BACKGROUND: Hepatic uptake of differently charged amphipathic endo- and xenobiotics is thought to occur via distinct carrier-mediated transport systems. Alternatively, a single rat organic anion transporting polypeptide (oatp) has recently been demonstrated to mediate hepatocellular uptake of differently charged amphipathic substrates. AIM: To investigate whether a cloned human liver organic anion transporting polypeptide (OATP) also can mediate charge- and class-independent hepatocellular uptake of amphipathic substrates. METHODS: Xenopus laevis oocytes were injected with OATP-cRNA. Sodium-independent uptake of estrone-3-sulfate, ouabain and the organic cation N-(4,4-azo-n-pentyl)-21-ajmalinium was compared in OATP-expressing and uninjected (or water injected) control oocytes. RESULTS: Our results indicate that OATP, in addition to bromosulfophthalein and bile salts, can also transport anionic estrone-3-sulfate (Km approximately 59 microM), neutral ouabain (K(m) approximately 5.5 mM) and cationic N-(4,4-azo-n-pentyl)-21-ajmalinium. For each of these compounds, OATP-mediated uptake was cis-inhibited by the OATP substrate taurochenodeoxycholate and the transport activities correlated well with the amounts of cRNA injected. CONCLUSION: Similar to the rat liver oatp, the human liver OATP can also mediate multispecific and charge-independent uptake of lipophilic amphipathic organic compounds. Thus, OATP may play an important role in the first pass clearance of drugs and other xenobiotics by the human liver.

Animals↗

PubMed1996

Polyspecific drug and steroid clearance by an organic anion transporter of mammalian liver.

An organic anion-transporting polypeptide that mediates sodium-independent uptake of negatively charged sulfobromophthalein and bile salts has recently been cloned from rat liver (Jacquemin et al., 1994). In this study we have extended the substrate specificity studies to neutral and positively charged organic compounds with use of the Xenopus laevis oocyte expression systems. We found that the same transporting polypeptide can also transport the neutral cardiac glycoside ouabain (apparent Km approximately 1.7mM); the endogenous steroids aldosterone (Km approximately 15nM), cortisol (Km approximately 13 microM) and dexamethasone; the anionic steroid-conjugates estrone-3-sulfate (Km approximately 4.5 microM) and estradiol-17-glucuronide (Km approximately 3.0 microM) and the exogenous amphipathic organic cation N-(4,4-azo-n-pentyl)-21-deoxyajmalinium, a permanently charged photolabile derivative of the antiarrhythmic drug N-propylajmaline. These data demonstrate that the previously cloned hepatic organic anion-transporting polypeptide can in fact transport a wide range of differently charged lipophilic organic compounds including exogenous and endogenous organic anions, neutral steroids and organic cations. Hence, a single sinusoidal (or basolateral) transporting polypeptide can account, at least in part, for charge-independent steroid and drug clearance in mammalian liver.

Aldosterone↗

PubMed1995

Mechanism of stimulation of microsomal UDP-glucuronosyltransferase by UDP-N-acetylglucosamine.

We propose the existence in rat liver endoplasmic reticulum (ER) of two asymmetric carrier systems. One system couples UDP-N-acetylglucosamine (UDPGlcNAc) transport to UDP-glucuronic acid (UDPGlcA) transport. When UDPGlcNAc was presented at the cytosolic side of the ER, it then acted as a weak inhibitor of UDPGlcA uptake. By contrast, UDPGlcNAc produced a forceful trans-stimulation of microsomal UDPGlcA uptake when it was present within the lumen of the ER. Likewise, cytosolic UDPGlcA strongly trans-stimulated efflux of intravesicular UDPGlcNAc, whereas cytosolic UDPGlcNAc was ineffective in trans-stimulating efflux of UDPGlcA. A second asymmetric carrier system couples UDPGlcNAc transport to UMP transport. Microsomal UDPGlcNAc influx was markedly stimulated by UMP present inside the microsomes. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP, indicating that UMP exerted its effect on UDPGlcNAc uptake by trans-stimulation from the lumenal side of the ER membrane. Contrariwise, extravesicular UMP only minimally trans-stimulated efflux of intramicrosomal UDPGlcNAc. It is widely accepted that UDPGlcNAc acts as a physiological activator of hepatic glucuronidation, but the mechanism of this effect has remained elusive. Based on our findings, we propose a model in which the interaction of two asymmetric transport pathways, i.e. UDPGlcA influx coupled to UDPGlcNAc efflux and UDPGlcNAc influx coupled to UMP efflux, combined with intravesicular metabolism of UDPGlcA, forms a mechanism that leads to stimulation of glucuronidation by UDPGlcNAc.

Animals↗

PubMed1994

Carrier-mediated transport of intact UDP-glucuronic acid into the lumen of endoplasmic-reticulum-derived vesicles from rat liver.

Uptake and metabolism of UDP-glucuronic acid (UDPGlcA) by rough-endoplasmic-reticulum (RER)-derived vesicles was studied. Analysis of the molecular species, double-labelling experiments and trans-stimulation experiments revealed that initial uptake represented entry into microsomes of predominantly intact UDPGlcA, concomitant with rapid hydrolysis of the internalized nucleotide sugar. The uptake constituted effective translocation from the medium into the lumen of the vesicles. Thus the amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space. Permeabilized microsomes were unable to retain UDPGlcA. The microsomal uptake of UDPGlcA met the criteria of bidirectional carrier-mediated translocation. Transport was time- and temperature-dependent, saturable, selective, capable of trans-stimulation, and operational against a concentration gradient. Microsomal uptake was inhibited by N-ethylmaleimide that was presented at the cytosolic side of the endoplasmic-reticulum (ER) membrane. Uptake studies performed in membrane preparations that were highly enriched in RER, smooth ER or Golgi revealed that UDPGlcA was taken up by the ER as well as by the Golgi apparatus. Our findings demonstrate the existence in rat liver ER of a carrier system mediating proper translocation of intact UDPGlcA across the membrane.

Animals↗

PubMed1994

Functional characterization of carrier-mediated transport of uridine diphosphate N-acetylglucosamine across the endoplasmic reticulum membrane.

Uptake and metabolism of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) by rough-endoplasmic-reticulum (rER)-derived vesicles was studied. Analysis of the molecular species, double-label experiments, cis-inhibition and trans-stimulation experiments revealed that uptake represented entry of intact UDP-GlcNAc into the microsomal lumen. The amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space, and permeabilized microsomes were unable to retain UDP-GlcNAc. These findings indicate that uptake constituted effective translocation from the medium into the lumen of the vesicles. The microsomal uptake of UDP-GlcNAc met the criteria of bidirectional, carrier-mediated translocation. Transport was time and temperature dependent, saturable, selective, capable of trans-stimulation and operational against a concentration gradient. Uptake studies performed in membrane preparations that were highly enriched in either rER, smooth ER, or Golgi revealed that UDP-GlcNAc was taken up by the ER and by the Golgi apparatus.

Animals↗

PubMed1993

Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes.

Activities of nucleotide-sugar:dolichyl phosphate glycosyltransferases (UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl 1-phosphotransferase, UDP-glucose:dolichyl phosphate glucosyltransferase and GDP-mannose:dolichyl phosphate mannosyltransferase) are not fully expressed in native microsomes and can be enhanced by pretreatment of the microsomes with detergent. To examine whether the latency of dolichyl phosphate glycosyltransferases in native microsomes reflects a lumenal orientation of the catalytic centre, we examined the effect of proteinase treatment of native microsomes on enzymic activity and investigated the relationship between enzymic activity and alteration of the permeability of the microsomal membrane barrier. The enzymic activities catalysing transfer of N-acetylglucosamine and glucose to lipid acceptors were proteinase-sensitive in native sealed microsomes. When various detergents were used to disrupt the membrane barrier, we found no relationship between activity of dolichyl phosphate glycosyltransferases and the latency of mannose-6-phosphatase, which is a marker of the permeability properties of the microsomal membrane. Permeabilization of the endoplasmic reticulum membrane by the pore-forming Staphylococcus aureus alpha-toxin did not affect glycosyltransferase activities. These results do not support the hypothesis that latency of the transferase activities is dependent on the permeability properties of the endoplasmic-reticulum membrane. Collectively our findings can best be explained by postulating that the active centres of the transferases are cytoplasmically oriented, while activation by detergent may be conformation-dependent.

Animals↗

PubMed1993

Effect of GTP on the dolichol pathway for protein glycosylation in rat liver microsomes.

Incubation of native rat liver microsomes with GTP resulted in enhanced incorporation of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc into lipid acceptors. The stimulation of GlcNAc transfer by GTP was specific for GTP; ATP exerted no effect. The GTP effect was blocked by a non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate, indicating that GTP hydrolysis was crucial. Though dolichyl pyrophosphate NN'-diacetylchitobiose [Dol-PP-(GlcNAc)2] was the main radiolabelled product formed upon incubation of GTP-treated microsomes with UDP-GlcNAc, GTP selectively stimulated UDP-GlcNAc:dolichyl phosphate (Dol-P) N-acetylglucosaminyl 1-phosphotransferase (N-acetylglucosaminyl 1-phosphotransferase). This conclusion was reached on the basis of experiments in which tunicamycin was used to selectively inhibit N-acetylglucosaminyl 1-phosphotransferase. The enhanced transformation of Dol-P to dolichyl pyrophosphate N-acetylglucosamine (Dol-PP-GlcNAc) by GTP ultimately led to enhanced protein glycosylation. GTP-induced stimulation of GlcNAc incorporation in lipid and protein by GTP was observed also in microsomes fully permeabilized with Staph. aureus alpha-toxin. These findings refute the previous proposal [Godelaine, Beaufay, Wibo and Ravoet (1983) J. Cell Biol. 97, 340-350] that increased membrane permeability constitutes the mechanism whereby GTP activates the reactions of the dolichol pathway.

Acetylglucosamine↗

PubMedDate not supplied

Evaluation of the immune response to pneumococcal capsular polysaccharides.

Antibody deficiency is the most common immunodeficiency. In 5% to 10% of the patients with recurrent infections that are evaluated for immunodeficiency, a specific deficiency in the immune response to capsular polysaccharides can be found. Patients with recurrent infections should therefore be tested for their capacity to produce antibodies against anti-capsular polysaccharides. As a clinical test, specific antibody levels are measured before and 14 days after immunization with the 23-valent pneumococcal polysaccharide vaccine. In this article we describe the indications, the method used to measure antibodies to capsular pneumococcal polysaccharide and the way the results have to be interpreted.

Adult↗