Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Sistemas de Transporte de Aminoácidos , Aminoácidos/metabolismo , Animais , Transporte Biológico , Glucose/metabolismo , Humanos , Insulina/fisiologia , Cinética , Proteínas de Membrana/metabolismo , Estrutura Molecular , Proteínas de Transporte de Monossacarídeos/ultraestrutura , Proteínas de Transporte de Nucleosídeos , Nucleosídeos/metabolismo , Purinas/metabolismoRESUMO
The characteristics of uridine transport were studied in rabbit intestinal brush-border membrane vesicles. Uridine was taken up into an osmotically active space in the absence of metabolism and there was no binding of uridine to the membrane vesicles. Uridine uptake was markedly enhanced by sodium, but showed no significant stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of uridine flux indicated a single system obeying Michaelis-Menten kinetics (Km value of 6.4 +/- 1.4 microM with a Vmax of 9.1 +/- 3.6 pmol/mg protein per s as measured under zero-trans conditions with a 100 mM NaCl gradient at 24 degrees C). A variety of purine and pyrimidine nucleosides were able to inhibit sodium-dependent uridine transport, suggesting that these nucleosides are also permeants for the same system. Consistent with this suggestion was the finding that these nucleosides also stimulated uridine efflux from the brush-border membrane vesicles. The sodium: uridine coupling stoichiometry was found to be 1:1 as measured by the activation method. From these results it is concluded that a broad specificity sodium-dependent nucleoside transporter is present at the brush-border membrane surface of rabbit enterocytes.
Assuntos
Absorção Intestinal , Mucosa Intestinal/metabolismo , Uridina/metabolismo , Animais , Transporte Biológico , Cátions/farmacologia , Sistema Livre de Células , Técnicas In Vitro , Cinética , Microvilosidades/metabolismo , Nucleosídeos/farmacologia , Concentração Osmolar , Coelhos , Sódio/farmacologiaRESUMO
The effects of hydrostatic pressure (0.1-50 MPa) on uridine transport mediated by the 'simple' facilitated nucleoside transporter of guinea-pig and human erythrocytes have been studied in an attempt to identify the volume changes which occur during transport. Pressure inhibited the zero-trans (influx or efflux) mode of uridine transport in guinea-pig cells significantly more (about 2.2- x) than equilibrium exchange. The equilibrium binding of 3H-nitrobenzylthioinosine, a potent specific inhibitor of nucleoside transport, to human red cells and ghosts, was not significantly altered by pressure suggesting that the permeation site was unperturbed. Thus pressure inhibited the transporter primarily by preventing the volume increase associated with the translocation step. Furthermore, the return of the 'empty' transporter was found to be rate-limiting because it required a larger increase in volume than when the transporter was loaded with substrate.
Assuntos
Proteínas de Transporte/metabolismo , Volume de Eritrócitos/fisiologia , Proteínas de Membrana/metabolismo , Uridina/metabolismo , Animais , Transporte Biológico , Cobaias , Humanos , Pressão Hidrostática , Técnicas In Vitro , Cinética , Proteínas de Transporte de NucleosídeosRESUMO
The involvement of protein kinase C (PKC) in the regulation of Na(+)-dependent and -independent hypoxanthine transport was investigated by exposing confluent monolayers of LLC-PK1 renal epithelia cells to the PKC activator, phorbol 12-myristate 13-acetate (PMA). Chronic exposure (> 2 h) of LLC-PK1 monolayers to 16 nM PMA resulted in approximately 75% inhibition of Na(+)-dependent hypoxanthine influx occurring maximally at 8 h and persisting for 72 h. In contrast, PMA had little effect on Na(+)-independent hypoxanthine influx at 8 h, but longer exposure resulted in stimulation of influx (approximately 3-fold) that peaked at 24 h and thereafter declined to control levels at 72 h. The effects of PMA were dose-dependent and were associated with changes in Vmax of transport (2-4-fold) with no significant change in apparent K(m). 4 alpha-Phorbol, a phorbol ester that does not activate PKC, had no effect on hypoxanthine transport by LLC-PK1 cells. The diacylglycerol kinase inhibitor, R59022 (10 microM), partially inhibited (28%) Na(+)-dependent hypoxanthine influx. In addition, the PMA-induced effects on hypoxanthine transport were reversed by Ro-31-8220 (1 and 5 microM) and calphostin C (50 nM), potent and selective inhibitors of PKC. The increase in Na(+)-independent hypoxanthine influx following exposure to PMA was blocked by the protein synthesis inhibitor, cycloheximide (20 microM), and correlated with an increase in LLC-PK1 cell proliferation. The PMA-induced decrease in Na(+)-dependent hypoxanthine transport was independent of PMA effects on cell proliferation and not dependent on protein synthesis. These results are consistent with the proposal that the PMA-induced effects on hypoxanthine transport are due to PKC activation.
Assuntos
Hipoxantina/metabolismo , Proteína Quinase C/metabolismo , Animais , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Cinética , Células LLC-PK1 , Sódio/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Timidina/metabolismoRESUMO
The characteristics of Na(+)-dependent and Na(+)-independent uridine uptake at 22 degrees C were determined for monolayers of OK renal epithelial cells. The majority of uridine influx in subconfluent to early confluent (day 1 postconfluency) OK monolayers was mediated via a facilitated-diffusion pathway (apparent Km 160 +/- 41 microM, Vmax 610 +/- 100 pmol/mg protein per min). This system was inhibited with high affinity by nitrobenzylthioinosine (NBMPR) (IC50 value 1.5 nM) and by purine and pyrimidine nucleosides. Specific [3H]NBMPR binding sites were detected in OK monolayers (apparent Kd 0.67 +/- 0.25 nM, Bmax 90 +/- 19 fmol/mg protein) yielding a turnover number for the carrier of 112 uridine molecules/site per s at 22 degrees C. Na(+)-dependent uridine uptake was minor in subconfluent OK monolayers, but increased 8-fold with time after confluency reaching a stable plateau at 8 days postconfluency. Inhibition of Na(+)-dependent 1 microM uridine uptake by inosine, guanosine, adenosine and uridine was biphasic with approx. 40% of the total uptake inhibited with high affinity (IC50 value 2 to 14 microM). Concentrations of thymidine and cytidine up to 1 mM had no effect on Na(+)-dependent uridine uptake and no Na(+)-dependent thymidine influx by confluent OK monolayers was detected. Using cell monolayers grown on a permeable filter support, Na(+)-dependent uridine uptake occurred preferentially from the apical surface. This high affinity component of Na(+)-dependent uridine uptake is suggested to represent the Na(+)-dependent purine preferring N1 nucleoside transporter. The Na+/uridine stoichiometry for this system was consistent with 1:1. The remaining component of Na(+)-dependent uridine uptake was inhibited by some nucleosides, such as guanosine and inosine, with low affinity (IC50 values of 0.6 to 5 mM). Other nucleosides showed little specific inhibition. We propose that this component of uridine uptake represents a mutated carrier that binds nucleosides but is defective in the translocation of permeant.
Assuntos
Túbulos Renais Proximais/metabolismo , Sódio/farmacologia , Uridina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Senescência Celular , Epitélio/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Cinética , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Nucleosídeos , Gambás , Nucleosídeos de Purina/metabolismo , Sódio/química , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Uridina/químicaRESUMO
Radiation inactivation analysis of the mazindol-sensitive binding of the dopamine transporter inhibitor, [3H]GBR-12935 to canine striatal membranes yielded a radiation inactivation target size of 278 +/- 16 kDa. This result, in conjunction with other findings in the literature demonstrating that the molecular mass of the dopamine transporter is approximately 70 kDa, suggests that the native form of the dopamine carrier in the neuronal membranes is a tetrameric assembly of identical 70 kDa subunits.
Assuntos
Proteínas de Transporte/química , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Piperazinas/farmacologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Corpo Estriado/metabolismo , Cães , Proteínas da Membrana Plasmática de Transporte de Dopamina , Peso Molecular , Piperazinas/metabolismo , Ligação ProteicaRESUMO
The characteristics of hypoxanthine transport were examined in opossum kidney (OK) epithelial cells and Xenopus laevis oocytes. In both cell types hypoxanthine influx was mediated by two distinct transport systems: a high-affinity Na+-dependent system and a Na+-independent transporter. Na+-dependent hypoxanthine transport in OK cells was saturable (Km 0.78+/-0.29 microM) and was inhibited by guanine, uracil, thymine and 5-fluorouracil (Ki values 0.5-7 microM), whereas adenine had no effect. Substitutions at the 2- and 4-position had a marked effect on the ability of uracil to inhibit Na+/hypoxanthine influx by OK cells revealing that an oxo group at both the 2- and 4-positions of uracil is required for interacting with the transporter. The properties of Na+-dependent hypoxanthine influx in oocytes were similar to those observed in OK cells. In particular, xanthine and oxypurinol inhibited hypoxanthine influx, a characteristic not observed previously for the Na+/nucleobase carrier in pig LLC-PK1 renal cells. Na+-independent hypoxanthine influx in OK cells and oocytes was of a lower affinity (Km 90-180 microM). Adenine and guanine inhibited Na+-independent hypoxanthine flux in OK cells, but had no effect in oocytes. Injection of LLC-PK1 mRNA into oocytes resulted in a 1.5-fold stimulation of Na+/hypoxanthine flux over water-injected oocytes. These results reveal further heterogeneity in Na+/nucleobase cotransporters.
Assuntos
Proteínas de Transporte/biossíntese , Hipoxantina/metabolismo , Rim/metabolismo , Oócitos/metabolismo , Uracila/análogos & derivados , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/genética , Células Epiteliais/metabolismo , Microinjeções , Gambás , RNA Mensageiro/biossíntese , RNA Mensageiro/farmacologia , Cloreto de Sódio/farmacologia , Especificidade por Substrato , Uracila/farmacologia , Xenopus laevisRESUMO
The transport of uridine into rat renal brush-border membrane vesicles was investigated using an inhibitor-stop filtration method. Uridine was not metabolized under these conditions. The rapid efflux of intravesicular uridine was prevented by adding 1 mM phloridzin to the ice-cold stop solution. In the presence of inwardly directed gradients of either Na+ or K+, zero-trans uridine uptake exhibited a transient overshoot phenomenon indicating active transport. The overshoot was much more pronounced with Na+ than K+ and it was not observed when either Na+ or K+ was at equilibrium across the membrane. The K+-induced overshoot was not due to the presence of a membrane potential alone, as an inwardly directed gradient of choline chloride failed to produce it. The amplitude of the overshoot was increased by raising either the Na+ or K+ concentration outside the membrane or by using more lipophilic anions (reactive order was NO3- greater than SCN- greater than Cl- greater than SO4(2-). Zero-trans efflux studies showed that the uridine transport is bidirectional. Li+ could substitute poorly for Na+ but not at all for K+. Stoichiometries of 1:1 and greater than 1:1 were observed for Na+: uridine and K+: uridine coupling, respectively. A preliminary analysis of the interactions between Na+ and K+ for uridine uptake showed complex interactions which can best be explained by the involvement of two different systems for nucleoside transport in the rat renal brush-border membrane, one requiring Na+ and the other K+ as transport coupler.
Assuntos
Córtex Renal/metabolismo , Uridina/metabolismo , Animais , Ânions/farmacologia , Transporte Biológico/efeitos dos fármacos , Técnicas In Vitro , Túbulos Renais Proximais/metabolismo , Cinética , Microvilosidades/metabolismo , Potássio/fisiologia , Ratos , Sódio/fisiologiaRESUMO
The human erythrocyte nucleoside and glucose transporters, identified previously as band 4.5 peptides (apparent Mr 66 000-45 000) on SDS-polyacrylamide gels, have been characterized in situ by radiation inactivation analysis. Target size analysis of lyophilized membranes indicates an apparent Mr of 110 000 +/- 12 000 and 124 000 +/- 11 000 for the nucleoside and glucose carriers, respectively. These data suggest that both transporters exist in the membrane as dimers.
Assuntos
Proteínas Sanguíneas/efeitos da radiação , Proteínas de Transporte/efeitos da radiação , Membrana Eritrocítica/efeitos da radiação , Proteínas de Membrana/efeitos da radiação , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Proteínas Sanguíneas/metabolismo , Relação Dose-Resposta à Radiação , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/sangue , Proteínas de Transporte de Nucleosídeos , Aceleradores de PartículasRESUMO
The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.
Assuntos
Córtex Renal/metabolismo , Medula Renal/metabolismo , Microvilosidades/metabolismo , Nucleosídeos/metabolismo , Uridina/metabolismo , Marcadores de Afinidade , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Dilazep/farmacologia , Epitélio/metabolismo , Cinética , Microvilosidades/efeitos dos fármacos , Nucleosídeos/farmacologia , Suínos , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Tioinosina/farmacologiaRESUMO
Pig erythrocytes are unable to metabolize glucose and their physiological energy source is unknown. These cells have a high-capacity nucleoside transport system with similar properties to that responsible for nucleoside transport in other species. Nucleoside transport is sufficiently rapid to allow the possibility that inosine and/or adenosine may represent major energy substrates for pig erythrocytes in vivo. Normal and adenosine deaminase-deficient pig erythrocytes have similar ATP levels, suggesting that adenosine is not important in this respect. However, it was calculated that an extracellular inosine concentration of only 40 nM could support the cells' entire energy requirement, a value 40-fold lower than plasma levels of this nucleoside.
Assuntos
Eritrócitos/metabolismo , Inosina/sangue , Adenosina Desaminase/deficiência , Trifosfato de Adenosina/sangue , Animais , Transporte Biológico/efeitos dos fármacos , Dipiridamol/farmacologia , Metabolismo Energético , Eritrócitos/efeitos dos fármacos , Cinética , Suínos , Tioinosina/análogos & derivados , Tioinosina/farmacologiaRESUMO
The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.
Assuntos
Proteínas de Transporte/isolamento & purificação , Eritrócitos/análise , Proteínas de Membrana/isolamento & purificação , Marcadores de Afinidade , Animais , Proteínas de Transporte/metabolismo , Cromatografia DEAE-Celulose , Eletroforese em Gel de Poliacrilamida , Glucosídeos , Guanosina/análogos & derivados , Guanosina/farmacologia , Proteínas de Membrana/metabolismo , Peso Molecular , Proteínas de Transporte de Nucleosídeos , Fotoquímica , Solubilidade , Suínos , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Tionucleosídeos/farmacologia , Uridina/metabolismoRESUMO
Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.
Assuntos
Membrana Eritrocítica/metabolismo , Inosina/análogos & derivados , Tioinosina/análogos & derivados , Uridina/sangue , Transporte Biológico , Relação Dose-Resposta à Radiação , Membrana Eritrocítica/efeitos da radiação , Congelamento , Humanos , Cinética , Tioinosina/sangueRESUMO
Gating of the yeast K+ channel encoded by the Saccharomyces cerevisiae gene TOK1, unlike other outward-rectifying K+ channels that have been cloned, is promoted by membrane voltage (inside positive-going) and repressed by extracellular K+. When expressed in Xenopus laevis oocytes, the TOK1p current rectified strongly outward, its activation shifting in parallel with the K+ equilibrium potential when the external K+ concentration ([K+]o) was increased above 3 mM. Analysis of the TOK1p current indicated that two kinetic components contributed to the conductance and the voltage sensitivity of the conductance. By contrast, the [K+]o sensitivity of the current was accommodated entirely within the slow-relaxing component; it was diminished near 1 mM [K+]o, and at submillimolar concentrations the voltage dependence of the TOK1p conductance was insensitive to [K+]o. External Rb+, the K+ channel blockers Cs+ and Ba2+--but not Na+, Ca2+ or Mg2+--substituted for K+ in control of TOK1p activation, indicating a specificity in cation interaction with the TOK1p gate. These and additional results indicate that external K+ acts as a ligand to inactivate the TOK1p channel, and they implicate a gating process mediated by a single cation binding site within the membrane electric field, but distinct from the permeation pathway.
Assuntos
Bário/farmacologia , Canais de Potássio/fisiologia , Potássio/farmacologia , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas Fúngicas/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Potenciais da Membrana , Oócitos , Saccharomyces cerevisiae , Xenopus laevisRESUMO
Nitrobenzylthioniosine (NBMPR), a potent and specific inhibitor of nucleoside transport, is bound reversibly by high affinity sites on nucleoside transporter proteins of erythrocyte membranes and, upon photoactivation, NBMPR molecules become covalently bonded to the sites. This study showed that [3H]NBMPR molecules reversibly bound to intact S49 and L5178Y mouse lymphoma cells became covalently bound upon exposure to UV light. Electrophoretic analysis of plasma membrane fractions from the labelled cells showed that 3H was present in polypeptides which migrated as a major band with an apparent Mr of 45000-65000.
Assuntos
Marcadores de Afinidade/metabolismo , Proteínas Sanguíneas/metabolismo , Inosina/análogos & derivados , Linfoma/metabolismo , Proteínas de Membrana/metabolismo , Tioinosina/análogos & derivados , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Membrana Eritrocítica/metabolismo , Leucemia L5178/metabolismo , Camundongos , Proteínas de Transporte de Nucleosídeos , Fotoquímica , Tioinosina/metabolismo , Raios UltravioletaRESUMO
The mechanism and inhibitor sensitivity of hypoxanthine transport by bloodstream forms of Trypanosoma brucei brucei was investigated. The dose response curve for the inhibition of hypoxanthine transport (1 microM) by guanosine was biphasic; approximately 90% of transport activity was inhibited with a Ki value of 10.8 +/- 1.8 microM, but 10% of the activity remained insensitive to concentrations as high as 2 mM. These two components of hypoxanthine transport are defined as guanosine-sensitive (H2) and guanosine-insensitive (H3). Hypoxanthine influx by both components was saturable, but there was a marked difference in their Km values (123 +/- 15 nM and 4.7 +/- 0.9 microM for H2 and H3, respectively) although the Vmax values (1.1 +/- 0.2 and 1.1 +/- 0.1 pmol (10[7] cells)[-1] s[-1], n = 3) were similar. Hypoxanthine uptake via the H2 carrier was inhibited by purine bases and analogues as well as by some pyrimidine bases and one nucleoside (guanosine), whereas the H3 transporter was sensitive only to inhibition by purine nucleobases. H2-mediated hypoxanthine uptake was inhibited by ionophores, ion exchangers and the potential H+-ATPase inhibitors, N,N'-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM). Measurements of the intracellular pH and membrane potential of bloodstream trypanosomes in the presence and absence of these agents established a linear correlation between protonmotive force and rate of [3H]hypoxanthine (30 nM) uptake. We conclude that hypoxanthine transport in bloodstream forms of T. b. brucei occurs by two transport systems with different affinities and substrate specificities, one of which, H2, appears to function as a H+-/hypoxanthine symporter.
Assuntos
Proteínas de Transporte/metabolismo , Hipoxantina/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Transporte Biológico , Inibidores Enzimáticos/farmacologia , Guanosina/farmacologia , Concentração de Íons de Hidrogênio , Hipoxantina/farmacologia , Ionóforos/farmacologia , Cinética , Potenciais da Membrana , Força Próton-Motriz , ATPases Translocadoras de Prótons/antagonistas & inibidores , Especificidade por SubstratoRESUMO
The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.
Assuntos
Proteínas de Transporte/metabolismo , Crithidia fasciculata/metabolismo , Proteínas de Membrana/metabolismo , Trypanosoma brucei brucei/metabolismo , Adenosina/antagonistas & inibidores , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Crithidia fasciculata/crescimento & desenvolvimento , Meios de Cultura , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Guanosina/farmacologia , Hipoxantina/metabolismo , Inosina/antagonistas & inibidores , Inosina/metabolismo , Proteínas de Transporte de Nucleosídeos , Nucleosídeos de Purina/metabolismo , Purinas/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Uracila/metabolismoRESUMO
1. The major toxicity associated with oral therapy with ribavirin is anaemia, which has been postulated to occur as a result of accumulation of ribavirin triphosphate interfering with erythrocyte respiration. The objective of this study was to determine the mechanism by which ribavirin enters into erythrocytes. 2. Entry into human erythrocytes was examined by measuring influx rates of [3H]-ribavirin alone and with the inhibitor nitrobenzylthioinosine (NBMPR), and by investigating the inhibitory effects of nucleoside and nucleobase permeants on ribavirin transport, by use of inhibitor oil-stop methods. Transport mechanisms were further characterized by assessment of substrates to cause countertransport of ribavirin in preloaded erythrocytes, and by measuring the effects of ribavirin on [3H]-NBMPR binding to erythrocyte membranes. 3. Human erythrocytes had a saturable influx mechanism for ribavirin (Km at 22 degrees C of 440+/-100 microM) which was inhibited by nanomolar concentrations of NBMPR (IC50 0.99+/-0.15 nM). Nucleosides also inhibited the influx of ribavirin (adenosine more effective than uridine) but the nucleobases hypoxanthine and adenine had no effect. In addition, uridine caused the countertransport of ribavirin in human erythrocytes. Entry of ribavirin into horse erythrocytes, a cell type that lacks the NBMPR-sensitive (es) nucleoside transporter, proceeded slowly and via a pathway that was resistant to NBMPR inhibition. Ribavirin was a competitive inhibitor of adenosine influx (mean Ki 0.48+/-0.14 mM) and also inhibited NBMPR binding to erythrocyte membranes (mean Ki 2.2+/-0.39 mM). 4. These data indicate that ribavirin is a transported permeant for the es nucleoside transporter of human erythrocytes. There was no evidence for ribavirin entering cells via a nucleobase transporter.
Assuntos
Antivirais/sangue , Proteínas de Transporte/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo , Eritrócitos/metabolismo , Proteínas de Membrana/metabolismo , Ribavirina/sangue , Adenosina/metabolismo , Animais , Membrana Eritrocítica/metabolismo , Cavalos , Humanos , Técnicas In Vitro , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Uridina/sangueRESUMO
Adenosine influx and formycin B influx and efflux were characterized in guinea-pig ventricular myocytes at 22 degrees. Transport by both modes was saturable and inhibited by nitrobenzylthioinosine (NBMPR), indicating the presence of an equilibrative NBMPR-sensitive nucleoside transporter in the cardiomyocytes. The kinetic constants for influx and efflux of formycin B, a non-metabolized nucleoside, were similar, suggesting that the nucleoside transporter exhibits symmetrical kinetics (apparent Km 490 +/- 160 and 700 +/- 140 microM; Vmax 6.5 +/- 1.7 and 3.5 +/- 0.3 nmol/10(6) cells per min for influx and efflux, respectively). No evidence was found of either NBMPR-insensitive equilibrative nucleoside transport or sodium-dependent concentrative nucleoside transport. Inhibition of adenosine influx (apparent Km100 +/- 33 microM), by lidoflazine and the analogues mioflazine, soluflazine and R73-335, gave average Ki values of 730, 100, 64 and 2.9 nM, respectively. These compounds also inhibited formycin B efflux with a similar potency to that of adenosine influx. NBMPR-sensitive nucleoside transport was associated with high affinity binding of NBMPR (apparent Kd approximately 1 nM; 9.6 x 10(5) sites/cell). Specific binding of NBMPR was also inhibited by lidoflazine and its analogues. Mioflazine and soluflazine were 20-30-fold more potent at inhibiting NBMPR-sensitive nucleoside influx in guinea-pig erythrocytes than ventricular myocytes, indicating that the potency of some of the compounds studied is tissue dependent.
Assuntos
Adenosina/metabolismo , Lidoflazina/análogos & derivados , Miocárdio/metabolismo , Marcadores de Afinidade , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Formicinas/metabolismo , Cobaias , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Técnicas In Vitro , Cinética , Lidoflazina/farmacologia , Tioinosina/análogos & derivados , Tioinosina/farmacologiaRESUMO
Human erythrocytes were shown to possess a saturable uptake mechanism for 2-chloroadenosine (apparent Km 23 microM, 22 degrees). Uptake by this route was inhibited by nitrobenzylthioinosine, uridine and adenosine, but adenine had no effect. In addition, uridine caused the countertransport of 2-chloroadenosine and vice versa. 2-Chloroadenosine was also shown to be an apparent competitive inhibitor of uridine influx (apparent Ki value of 33 microM) and high-affinity nitrobenzylthioinosine binding (apparent Ki 0.18 mM). The apparent Ki value for inhibition of uridine influx was close to the apparent Km value for 2-chloroadenosine uptake. Previous studies [Jarvis et al., Biochem. J. 208, 83 (1982)] have demonstrated that dog erythrocytes do not possess a saturable transport system for uridine and adenosine. Similarly, in the present study, the entry of 2-chloroadenosine into dog erythrocytes was slow and linear with concentration. Nitrobenzylthioinosine (NBMPR) had no effect on the uptake of 2-chloroadenosine into dog erythrocytes. These results demonstrate that 2-chloroadenosine enters human erythrocytes by the same nucleoside carrier as other nucleosides. It is suggested from these data that the previous explanation that the inability of nucleoside transport inhibitors to potentiate the pharmacological effects of 2-chloroadenosine was due to the failure of the nucleoside carrier to accept 2-chloroadenosine as a permeant may have to be reassessed.