RESUMO
Diamidine drugs such as pentamidine and berenil (diminazene aceturate) are vital drugs for the treatment of early stage human African trypanosomiasis and the corresponding veterinary condition, respectively. The action of diamidines on trypanosomes is critically dependent on their efficient uptake by the parasite. We have therefore investigated the mode of uptake of pentamidine by Trypanosoma brucei brucei, using [(125)I]iodopentamidine as a permeant. [(125)I]Iodopentamidine uptake was linear for up to 15 min and inhibited by adenosine with a K(i) value of 0.64+/-0.03 microM to a maximum of 50-70%. The adenosine-sensitive flux was also inhibited by adenine with a K(i) value of 0.44+/-0.04 microM. Iodopentamidine uptake was saturable, with the adenosine-insensitive flux displaying a K(m) of 22+/-2 microM and a V(max) of 2.2+/-0.9 pmol(10(7) cells)(-1)s(-1), whereas the adenosine-sensitive flux was inhibited by much lower iodopentamidine concentrations. These results clearly demonstrate that iodopentamidine is taken up by at least two different T. b. brucei transporters, an adenosine-sensitive pentamidine transporter (ASPT1) and a low-affinity pentamidine transporter (LAPT1). The identity of these transporters was investigated, and their significance for drug uptake and resistance in African trypanosomes is discussed.
Assuntos
Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Pentamidina/metabolismo , Tripanossomicidas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Transporte Biológico , Resistência a Medicamentos , Proteínas de Transporte de Nucleosídeos , Pentamidina/farmacologia , Ratos , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
Vitamin C is essential for many enzymatic reactions and also acts as a free radical scavenger. Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, L-ascorbic acid, across biological membranes. Dehydroascorbic acid uptake is via the facilitated-diffusion glucose transporters, GLUT 1, 3 and 4, but under physiological conditions these transporters are unlikely to play a major role in the uptake of vitamin C due to the high concentrations of glucose that will effectively block influx. L-ascorbic acid enters cells via Na+-dependent systems, and two isoforms of these transporters (SVCT1 and SVCT2) have recently been cloned from humans and rats. Transport by both isoforms is stereospecific, with a pH optimum of approximately 7.5 and a Na+:ascorbic acid stoichiometry of 2:1. SVCT2 may exhibit a higher affinity for ascorbic acid than SVCT1 but with a lower maximum velocity. SVCT1 and SVCT2 are predicted to have 12 transmembrane domains, but they share no structural homology with other Na+ co-transporters. Potential sites for phosphorylation by protein kinase C exist on the cytoplasmic surface of both proteins, with an additional protein kinase A site in SVCT1. The two isoforms also differ in their tissue distribution: SVCT1 is present in epithelial tissues, whereas SVCT2 is present in most tissues with the exception of lung and skeletal muscle.
Assuntos
Ácido Ascórbico/metabolismo , Simportadores , Sequência de Aminoácidos , Animais , Transporte Biológico , Células COS , Cátions , Membrana Celular/metabolismo , Clonagem Molecular , Ácido Desidroascórbico/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Pulmão/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Fosforilação , Filogenia , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Sódio/metabolismo , Transportadores de Sódio Acoplados à Vitamina C , Fatores de Tempo , Distribuição Tecidual , XenopusRESUMO
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/metabolismoAssuntos
Melarsoprol/farmacologia , Purinas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Antiprotozoários/farmacologia , Arsenicais/farmacologia , Transporte Biológico Ativo , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Camundongos , Camundongos Endogâmicos BALB C , Repetições Minissatélites , Trypanosoma brucei brucei/genéticaRESUMO
Adenosine influx by Trypanosoma brucei brucei P1 and P2 transporters was kinetically characterized. The P1 transporter displayed a higher affinity and capacity for adenosine (K(m) = 0.38 +/- 0.10 microM, V(max) = 2.8 +/- 0.4 pmol x 10(7) cells(-1) x s(-1)) than the P2 transporter (K(m) = 0.92 +/- 0.06 microM, V(max) = 1.12 +/- 0.08 4 pmol x 10(7) cells(-1) x s(-1)). To formulate a structure-activity relationship for the interaction of adenosine with the transporters, a series of analogs were evaluated as potential inhibitors of adenosine transport, and the K(i) values were converted to binding energy. The P1 transporter was found to be selective inhibited by purine nucleosides (K(i) approximately 1 microM for inosine and guanosine), but nucleobases and pyrimidines had little effect on P1-mediated transport. The P1 transporter appears to form hydrogen bonds with N3 and N7 of the purine ring as well as with the 3' and 5' hydroxyl groups of the ribose moiety, with apparent bond energies of 12.8 to 15.8 kJ/mol. The P2 transporter, in contrast, had high-affinity (K(i) = 0.2-4 microM) for 6-aminopurines, including adenine, 2'-deoxyadenosine, and tubercidin, but not for any oxopurines. The main interaction of adenosine with the P2 transporter is suggested to be via hydrogen bonds to N1 and the 6-amino group. Additional pi-pi interactions of the purine ring and electrostatic interactions with N9 may also be important. The predicted substrate recognition motif of P2, but not of P1, corresponds to parts of the melaminophenylarsenical and diamidine molecules, confirming the potent inhibition observed with these trypanocides for P2-mediated adenosine transport (K(i) = 0.4-2.4 microM).
Assuntos
Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Nucleares/metabolismo , Proteínas de Protozoários , Receptores Purinérgicos P1/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Pentamidina/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
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
Adenosine transport at 22 degrees C in procyclic forms of Trypanosoma brucei brucei was investigated using an oil-inhibitor stop procedure for determining initial rates of adenosine uptake in suspended cells. Adenosine influx was mediated by a single high affinity transporter (Km 0.26 +/- 0.02 microM, Vmax 0.63 +/- 0.18 pmol/10(7) cells s-1). Purine nucleosides, with the exception of tubercidin (7-deazaadenosine), and dipyridamole inhibited adenosine influx (Ki 0.18-5.2 microM). Purine nucleobases and pyrimidine nucleosides and nucleobases had no effect on adenosine transport. This specificity of the transporter appears to be similar to the previously described P1 adenosine transporter in bloodstream forms of trypanosomes. Uptake of adenosine was Na+-independent, but ionophores reducing the membrane potential and/or the transmembrane proton gradient (monitored with the fluorescent probes bis-(1,3-diethylthiobarbituric acid)-trimethine oxonol and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, respectively) inhibited adenosine transport. Similarly, an increase in extracellular pH from 7.3 to 8.0 reduced adenosine influx by 30%. A linear correlation was demonstrated between the rate of adenosine transport and the protonmotive force. Adenosine uptake was accompanied by a proton influx in base-loaded cells and was also shown to be electrogenic. These combined results indicate that transport of adenosine in T. brucei brucei procyclics is protonmotive force-driven and strongly suggest that the adenosine transporter functions as an H+ symporter.
Assuntos
Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Protozoários/metabolismo , Nucleosídeos de Purina/farmacologia , Trypanosoma brucei brucei/fisiologia , Trifosfato de Adenosina/metabolismo , Amilorida/farmacologia , Animais , Ligação Competitiva , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Proteínas de Transporte/isolamento & purificação , Dicicloexilcarbodi-Imida/farmacologia , Corantes Fluorescentes , Gramicidina/farmacologia , Concentração de Íons de Hidrogênio , Inosina/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Nigericina/farmacologia , Proteínas de Protozoários/isolamento & purificação , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tubercidina/farmacologiaRESUMO
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
The inward transport of two purines, adenosine and hypoxanthine, at 37 degrees C by horse erythrocytes was compared. No mediated transport of adenosine was detected in horse erythrocytes, nor was saturable, high-affinity binding of the potent facilitated-diffusion inhibitor nitrobenzylthioinosine demonstrable in horse erythrocyte membranes. In contrast, erythrocytes from most horses possessed a saturable sodium-dependent hypoxanthine transporter (apparent K(m), 100 +/- 28 microM; Vmax, 0.20 +/- 0.08 mmol (l cells)-1 h-1; means +/- S.E.M., n = 5). Guanine inhibited hypoxanthine influx (apparent Ki, 24 +/- 6 microM), but adenine and xanthine had no effect. Unlike human erythrocytes, no sodium-independent hypoxanthine transporter was detected in horse erythrocytes. There are, however, a small number of animals (approximately 15%) whose erythrocytes fail to transport hypoxanthine. This variation appears to be under genetic control, but the precise nature of the control is unknown.
Assuntos
Adenosina/sangue , Eritrócitos/metabolismo , Hipoxantina/sangue , Proteínas de Membrana Transportadoras , Animais , Proteínas de Transporte/sangue , Membrana Eritrocítica/metabolismo , Cavalos , Cinética , Ligação Proteica , Sódio/sangue , Tioinosina/análogos & derivados , Tioinosina/sangueRESUMO
The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.
Assuntos
Proteínas de Transporte/química , Nucleosídeos , Trypanosoma brucei brucei/química , Uracila/metabolismo , Animais , Relação Dose-Resposta a Droga , Cinética , Fatores de Tempo , Uracila/antagonistas & inibidoresRESUMO
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 mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22 degrees C was rapid and saturable, exhibiting an apparent Km of 9.3 +/- 2.0 microM and a Vmax of 4.5 +/- 0.8 pmol x (10(7) cells)(-1) x s(-1). All the natural purine nucleobases tested (Ki 1.8-7.2 microM), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2',7'-bis-(carboxyethyl)-5,6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi), the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH 7 after base-loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electrogenic. These data show that hypoxanthine uptake in T. b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.
Assuntos
Proteínas de Transporte/metabolismo , Hipoxantina/metabolismo , Purinas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Concentração de Íons de Hidrogênio , Cinética , Potenciais da Membrana , Concentração Osmolar , Prótons , Especificidade por Substrato , Trypanosoma brucei brucei/citologiaAssuntos
Hipoxantina/metabolismo , Purinas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Etilmaleimida/farmacologia , Cinética , Monensin/farmacologia , Purinas/farmacologia , Pirimidinas/farmacologiaAssuntos
Proteínas de Transporte/biossíntese , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras , Sódio/metabolismo , Uracila/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Feminino , Hipoxantina/metabolismo , Jejuno/metabolismo , Mamíferos , Oócitos/fisiologia , Reação em Cadeia da Polimerase , Coelhos , Transcrição Gênica , Xenopus laevisRESUMO
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
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 transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.
Assuntos
Proteínas de Transporte/efeitos dos fármacos , Endotélio Vascular/metabolismo , Hipoxantinas/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Tioinosina/análogos & derivados , Adenosina/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Linhagem Celular , Endotélio Vascular/enzimologia , Humanos , Hipoxantina , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Nucleosídeos , Tioinosina/farmacologiaRESUMO
1. In mammals, nucleoside transport is an important determinant of the pharmacokinetics, plasma and tissue concentration, disposition and in vivo biological activity of adenosine as well as nucleoside analogues used in antiviral and anticancer therapies. 2. Two broad types of adenosine transporter exist, facilitated-diffusion carriers and active processes driven by the transmembrane sodium gradient. 3. Facilitated-diffusion adenosine carriers may be sensitive (es) or insensitive (ei) to nanomolar concentrations of the transport inhibitor nitrobenzylthioinosine (NBMPR). Dipyridamole, dilazep and lidoflazine analogues are also more potent inhibitors of the es carrier than the ei transporter in cells other than those derived from rat tissues. 4. The es transporter has a broad substrate specificity (apparent Km for adenosine approximately 25 microM in many cells at 25 degrees C), is a glycoprotein with an average apparent Mr of 57,000 in human erythrocytes that has been purified to near homogeneity and may exist in situ as a dimer. However, there is increasing evidence to suggest the presence of isoforms of the es transporter in different cells and species, based on kinetic and molecular properties. 5. The ei transporter also has a broad substrate specificity with a lower affinity for some nucleoside permeants than the es carrier, is genetically distinct from es but little information exists as to the molecular properties of the protein. 6. Sodium-dependent adenosine transport is present in many cell types and catalysed by four distinct systems, N1-N4, distinguished by substrate specificity, sodium coupling and tissue distribution. 7. Two genes have been identified which encode sodium-dependent adenosine transport proteins, SNST1 from the sodium/glucose cotransporter (SGLT1) gene family and the rat intestinal N2 transporter (cNT1) from a novel gene family including a bacterial nucleoside carrier (NupC). Transcripts of cNT1, which encodes a 648-residue protein, are found in intestine and kidney only. 8. Success in cloning the remaining adenosine transporter genes will improve our understanding of the diversity of nucleoside transport processes, with a view to better targeting of therapeutic nucleoside analogues and protective use of transport inhibitors.
Assuntos
Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Animais , Humanos , Proteínas de Transporte de Nucleosídeos , Ratos , Sódio/fisiologiaRESUMO
The subcellular distributions of the mammalian passive glucose transporter isoforms GLUT1, GLUT3 and GLUT4, in the human placenta, were investigated using isoform-specific anti-peptide antibodies. On western blots of both basal and brush-border plasma membranes isolated from the syncytiotrophoblast, antibodies specific for GLUT1 labelled a broad band (apparent Mr 55,000) that co-migrated with the human erythrocyte GLUT1 glucose transporter. In contrast, no labelling was detectable when blots were probed with antibodies specific for the GLUT3 or GLUT4 isoforms. Densitometric analysis of blots showed that GLUT1 accounts for approximately 90 and 65 per cent of the D-glucose-sensitive cytochalasin B binding sites present in brush-border and basal membranes, respectively. Confocal immunofluorescence microscopy of fixed placental tissue showed that GLUT1 is abundant at both maternal- and fetal-facing surfaces of the syncytiotrophoblast whereas it was undetectable at the fetal capillary endothelium. In parallel experiments, no staining by antibodies against either the GLUT3 or the GLUT4 isoforms was detected in placental tissue. These results indicate that GLUT1 is the major isoform responsible for glucose transfer from mother to fetus. The absence of GLUT4 is consistent with the lack of insulin-sensitive glucose transport across the placenta.