RESUMEN
BACKGROUND: The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes. METHODS: We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-(32)P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation. RESULTS: This activity was linear with time up to 20min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5mM MgCl(2) was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN(3)) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN(3). The dose-response of ATP revealed a hyperbolic profile with maximal velocity of 25.2±1.2nmol Pixmg(-1)xmin(-1) and K(0.5) of 0.07±0.01mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60min of ischemia. CONCLUSION: Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia. GENERAL SIGNIFICANCE: This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Isquemia/fisiopatología , Túbulos Renales Proximales/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Células Cultivadas , Concentración de Iones de Hidrógeno , Hidrólisis , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Cinética , L-Lactato Deshidrogenasa/metabolismo , Células LLC-PK1 , Suramina/farmacología , PorcinosRESUMEN
We compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated alpha-L-iduronic acid and 4-O-sulfated N-acetyl-beta-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated alpha-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-beta-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.
Asunto(s)
Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Dermatán Sulfato/farmacología , Hemorragia/inducido químicamente , Trombosis/tratamiento farmacológico , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/química , Bovinos , Dermatán Sulfato/administración & dosificación , Dermatán Sulfato/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Femenino , Cofactor II de Heparina/metabolismo , Masculino , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Especificidad de la Especie , Trombina/antagonistas & inhibidores , Trombina/metabolismo , Trombosis/prevención & control , UrocordadosRESUMEN
The antithrombotic activity of a fucosylated chondroitin sulfate extracted from the body wall of a sea cucumber was assessed using a stasis thrombosis model in rats. Intravenous administration of the polysaccharide reduced thrombosis in a dose-dependent manner. We also compared the antithrombotic action of the sea cucumber chondroitin sulfate with that of standard mammalian glycosaminoglycans, mainly heparin and dermatan sulfate. Intravascular injection of fucosylated chondroitin sulfate at the dose totally preventing thrombus formation produced a much more intense modification of the plasma anticoagulant activity than antithrombotic doses of unfractionated heparin, low-molecular-weight heparin and mammalian dermatan sulfate. Thus, it is possible that the mechanism of antithrombotic action of these polysaccharides are different. For fucosylated chondroitin sulfate, it depends mostly on modifications of the plasma anticoagulant activity, but it may involve additional effects in the case of mammalian glycosaminoglycans, perhaps modifications induced in the cells of the vessel wall. The anticoagulant and possibly the antithrombotic actions of fucosylated chondroitin sulfate are mostly dependent on heparin cofactor II activity, and both are markedly reduced with the decrease of the chain size of the polymer. Overall, the sulfated polysaccharide from the invertebrate revealed an unequivocal effect in preventing experimental venous thrombosis, is a useful tool to investigate the antithrombotic action in mammals and may offer an alternative for future development of a new therapeutic agent.