RESUMEN

Amino acids and polyamines are involved in relevant processes for the parasite Trypanosoma cruzi, like protein synthesis, stress resistance, life cycle progression, infection establishment and redox balance, among others. In addition to the biosynthetic routes of amino acids, T. cruzi possesses transport systems that allow the active uptake from the extracellular medium; and in the case of polyamines, the uptake is the unique way to obtain these compounds. The TcAAAP protein family is absent in mammals and its members are responsible for amino acid and derivative uptake, thus the TcAAAP permeases are not only interesting and promising therapeutic targets but could also be used to direct the entry of toxic compounds into the parasite. Although there is a treatment available for Chagas disease, its limited efficacy in the chronic stage of the disease, as well as the side effects reported, highlight the urgent need to develop new therapies. Discovery of new drugs is a slow and cost-consuming process, and even during clinical trials the drugs can fail. In this context, drug repositioning is an interesting and recommended strategy by the World Health Organization since costs and time are significantly reduced. In this article, amino acids and polyamines transport and their potential as therapeutic targets will be revised, including examples of synthetic drugs and drug repurposing.

Asunto(s)

Sistemas de Transporte de Aminoácidos/antagonistas & inhibidores , Proteínas de Transporte de Catión/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Reposicionamiento de Medicamentos , Poliaminas/metabolismo

RESUMEN

Leishmania amazonensis promastigotes are known to express furosemide (Lasix®)-sensitive P-type membrane Na+-ATPase. In the present study, furosemide activity was studied in intracellular amastigotes and infected BALB/c mice to investigate its efficacy in cutaneous leishmaniasis (CL). Intracellular parasites, but not macrophages, were found to be sensitive to killing by furosemide (IC50 = 87 µ m vs CC50 ≫ 1000 µ m, respectively). Although furosemide did not induce nitric oxide production or intracellular pH changes in infected macrophages, it led to a significant reactive oxygen species (ROS) burst. Freshly isolated tissue parasites expressed a high degree of Na+-ATPase activity that decreased with culture, indicative of a higher enzyme expression in amastigotes than in promastigotes. Both intraperitoneal and oral treatment of L. amazonensis-infected mice with furosemide dosages equivalent to that prescribed as a diuretic significantly reduced the parasite's growth compared with the situation in untreated mice. Combination with oral furosemide increased the efficacy and safety of intraperitoneal treatment with sodium stibogluconate (SSG). To summarize, furosemide control of intracellular leishmanial growth by means of parasite Na+-ATPase inhibition, and macrophage ROS activation may help explain its sole and SSG-combined therapeutic effect against murine CL.

Asunto(s)

Furosemida/farmacología , Leishmania/efectos de los fármacos , Tripanocidas/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Diuréticos/farmacología , Femenino , Leishmaniasis Cutánea , Ratones , Ratones Endogámicos BALB C

RESUMEN

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 µM Ca(2+), whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Hígado/enzimología , Proteína Quinasa C/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Western Blotting , Proteínas de Transporte de Catión/antagonistas & inhibidores , Cobre/farmacología , ATPasas Transportadoras de Cobre , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Isoenzimas , Hígado/efectos de los fármacos , Datos de Secuencia Molecular , Naftalenos/farmacología , Ésteres del Forbol/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Pirrolidinonas/farmacología , Homología de Secuencia de Aminoácido , Porcinos

RESUMEN

The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain largely unknown. The present study 1)investigates whether ANG-(3-4) modulates ouabain-resistant Na(+)-ATPase resident in proximal tubule cells and 2) verifies whether its possible action on pumping activity, considered the fine tuner of Na(+) reabsorption in this nephron segment, depends on blood pressure. ANG-(3-4) inhibited Na(+)-ATPase activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar concentrations, with no effect in Wistar-Kyoto (WKY) rats or on Na(+)-K(+)-ATPase. PD123319 (10(-7) M) and PKA(5-24) (10(-6) M), an AT2 receptor (AT2R) antagonist and a specific PKA inhibitor, respectively, abrogated this inhibition, indicating that AT2R and PKA are central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in WKY rats, the peptide (10(-8) M) completely blocked stimulation of Na(+)-ATPase induced by 10(-10) M ANG II in normotensive rats through a mechanism that also involves AT2R and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R heterodimers, which remain associated in 10(-10) M ANG II and dissociate to a very low dimerization state upon addition of 10(-8) M ANG-(3-4). This lower level of heterodimers was that found in SHR, and heterodimers did not dissociate when the same concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg body mass) increased urinary Na(+) concentration and urinary Na(+) excretion with a simultaneous decrease in systolic arterial pressure in SHR, but not in WKY rats. Thus the influence of ANG-(3-4) on Na(+) transport and its hypotensive action depend on receptor association and on blood pressure.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas de Transporte de Catión/antagonistas & inhibidores , Dipéptidos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Bloqueadores del Receptor Tipo 2 de Angiotensina II/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Hipertensión/fisiopatología , Imidazoles/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Ouabaína/farmacología , Piridinas/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor de Angiotensina Tipo 2/efectos de los fármacos , Receptor de Angiotensina Tipo 2/fisiología , Sodio/orina , ATPasa Intercambiadora de Sodio-Potasio/metabolismo

RESUMEN

BACKGROUND: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. METHODS: We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. RESULTS: T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. CONCLUSIONS: Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. GENERAL SIGNIFICANCE: This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.

Asunto(s)

Fosfatos/metabolismo , Rhodnius/parasitología , Sodio/metabolismo , Trypanosoma/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Animales , Transporte Biológico , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Macrólidos/farmacología , Ouabaína/farmacología , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhodnius/metabolismo , Trypanosoma/crecimiento & desarrollo

RESUMEN

There is some evidence to show a possible role of guanosine in the modulation of cellular function, in particular, in the neuronal system. However, nothing is known about the role of guanine in renal function. The aim of the present work was to investigate the role of guanine on modulation of Na+-ATPase activity in isolated basolateral membrane (BLM) of the renal cortex. Guanine inhibited the enzyme activity in a dose-dependent manner with maximal effect (56%) obtained at 10⁻6 M. This effect was reversed by DPCPX (8-cyclopentyl-1,3-dipropylxanthine), an antagonist of A1 receptors, but it was not changed by 10⁻8 M DMPX (3,7-dimethyl-1-propargylxanthine) or 10⁻8 M MRS (2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate), antagonists of A2 and A3 receptors, respectively. Furthermore, it was observed that guanine increased [γ-³5S]GTP-specific binding with the maximal effect observed at 10⁻6 M and this effect was abolished by 10⁻6 M GDPßS. The inhibitory effect of 10⁻6 M guanine on Na+-ATPase activity was reversed by 10⁻6 M GDPßS, 10⁻6 M forskolin, 10⁻6 M pertussis toxin and 10⁻8 M cholera toxin. These results indicate that guanine binds to a DPCPX-sensitive receptor promoting the activation of Gi protein and leading to a decrease in cAMP level and, consequently, inhibition of BLM Na+-ATPase activity.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas de Transporte de Catión/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Guanina/farmacología , Corteza Renal/metabolismo , Antagonistas del Receptor de Adenosina A1/farmacología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Guanina/metabolismo , Técnicas In Vitro , Corteza Renal/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Cinética , Porcinos , Xantinas/farmacología

RESUMEN

Previous data showed that prostaglandin E2 (PGE2) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na+-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE2 on PT Na+-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE2 on PT Na+-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE2 increased [³5S]GTPγS binding; (2) GDPßS abolished the inhibitory effect of PGE2; (3) PGE2 increased PKA activity; (4) the inhibitory effect of PGE2 was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE2 on PT Na+-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE2 stimulates Ca²+-independent phospholipase A2 activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE2 on proximal tubule sodium reabsorption.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinoprostona/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/enzimología , Proteína Quinasa C/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Bradiquinina/farmacología , Proteínas de Transporte de Catión/antagonistas & inhibidores , Dinoprostona/metabolismo , Inhibidores Enzimáticos/farmacología , Retroalimentación Fisiológica/efectos de los fármacos , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Técnicas In Vitro , Túbulos Renales Proximales/metabolismo , Fosfolipasas A2 Calcio-Independiente/metabolismo , Multimerización de Proteína/efectos de los fármacos

RESUMEN

The basolateral membranes of kidney proximal tubule cells have (Na(+)+K(+))-ATPase and Na(+)-ATPase activities, involved in Na(+) reabsorption. We showed that ceramide (Cer) modulates protein kinase A (PKA) and protein kinase C (PKC), which are involved in regulating ion transporters. Here we show that ceramide, promotes 60% inhibition of Na(+)-ATPase activity (I(50) approximately 100nM). This effect was completely reversed by inhibiting PKA but did not involve the classic PKC signaling pathway. In these membranes we found the Cer-activated atypical PKC zeta (PKCzeta) isoform. When PKCzeta is inhibited, Cer ceases to inhibit the Na(+)-ATPase, allowing the cAMP/PKA signaling pathway to recover its stimulatory effect on the pump. There were no effects on the (Na(+)+K(+))-ATPase. These results reveal Cer as a potent physiological modulator of the Na(+)-ATPase, participating in a regulatory network in kidney cells and counteracting the stimulatory effect of PKA via PKCzeta.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas de Transporte de Catión/antagonistas & inhibidores , Ceramidas/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Proteína Quinasa C/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/enzimología , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Porcinos

RESUMEN

We have previously demonstrated that adenosine is deaminated to inosine in the isolated basolateral membrane (BLM) of kidney proximal tubules. This work investigates the possible effect of inosine on proximal tubule Na(+)-ATPase activity. Inosine reduced Na(+)-ATPase activity by 70%. This effect of inosine was completely attenuated by 10(-8) M DPCPX, an A(1) receptor-selective antagonist, but it was not affected by either 10(-8) M DMPX or 10(-7) M MRS1523, A(2) and A(3) receptor-selective antagonists, respectively. The inhibitory effect of inosine was blocked by: (1) 10(-6) M GDPbetaS, a trimeric G protein inhibitor; (2) 1microg/ml pertussis toxin, a Gi protein inhibitor; (3) 10(-6) M forskolin, an adenylyl cyclase activator; (4) 10(-9) M cholera toxin, a Gs protein activator; (5) 10(-6)M cAMP. Our results demonstrate that the inhibitory effect of inosine on the sodium pump is mediated by the A(1) receptor/Gi/cAMP pathway.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , AMP Cíclico/metabolismo , Inosina/farmacología , Túbulos Renales Proximales/enzimología , Receptor de Adenosina A1/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Antagonistas del Receptor de Adenosina A1 , Antagonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A3 , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Receptor de Adenosina A3/metabolismo , Receptores de Adenosina A2/metabolismo , Porcinos , Xantinas/farmacología

RESUMEN

The Arabidopsis thaliana AtHMA1 protein is a member of the P(IB)-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca(2+) homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wild-type yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd(2+) detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca(2+) transport mediated by AtHMA1. Ca(2+) transport displayed high affinity with an apparent K(m) of 370 nm and a V(max) of 1.53 nmol mg(-1) min(-1). This activity was strongly inhibited by thapsigargin (IC(50) = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca(2+)/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Inhibidores Enzimáticos/farmacología , Metales Pesados/metabolismo , Tapsigargina/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Arabidopsis , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Prueba de Complementación Genética , Homeostasis/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Homología de Secuencia de Aminoácido

RESUMEN

We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Inhibidores Enzimáticos/farmacología , Leishmania mexicana/enzimología , Leishmania mexicana/crecimiento & desarrollo , Ouabaína/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/efectos de los fármacos , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Furosemida/farmacología , Humanos , Concentración de Iones de Hidrógeno , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Cutánea Difusa/parasitología , Sodio/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Vanadatos/farmacología

RESUMEN

In the present work, we demonstrate that adenine reduced Na(+)-ATPase activity in isolated basolateral membrane (BLM) of proximal tubule in a dose-dependent manner. Adenine metabolism was ruled out by TLC analysis of the potential [(3)H]adenine derived-metabolites. Specific binding of [(3)H]adenine to isolated BLM was observed in a dose-dependent manner with K(d) and B(max) of 242.6+/-27.6 nM and 2749.9+/-104.9 fmolmg(-1), respectively. Adenine increased the [(35)S]GTPgammaS specific binding and it was completely abolished by 10(-6)M GDPbetaS (G protein inhibitor) but it was not modified by DPCPX, DMPX and MRS1523, selective antagonists for A(1), A(2) and A(3) receptors, respectively. Furthermore, the inhibitory effect of adenine on the Na(+)-ATPase activity was blocked by 10(-6)M GDPbetaS, 1 microg/ml pertussis toxin (Gi protein inhibitor), 10(-6)M foskolin (adenylyl cyclase activator) and 10(-8)M cAMP. These data demonstrate that adenine inhibits the proximal tubule Na(+)-ATPase activity through the Gi protein-coupled receptor.

Asunto(s)

Adenina/administración & dosificación , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Túbulos Renales Proximales/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Túbulos Renales Proximales/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio , Porcinos

RESUMEN

Our aim was to determine the presence of sodium pumps in Entamoeba histolytica. It is shown through the measurement of ouabain-sensitive ATPase activity and immunoblotting that E. histolytica does not express (Na(+)+K(+))ATPase. On the other hand, we observed a Na(+)-ATPase with the following characteristics: (1) stimulated by Na(+) or K(+), but these effects are not addictive; (2) the apparent affinity is similar for Na(+) and K(+) (K(0.5) = 13.3 +/- 3.7 and 15.4 +/- 3.1mM, respectively), as well as the V(max) (24.9 +/- 1.5 or 27.5 +/- 1.6 nmol Pi mg(-1)min(-1), respectively); (3) insensitive up to 2mM ouabain; and (4) inhibited by furosemide with an IC(50) of 0.12 +/- 0.004 mM. Furthermore, this enzyme forms a Na(+)- or K(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Entamoeba histolytica/enzimología , Inhibidores Enzimáticos/farmacología , Ouabaína/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/efectos de los fármacos , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Entamoeba histolytica/efectos de los fármacos , Furosemida/farmacología , Immunoblotting , Corteza Renal/enzimología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos

Asunto(s)

Cardiomegalia/fisiopatología , Proteínas de Transporte de Catión/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Amilorida/farmacología , Amilorida/uso terapéutico , Angiotensina II/fisiología , Animales , Señalización del Calcio , Anhidrasa Carbónica II/fisiología , Cardiomegalia/prevención & control , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/química , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Endotelinas/fisiología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Hormonas/fisiología , Humanos , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/prevención & control , Sistema de Señalización de MAP Quinasas , Ratones , Mitocondrias Cardíacas/efectos de los fármacos , Modelos Cardiovasculares , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Conejos , Ratas , Ratas Endogámicas SHR , Especies Reactivas de Oxígeno , Transducción de Señal , Sodio/metabolismo , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/química , Estrés Mecánico , Porcinos

RESUMEN

As consequence of glomerular filtration the viscosity of blood flowing through the efferent arteriole increases. Recently, we found that shear stress modulates proximal bicarbonate reabsorption and nitric oxide (NO.) was the chemical mediator of this effect. In the present work, we found that agonists of NO. production affected basolateral membrane potential (V (blm)) of the proximal convoluted tubule (PCT) epithelium. Using paired micropuncture experiments, we perfused peritubular capillaries with solutions with different viscosity while registering the V (blm). Our results showed that a 50% increment in the viscosity, or the addition of bradykinin (10(-5) M) to the peritubular perfusion solution, induced a significant and similar hyperpolarization of the V (blm) at the PCT epithelium of 6 +/- 0.7 mV (p < 0.05). Both hyperpolarizations were reverted by L-NAME (10(-4) M). Addition of 2,2'-(hydroxynitrosohydrazino) bis-ethanamine (NOC-18) 3 x 10(-4) M to the peritubular perfusion solution induced a hyperpolarization of the same magnitude of that high viscosity or bradykinin. These results strongly suggest the involvement of NO. in the effect of high viscosity solutions. This effect seems to be mediated by activation of K+(ATP) channels as glybenclamide (5 x 10(-5) M) added to peritubular solutions induced a larger depolarization of the V (blm) with high viscosity solutions. Acetazolamide (5 x 10(-5) M) added to high viscosity solutions induced a larger hyperpolarization (8 +/- 1 mV; p < 0.05), suggesting that depolarizing current due to HCO(-)3 exit across the basolateral membrane damps the hyperpolarizing effect of high viscosity. Considering that Na(+) and consequently water reabsorption is highly dependent on electrical gradient, the present data suggest that the endothelium of kidney vascular bed interacts in paracrine fashion with the epithelia, affecting V (blm) and thus modulating PCT reabsorption.

Asunto(s)

Endotelio Vascular/fisiología , Células Epiteliales/fisiología , Túbulos Renales Proximales/fisiología , Acetazolamida/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Bradiquinina/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Proteínas de Transporte de Catión/antagonistas & inhibidores , Electrofisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Gliburida/farmacología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Compuestos Nitrosos/farmacología , Perfusión , Ratas , Ratas Wistar , Estrés Mecánico

RESUMEN

Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen: iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes.

Asunto(s)

Terapia Genética/métodos , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Absorción Intestinal , Hierro/metabolismo , Proteínas de la Membrana/genética , Adenoviridae/genética , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Vectores Genéticos , Hemocromatosis/terapia , Proteína de la Hemocromatosis , Humanos , Hierro/antagonistas & inhibidores , ARN sin Sentido/uso terapéutico

RESUMEN

The molecular mechanisms involved in the Ang-(1-7) [angiotensin-(1-7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1-7) has a biphasic effect on the proximal tubule Na+-ATPase activity, with the stimulatory effect mediated by the AT1 receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na+-ATPase by Ang-(1-7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT1 receptor-mediated stimulation. In this condition, Ang-(1-7) at 0.1 nM inhibited the Na+-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT2 receptor, and by 1 muM GDP[beta-S] (guanosine 5'-[beta-thio]diphosphate), an inhibitor of G protein. Ang-(1-7) at 0.1 M induced [35S]GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and 1 mug/ml pertussis toxin, an inhibitor of G(i/o) protein, reversed the Ang-(1-7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1-7) on the Na+-ATPase activity was completely reversed by 0.1 microM LY83583, an inhibitor of guanylate cyclase, and by 2 muM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1-7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 microM LY83583. Taken together, these data indicate that Ang-(1-7) inhibits the proximal tubule Na+-ATPase by interaction with the AT2 receptor that subsequently activates the G(i/o) protein/cGMP/PKG pathway.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Angiotensina I/farmacología , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Receptor de Angiotensina Tipo 2/metabolismo , Adenosina Trifosfatasas/metabolismo , Bloqueadores del Receptor Tipo 2 de Angiotensina II , Animales , Proteínas de Transporte de Catión/metabolismo , Corteza Renal/efectos de los fármacos , Corteza Renal/enzimología , Corteza Renal/metabolismo , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/metabolismo , Transducción de Señal/efectos de los fármacos , Porcinos

RESUMEN

Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen; iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes.

Asunto(s)

Humanos , Terapia Genética/métodos , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Absorción Intestinal , Hierro/metabolismo , Proteínas de la Membrana/genética , Adenoviridae/genética , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Vectores Genéticos , Hemocromatosis/terapia , Hierro/antagonistas & inhibidores , ARN sin Sentido/uso terapéutico

RESUMEN

In the present paper we studied the effect of urodilatin and atrial natriuretic peptide (ANP) on the proximal tubule Na+-ATPase and (Na+K+)ATPase activities. Urodilatin and ANP inhibit the Na+-ATPase activity but not the (Na+K+)ATPase activity. Maximal effect was observed at a concentration of 10(-11) M for both peptides. In this condition, the enzyme activity decreases from 10.8 +/- 1.6 (control) to 5.7 +/- 0.9 or 6.1 +/- 0.7 nmol Pi mg(-1) min(-1) in the presence of urodilatin or ANP, respectively. This effect was completely reversed by 10(-6) M LY83583, a guanylyl cyclase inhibitor, and mimicked by 10 nM cGMP. Furthermore, both ANP and urodilatin increase cGMP production by 33% and 49%, respectively. This is the first demonstration that it was shown that urodilatin and ANP directly modulate primary active sodium transport in the proximal tubule. The data obtained indicate that this effect is mediated by the activation of the NPR-A/guanylate cyclase/cGMP pathway.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Factor Natriurético Atrial/farmacología , Proteínas de Transporte de Catión/metabolismo , Túbulos Renales/enzimología , Fragmentos de Péptidos/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , GMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Epitelio/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Túbulos Renales/efectos de los fármacos , Ouabaína/farmacología , Receptores del Factor Natriurético Atrial/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos

RESUMEN

Despite important advances in the understanding of copper secretion and excretion, the molecular components of intestinal copper absorption remain a mystery. DMT1, also known as Nramp2 and DCT1, is the transporter responsible for intestinal iron uptake. Electrophysiological evidence suggests that DMT1 can also be a copper transporter. Thus we examined the potential role of DMT1 as a copper transporter in intestinal Caco-2 cells. Treatment of cells with a DMT1 antisense oligonucleotide resulted in 80 and 48% inhibition of iron and copper uptake, respectively. Cells incorporated considerable amounts of copper as Cu(1+), whereas Cu(2+) transport was about 10-fold lower. Cu(1+) inhibited apical Fe(2+) transport. Fe(2+), but not Fe(3+), effectively inhibited Cu(1+) uptake. The iron content of the cells influenced both copper and iron uptake. Cells with low iron content transported fourfold more iron and threefold more copper than cells with high iron content. These results demonstrate that DMT1 is a physiologically relevant Cu(1+) transporter in intestinal cells, indicating that intestinal absorption of copper and iron are intertwined.

Asunto(s)

Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Proteínas de Unión a Hierro/metabolismo , Animales , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Células CACO-2 , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Humanos , Mucosa Intestinal/citología , Hierro/metabolismo , Proteínas de Unión a Hierro/antagonistas & inhibidores , Proteínas de Unión a Hierro/genética , Oligonucleótidos Antisentido/metabolismo