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Ouabain-insensitive Na+-ATPase activity of Malpighian tubules from Rhodnius prolixus
Abstract
In the present paper, we show the existence of a furosemide-sensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in cell lysates of Malpighian tubular cells from Rhodnius prolixus, which could be the biochemical expression of the Na(+)-pump. The main characteristics of this activity are: (1) K0.5 for Na+ = 1.49 +/- 0.18 mM, (2) Vmax = 2.8 +/- 0.1 nmol inorganic orthophosphate (Pi).mg prot-1.min-1, (3) it is fully abolished by 2 mM furosemide, (4)it is insensitive to ouabain concentrations up to 10(-2) M, (5) it is sensitive to the presence of vanadate in the incubation medium indicating it to be a P-type ATPase, and (6) it is stimulated by nanomolar concentrations of Ca2+ in the incubation medium.
... Besides the (Na + + K + )ATPase, another sodium pump was found in Malpighian tubule of Rhodnius prolixus (Caruso-Neves et al. 1998b). This Na + -stimulated ATPase activity has the following characteristics: 1) K 0.5 for Na + = 1.49 ...
... This Na + -ATPase has been described in several cell types (Proverbio et al. 1989, Moretti et al. 1991, Caruso-Neves et al. 1997, 1998b, Rangel et al. 1999). It was initially described in aged microsomal fractions from guinea-pig kidney cortex as an active Na + transporter not stimulated by K + (Proverbio et al. 1989). ...
... It was initially described in aged microsomal fractions from guinea-pig kidney cortex as an active Na + transporter not stimulated by K + (Proverbio et al. 1989). This pump has same distribution that (Na + + K + )ATPase, and is only found in the plasma membrane (Proverbio et al. 1989, Caruso-Neves et al. 1997, 1998b, Rangel et al. 1999. The Na + -ATPase of the Malpighian tubule cells from Rhodnius prolixus is inhibited by KCl in a dose-dependent manner with maximal effect observed at 5 mM ( Figure 1). ...
Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+)ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+)ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+)ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+)ATPase activity. Taken together these data indicate that (Na+ + K+)ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.
... Besides the well-known (Na + + K + )ATPase, a second sodium pump, the Na + -ATPase, has been described [1][2][3][4][5]. This enzyme is a transport ATPase that catalyzes the transduction of chemical energy from ATP into ionic gradients across biological membranes [6,7]. ...
... This Na + transport against electrochemical gradient is coupled to the ATP hydrolysis and occurs in the presence of 7 mM ouabain and in the absence of K + as shown in BLM vesicles isolated from murine renal proximal tubules [7]. Several studies have shown biochemical differences between the classical (Na + + K + )ATPase and the Na + -ATPase, including the sensitivity to SDS and trypsin [2,4,10,11]. Recently, it was shown that the Na + -ATPase has a molecular weight of about 100 kD and is a P-type enzyme, i.e is able to form phosphorylated intermediate during its catalytic cycle, where phosphate is bound to an aspartyl residue at the enzyme's substrate site [1]. ...
... Recently, it was shown that the Na + -ATPase has a molecular weight of about 100 kD and is a P-type enzyme, i.e is able to form phosphorylated intermediate during its catalytic cycle, where phosphate is bound to an aspartyl residue at the enzyme's substrate site [1]. The expression of this enzyme was demonstrated in different tissues of several species and its widespread and parallel distribution with the (Na + + K + )ATPase has been verified [2][3][4][5][6][12][13][14][15]. ...
In addition to the (Na(+)+K(+))ATPase another P-ATPase, the ouabain-insensitive Na(+)-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg(2+), MgATP and furosemide on the Na(+)-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na(+)-ATPase at pH 7.0 are: (a) K(0.5) for Na(+)=8.9+/-2.2mM; (b) K(0.5) for MgATP=1.8+/-0.4mM; (c) two sites for free Mg(2+): one stimulatory (K(0.5)=0.20+/-0.06 mM) and other inhibitory (I(0.5)=1.1+/-0.4 mM); and (d) I(0.5) for furosemide=1.1+/-0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na(+) (K(0.5)=19.5+/-0.4) and MgATP (K(0.5)=3.4+/-0.3 mM) but increases the apparent affinity for furosemide (0.18+/-0.02 mM) and Mg(2+) (0.05+/-0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na(+) (K(0.5)=18.7+/-1.5 mM) and furosemide (I(0.5)=3.04+/-0.57 mM) but does not change the apparent affinity to MgATP and Mg(2+). The data presented in this paper indicate that the modulation of the Na(+)-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg(2+) and MgATP complex may play an important role in the Na(+)-ATPase physiological regulatory mechanisms.
... Some biochemical characteristics led some authors to postulate that Na þ-ATPase belongs to the P-ATPase family [1,10]. P-type ATPases comprise an important family of homologous enzymes that have at least two main conformational states: E 1 and E 2 [11]. ...
... The Na þ concentration that promoted half-maximal stimulation (K 0.5 ) of Na þ-ATPase was 7.2 AE 1.4 and 7.5 AE 1.5 mM, and the maximal rate (V max ) was 4.4 AE 0.2 and 23.12 AE 1.1 nmol Pi/mg per min in homogenate and the microsomal fraction, respectively. The K 0.5 for Na þ is in agreement with that found for Na þ-ATPase in other organisms [1,2] and it is different from that observed in dog and sheep kidney outer medulla (Na þ þ K þ )ATPase, measured in the absence of K þ (34e35.5 mM) [22]. ...
... Previous data indicate that ouabain-insensitive Na þ-ATPase belongs to the P-type ATPase family [1,10]. One characteristic of P-ATPases is their sensitivity to vanadate, which blocks the phosphorylation of these enzymes by ATP, impairing the formation of the acylphosphate E-P [1,11]. ...
We show that MDCK I cells express, besides the classical (Na(+)+K(+))ATPase, a Na(+)-stimulated ATPase activity with the following characteristics: (1) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 23.12+/-1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, respectively). This enzyme forms a Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na(+)+K(+))ATPase with monoclonal anti-alpha(1) antibody reduced its activity in the supernatant by 90%; the Na(+)-ATPase activity was completely maintained. In addition, the formation of the Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na(+)-ATPase and (Na(+)+K(+))ATPase activities are independent, with Na(+)-ATPase belonging to a different enzyme entity.
... La bumetanida a mayores concentraciones no mostró un efecto mayor (datos no mostrados). Estos dos últimos compuestos también inhiben la segunda bomba de Na + , cuya actividad ha sido reportada en homogenatos de UMT 10,51 . ...
... y Bumetanida (50 ?M) inhibió la secreción de fluido en un 82% (p?0,03). Como ya se mencionó, además de inhibir al cotransportador Na + -K + -2Cl -, estos dos últimos compuestos también inhiben la segunda bomba de Na + , cuya actividad ha sido reportada en homogenatos de UMT 10,51 . DIOA (10?M) inhibió Jv en un 96% (p?0,001), en contraste agudo con su acción en el baño. ...
RESUMEN. Se midió la tasa de secreción y los cambios en la concentración interna de sodio y protones de los túbulos de malpighi (UMT) en Rhodnius prolixus bajo estimulación con 5-OH-Triptamina. Para medir secreción utilizamos un sistema de doble perfusión para tener acceso por separado a las membranas basolateral y/o apical de la célula. Los flujos iónicos se midieron por microfluorometría. Se aplicaron hasta trece agentes farmacológicos: Ouabaina, Bafilomicina A1, Furosamida, Bumetanida, DIOA, Probenecina, SITS, Acetazolamida, Amilorida, DPC, BaCl 2, pCMBS y DTT. Estos agentes son bloqueadores conocidos de diferentes funciones de transporte de iones, conocidos como ATPasas, co-y/o contratransportadores asi como de canales iónicos y de agua. La asunción básica es que los cambios en los movimientos de agua reflejan los cambios en los mecanismos del transporte de iones que se localizan de la siguiente manera: (i) en la membrana basolateral de la célula, los principales son un cotransportador de Na +-K +-2Cl – y el intercambiador de Cl-HCO-3 ; la ATPasa de Na +-K + y la ATPasa de Na + y los canales de Cl-y de agua (Rp-MIP) son de importancia intermedia, mientras que los canales de K + son menos importantes: (ii) en la membrana celular apical, la mayor importancia la tiene un cotransportador de K +-Cl-, que ha sido localizado por primera vez, una ATPasa de H + tipo V, y un intercambiador de Na +-H + ; el intercambiador urato-anión y los canales de K + son importantes mientras que los canales de Cl-no lo son. En este trabajo presentamos un modelo actualizado que explica la secreción del UMT. Palabras claves: Secreción en insectos-Transporte transepitelial de iones, Transporte de iones en insectos, Secreción de fluidos. ABSTRACT. We have measured fluid secretion rate and intracelular concentration of sodium and protons, in Rhodnius prolixus UMT stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. The ionic fluxes has been measured using microfluorimetry. 13 pharmacological agents were applied: Ouabain, Bafilomycin A 1 , Furosemide, Bumetanide, DIOA, Probenecid, SITS, Acetazolamide, Amiloride, DPC, BaCl 2 , pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co-and/or counter-transporters and ionic and water channels. The basic assumption is that water movement changes reflect changes on ion transport mechanisms which we localize as follows: (i) At the basolateral cell membrane: a Na +-K +-2Cl-cotransport; a Cl-HCO 3-exchange; the Na +-K +-ATPase, Cl-channels and Rp-MIP water channels are fundamental; K + channels play a lesser role. (ii) At the apical cell membrane, a K +-Cl-cotransport that is located for the first time; a V-H +-ATPase; an Na + – H + exchange; an urate-anion exchange and K + channels are important, while Cl-channels are not. The present experiments allow us to build a tentative model for the function of the UMT cell, which includes a paracellular pathway for fluid flow. In this work we propose a new actualized model to explain the UMT secretion.
... The ouabaininsensitive N a+-ATPase activity was initially de scribed in aged microsomal fractions from guineapig kidney cortex and was so-called the second so dium pump (Proverbio et al., 1989). Later, the presence of this enzyme was demonstrated in plasma membranes from several different tissues of different species (M oretti et al., 1991;Caruso-Neves et al., 1998b). The Na+-ATPase transports N a+ against an electrochemical gradient and is not stimulated by K+ (Proverbio et al., 1989). ...
... It is known that the Na+-ATPase is inhibited specifically by furosemide (Proverbio et al., 1989;Caruso-Neves et al., 1998a;1998b). Fig. 2 shows the effect of furosemide on the Na+-stimulated ATPase activity in the presence of 2 m M ouabain. The increase in furosemide concentration from 0.1 to 2.0 mM completely inhibited the Na+-stimulated ATPase activity in a dose-dependent manner, with maximal effect observed in the presence of 2 m M furosemide in both cell types. ...
In the present paper, the presence of a ouabain-insensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in T. cruzi epimastigotes CL14 clone and Y strain was investigated. The increase in Na+ concentration (from 5 to 170 mM), in the presence of 2 mM ouabain, increases the ATPase activity in a saturable manner along a rectangular hyperbola. The Vmax was 18.0 +/- 1.0 and 21.1 +/- 1.1 nmoles Pi x mg-1 x min-1 and the half-activation value (K50) for Na+ was 34.3 +/- 5.8 mM and 37.7 +/- 5.3 in CL14 clone and in Y strain, respectively. The Na(+)-stimulated ATPase activity was inhibited by 5-[aminosulfonyl]-4-chloro-2-[(2-furanylmethyl)-amino] benzoic acid (furosemide) in a dose-dependent manner. The half-inhibition value (I50) was 0.22 +/- 0.03 and 0.24 +/- 0.07 mM, and the Hill number (n) was 0.99 +/- 0.2 and 2.16 +/- 0.29 for CL14 clone and Y strain, respectively. These data indicate that both cell types express the ouabain-insensitive Na(+)-ATPase activity, which might be considered the biochemical expression of the second Na+ pump.
... However, the presence of ouabain in the incubation medium was able to prevent 14 C-MeAIB uptake in hepatopancreas at 24 h of hyperosmotic stress, whereas it did not prevent 14 C-MeAIB uptake in jaw muscle at the same time (24 h). These findings suggest that there is another ouabain-insensitive type of Na + -ATPase in C. granulata jaw muscle and hepatopancreas, as was found in Malpighian tubular cells from Rhodnius (Caruso-Neves et al., 1998). In this insect, Caruso-Neves et al. (1998) found a furosemide-sensitive Na + -ATPase and suggested its involvement in cell volume regulation as found in other animal tissues (Moretti et al., 1991;Proverbio et al., 1988). ...
... These findings suggest that there is another ouabain-insensitive type of Na + -ATPase in C. granulata jaw muscle and hepatopancreas, as was found in Malpighian tubular cells from Rhodnius (Caruso-Neves et al., 1998). In this insect, Caruso-Neves et al. (1998) found a furosemide-sensitive Na + -ATPase and suggested its involvement in cell volume regulation as found in other animal tissues (Moretti et al., 1991;Proverbio et al., 1988). Nevertheless, in C. granulata gills, Castilho et al. (2001) demonstrated that the Na + -K + ATPase was blocked by 70% with 1 M of ouabain. ...
We investigated the transport of (14)C-methylaminoisobutyric acid ((14)C-MeAIB) and (14)C-alanine oxidation in hepatopancreas and jaw muscle of Chasmagnathus granulata submitted to 24, 72, and 144 h of hypo- or hyperosmotic stress. While (14)C-MeAIB uptake increased in jaw muscle and hepatopancreas from crabs submitted to hyperosmotic stress, it did not change in tissues from animals submitted to hypo-osmotic stress. Incubation of jaw muscle and hepatopancreas from control groups with 1 mM ouabain did not decrease (14)C-MeAIB uptake. However, ouabain prevented (14)C-MeAIB uptake in hepatopancreas at 24 h of hyperosmotic stress. In contrast, in jaw muscle from crabs submitted to the same conditions, (14)C-MeAIB uptake was not prevented by ouabain in the incubation medium. Jaw muscle from the control group produced four times more (14)CO(2) from (14)C-alanine than the hepatopancreas. During hypo-osmotic stress, amino acid oxidation does not seem to be one of the pathways implicated in the decrease of the amino acid pools in hepatopancreas and jaw muscle. In contrast, during hyperosmotic stress the reduction in (14)C-alanine oxidation appears to be one of the mechanisms involved in the increase of the amino acid pool in the hepatopancreas.
... A bomba de Ca 2+ possui sua atividade dependente de Ca 2+ e estimulada por calmodulina, e in vitro, na ausência de calmodulina, é estimulada por tratamentos como a exposição à fosfolipídeos (incluindo os derivados fosforilados do fosfatidil-inositol), por tratamento controlado com proteases (tripsina ou calpaína) e por fosforilação por proteína kinase A ou C (Carafoli, 1997). têm sido descritas, principalmente em parasitos como Tripanosoma cruzi (Caruso Neves et al., 1998; Iizumi et al., 2006), Entamoeba histolytica (De Souza et al., 2007) e Leishmania amazonensis (De Almeida-Amaral et al., 2008). Esta ATPase é importante para parasitos pois bombeia Na + para fora da célula como uma alternativa a Na + -K + -ATPase, permitindo a adaptação ao ambiente do hospedeiro que muitas vezes possui concentrações de Na + de cerca de 135–146 mmol.L -1 (Iizumi et al., 2006; De Almeida-Amaral et al., 2008). ...
Miosina pode ser precipitada pelo congelamento e descongelamento de fração
solúvel de diferentes tecidos de mamíferos. Nesse trabalho, o objetivo foi
precipitar ATPase similar à miosina a partir de fração solúvel de larva de P.
nucleorum. A fração solúvel foi congelada a -20 oC e descongelada após 48
horas. O precipitado foi recuperado por centrifugação a 45.000 g x 40 minutos e
foi lavado sucessivamente com solução tampão em pH 7,5 e 9,0. Posteriormente,
ATPase foi solubilizada pelo tratamento do precipitado com solução tampão pH
9,0 contendo 0,5 mol.L-1 de NaCl. Os principais polipeptídeos da fração ATPase
apresentaram Mr de 205 e 45 kDa. Essa fração expressou alta atividade CaATPase, mas não apresentou atividade Mg2+-ATPase e nem K+/EDTA-ATPase. A
atividade Ca2+-ATPase não foi inibida por tapsigargina 140 µ mmol.L-1, ouabaína
1,7 mmol.L-1 ou azida 1 mmol.L-1. A atividade foi sensivelmente inibida por
magnésio, mas praticamente não foi inibida por cobre ou zinco. A enzima não
hidrolisou AMP e nem PPi e hidrolisou muito pouco ADP e GTP. Nesse trabalho,
foi isolado um polipeptídeo de 205 kDa de larva de P. nucleorum, que co-purificou
com atividade Ca2+-ATPase.
... Spontaneous hydrolysis of [γ 32 P]ATP was measured simultaneously by adding protein to some tubes after the addition of activated charcoal. The Na + / K + ATPase activity was calculated as the difference between [ 32 Pi] released in the absence and in the presence of 1 mM ouabain [34]. ...
Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2) generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.
... This basolateral active Na + -transport, coupled to the passive Na + entry through apical co-transporters, energizes the absorption of osmotically active solutes followed by water. This process generates isosmotic absorption of sodium, solutes and water [24, 25]; Trypanosoma cruzi epimastigotes [21, 145]; cultured MDCK I cells [39]; Entamoeba histolytica [38]; Leshmania amazonensis [36]; and pig kidney [79] . Recently, the Na + - ATPase activity has been reported in homogenates of several rat tissues [136]. ...
Transepithelial Na(+) transport is mediated by passive Na(+) entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na(+)/K(+) pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na(+)/K(+)-ATPase and the ouabain-insensitive, furosemide-inhibitable Na(+)-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na(+)-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na(+)- and Na(+)/K(+)-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na(+)-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na(+)-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na(+)-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na(+) transport and cellular Na(+) homeostasis leads us to reconsider its role in health and disease.
... The second Na + pump is another ATPase which participates in Na + transport across the membrane; it is ouabain insensitive but sensitive to bumetanide and furosemide (Whittembury and Proverbio, 1970;Proverbio et al., 1975;Caruso-Neves et al., 1998;del Castillo and Robinson, 1982). It has been isolated and cloned (Rocafull et al., 2011). ...
... Spontaneous hydrolysis of [c 32 P]ATP was measured simultaneously by adding protein to some tubes after the addition of activated charcoal. The (Na + + K + )ATPase activity was calculated as the difference between [ 32 P] inorganic phosphate ([ 32 P]Pi) released in the absence, and in the presence, of 1 mM ouabain (Caruso-Neves et al., 1998). ...
In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na(+)+K(+))ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH(3) and U73122, specific inhibitors of PI-PLC. (Na(+)+K(+))ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50nM. This effect was completely reversed by 10nM calphostin C, an inhibitor of PKC. Thus, the effect of 50nM heme on (Na(+)+K(+))ATPase activity is completely abolished by ET-18-OCH(3) and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na(+)+K(+))ATPase activity through a PI-PLC/PKC signaling pathway.
... There is a prominent expression of Na + ,K + -ATPase in renal proximal tubule cells which provides the energy gradient that supplies the transcellular Na + reabsorption in this segment of the nephron. In the past decades, a second Na + -ATPase, which is insensitive to ouabain and sensitive to furosemide, was described in several animal tissues (23)(24)(25)(26)(27)(28). This Na + -ATPase transports Na + against an electrochemical gradient and is not stimulated by K + . ...
For several years it was believed that angiotensin II (Ang II) alone mediated the effects of the renin-angiotensin system. However, it has been observed that other peptides of this system, such as angiotensin-(1-7) (Ang-(1-7)), present biological activity. The effect of Ang II and Ang-(1-7) on renal sodium excretion has been associated, at least in part, with modulation of proximal tubule sodium reabsorption. In the present review, we discuss the evidence for the involvement of Na+-ATPase, called the second sodium pump, as a target for the actions of these compounds in the regulation of proximal tubule sodium reabsorption.
... Therefore, to discard the possibility that the ATP hydrolysis stimulated by MgCl 2 was due to phosphatase or other type of ATPases with internal ATP binding sites, different inhibitors for those enzymes were tested. Table 1 shows that the Mg 2+-dependent ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase (Meyer-Fernandes et al., 1997); bafilomycin A 1 , a V-ATPase inhibitor (Browman et al., 1988); ouabain, a Na + + K +-ATPase inhibitor (Caruso-Neves et al., 1998a); furosemide, a Na +-ATPase inhibitor (Caruso-Neves et al., 1998b) and vanadate, a potent inhibitor of P-ATPases and acid phosphatases ( Fernandes et al., 1997;Dutra et al., 1998;Meyer-Fernandes et al., 1999). However, molybdate, which is a phosphatase inhibitor ( Dutra et al., 1998), and vanadate inhibited the Mg 2+-independent ecto-ATPase, as well as the phosphatase activities ( Table 1), suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. ...
In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl(2) to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46mM MgCl(2). The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2), but not by SrCl(2). The Mg(2+)-dependent ATPase presents two apparent K(m) values for Mg-ATP(2-) (K(m1)=0.03 mM and K(m2)=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg(2+)-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg(2+)-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg(2+)-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2',2'-disulfonic acid (DIDS) as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50mM D-galactose. Since previous results showed that D-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg(2+)-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.
... The Mg 2+ -stimulated cryptococcal ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg 2+ -ATPases [42]. As demonstrated in Table 1, similar results were observed when ATPase activity was determined in the presence of bafilomycin A 1 (V-ATPase inhibitor [52]), ouabain (Na + /K + -ATPase inhibitor [53]), furosemide (Na + -ATPase inhibitor [54]), sodium vanadate (P-ATPase inhibitor [55]), inorganic phosphate (Pi, the product of the ATP hydrolysis reaction), dipyridamole (a nucleoside transporter antagonist [56]), and concanavalin A (activator of some ecto-ATPases [57,58]). ...
Cryptococcus neoformans is the causative agent of pulmonary cryptococcosis and cryptococcal meningoencephalitis, which are major clinical manifestations in immunosuppressed patients. In the present study, a surface ATPase (ecto-ATPase) was identified in C. neoformans yeast cells. Intact yeasts hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 29.36+/-3.36nmol Pi/hx10(8) cells. In the presence of 5 mM MgCl(2), this activity was enhanced around 70 times, and an apparent K(m) for Mg-ATP corresponding to 0.61mM was determined. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases, V-ATPases, Na(+)-ATPases or P-ATPases had no effect on the cryptococcal ATPase, but extracellular impermeant compounds reduced enzyme activity in living cells. ATP was the best substrate for the cryptococcal ecto-enzyme, but it also efficiently hydrolyzed inosine 5'-triphosphate (ITP), cytidine 5'-triphosphate (CTP), guanosine 5'-triphosphate (GTP) and uridine-5'-triphosphate (UTP). In the presence of ATP, C. neoformans became less susceptible to the antifungal action of fluconazole. Our results are indicative of the occurrence of a C. neoformans ecto-ATPase that may have a role in fungal physiology.
... Data are means § SE of three determinations with diVerent cell suspensions. 1988); ouabain, a Na + +K + -ATPase inhibitor (Caruso-Neves et al., 1998a); furosemide, a Na + -ATPase inhibitor (Caruso-Neves et al., 1998b); sodium Xuoride and ammonium molybdate, two potent inhibitors of acid phosphatase activity (Dutra et al., 2000) and sodium orthovanadate, a potent inhibitor of P-ATPases and acid phosphatases (Dutra et al., 2001;Meyer-Fernandes et al., 1999). Levamizole, an inhibitor of alkaline phosphatase (Van Belle, 1976), and dipyridamole, a nucleoside transporter antagonist (Lemmens et al., 1996) also failed to inhibit the ATPase activity (Table 1), as well as p-nitrophenylphosphate (p-NPP), -naphtylphosphate andglycerophosphate, substrates for phosphatase activities and 5ЈAMP, a substrate for 5Ј-nucleotidase. ...
In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.
... During this period of interaction with fungi, epithelial cells were not damaged (data not shown). Sodium orthovanadate can affect different biological processes (Klarlund, 1985) and inhibit ATPases involved in cation transport (Caruso-Neves et al., 1998;Sodre et al., 2000). However, its major biological activity in living cells is over the cell surface, since the oxidation -reduction reactions that take place in the cytoplasm diminish its inhibitory effect (Cantley & Aisen, 1979). ...
There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.
... activity (Table 1 ), indicated that this enzyme did not contribute to the observed ATP hydrolysis. The Mg-dependent ATPase activity was insensitive to oligomycin, one inhibitor of mitochondrial Mg-ATPase (Meyer-Fernandes et al., 1997); baWlomycin, a V-ATPase inhibitor (Bowman et al., 1988); ouabain, a Na + /K + -ATPase inhibitor (Caruso-Neves et al., 1998a); furosemide, a Na + -ATPase inhibitor (Caruso Neves et al., 1998b); as well as to vanadate, which is a potent inhibitor of P-ATPases (Sodre et al., 2000 ). Dipyridamole , a nucleoside transporter antagonist (Lemmens et al., 1996) also failed to inhibit the ATPase activity (Table 1). ...
In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.
... Previous data showed that ouabain-insensitive Na +ATPase belongs to the P-type ATPase family (CarusoNeves et al., 1998b;Lizumi et al., 2006). One characteristic of the P-ATPases is their sensitivity to sodium orthovanadate, which blocks the phosphorylation of these enzymes by ATP, impairing the formation of the acylphosphate phosphorylated intermediate (Caruso-Neves et al., 1998b;Moller et al., 1996).Fig. 3bshows that the Na +-ATPase activity decreased in a dose-dependent manner in the presence of sodium orthovanadate (0.01?1 lM). ...
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.
... The Mg 2+ -dependent ATPase activity was insensitive to oligomycin (10.0 lg/mL) and sodium azide (10.0 mM), two mitochondrial Mg-ATPase inhibitors (Meyer-Fernandes et al., 1997). The same lack of response was observed to bafilomycin A 1 (10.0 lM), a V-ATPase inhibitor (Bowman et al., 1988), ouabain (1.0 mM), a Na + / K + -ATPase inhibitor (Caruso-Neves et al., 1998a) and furosemide (1.0 mM), a Na + -ATPase inhibitor (Caruso-Neves et al., 1998b). Similarly, 5 AMP and p-NPP, substrates for 5 0 nucleotidase and phosphatase activities, respectively, also failed to inhibit the ecto-ATPase activity. ...
In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78+/-0.08 nmol Pi x h(-1)x10(-6) cells). The ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53+/-0.07 mM. ATP was the best substrate for this enzyme. The apparent K(m) for ATP was 0.21+/-0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg(2+)-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A(1) (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5 AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl(2). Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.
... In our test, a little difference in toxicity to adult zebrafish between R-metalaxyl and rac-metalaxyl were observed according to LC 50 , but a statistical difference (P [ 0.05) was not detected. Na 1 ,K 1 -ATPase has been found in a variety of tissues from different animals including aquatic organisms (Caruso-Neves et al., 1998). This plasma membrane-bound enzyme plays an important role in maintaining intracellular Na 1 ,K 1 homeostasis at the expense of ATP (Sancho et al., 2003). ...
Chiral pesticides are used widely in the world, and at present, older racemic products are being replaced by enantiopure products because of accelerated development of asymmetry synthesis techniques. Pesticides as xenobiotic released into environment impose a great stress on nontarget organisms. Although it is a necessary procedure for pesticides to have a registration based on toxicological data from nontarget organism, until now ecological risk assessment about metalaxyl only depend on racemic products. Hence, we investigated the acute, chronic, and sublethal toxicity of R-metalaxy and rac-metalaxyl on aquatic organisms such as D. magna (Daphnia magna), algae (Scenedesmus quadricanda), and adult zebrafish (Danio rerio). The results showed a significant difference in toxicity between R-metalaxyl and rac-metalaxyl. R-Metalaxy was about 20-fold more toxic to algae than rac-metalaxyl with IC(50) of 222.89 +/- 1.18 mg/L and 19.95 +/- 1.12 mg/L, respectively. Similarly, R-metalaxyl was about fourfold toxic to D. magna than rac-metalaxyl according to the individual 24-h-LC(50) values, and sixfold toxic than rac-metalaxyl based on 24-h-EC(50) values. In the light of 48-h-LC(50) and EC(50), this difference in toxicity was more significant. As for adult zebrafish, there was no pronounced difference in acute toxicity, in addition, at sublethal level a different pattern in inducing Na(+),K(+)-ATPase activity between them was found. In general, R-metalaxyl seemed more toxic to aquatic organisms than rac-metalaxyl.
Metastasis is a major cause of death in patients with breast cancer. A growing body of evidence has demonstrated the antitumour effects of resveratrol, a non‐flavonoid polyphenol. Resveratrol inhibits metastatic processes such as the migration and invasion of cancer cells. In several cancer types, the importance of inorganic phosphate (Pi) for tumour progression has been demonstrated. The metastatic process in breast cancer is associated with Na⁺‐dependent Pi transporters. In this study, we demonstrate, for the first time, that resveratrol inhibits the Na⁺‐dependent Pi transporter. Results from kinetic analysis shows that resveratrol inhibits Na⁺‐dependent Pi transport non‐competitively. Resveratrol also inhibits adhesion/migration in MDA‐MB‐231 cells, likely related to inhibition of the Na⁺‐dependent Pi transporter.
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We have measured fluid secretion rate and intracelular concentration of sodium and protons, in Rhodnius prolixus UMT stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. The ionic fluxes has been measured using microfluorimetry. 13 pharmacological agents were applied: Ouabain, Bafilomycin A1, Furosemide, Bumetanide, DIOA, Probenecid, SITS, Acetazolamide, Amiloride, DPC, BaCl2, pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter- transporters and ionic and water channels. The basic assumption is that water movement changes reflect changes on ion transport mechanisms which we localize as follows: (i) At the basolateral cell membrane: a Na + - K+ - 2Cl- cotransport; a Cl- - HCO3 - exchange; the Na+ - K+ - ATPase, Cl- channels and Rp-MIP water channels are fundamental; K+ channels play a lesser role. (ii) At the apical cell membrane, a K+ - Cl- cotransport that is located for the first time; a V - H+ - ATPase; an Na+- H+ exchange; an urate - anion exchange and K + channels are important, while Cl- channels are not. The present experiments allow us to build a tentative model for the function of the UMT cell, which includes a paracellular pathway for fluid flow. In this work we propose a new actualized model to explain the UMT secretion.
The interaction of nicotinamide (NA) with NH4VO3 at the physiological pH (pH 7.4) was studied by voltammetry and UV-Vis spectroscopy. The reduction of V(V) ions complexed with NA in phosphate buffer (pH 7.4) was observed as a well-defined reversible reduction peak at -0.35 V. The stoichiometric (metal:ligand) ratio of the complex of V(V) with NA was determined as 1:2 by the mole ratio method.
Ecto-enzymes can be defined as membrane-bound proteins that have their active site facing the extracellular millieu. In trypanosomatids, the physiological roles of these enzymes remain to be completed elucidated; however, many important events have already been related to them, such as the survival of parasites during their complex life cycle and the successful establishment of host infection. This chapter focuses on two remarkable classes of ecto-enzymes: ecto-nucleotidases and ecto-phosphatases, summarizing their occurrence and possible physiological roles in Leishmania and Trypanosoma genera. Ecto-nucleotidases are characterized by their ability to hydrolyze extracellular nucleotides, playing an important role in purinergic signaling. By the action of these ecto-enzymes, parasites are capable of modulating the host immune system, which leads to a successful parasite infection. Furthermore, ecto-nucleotidases are also involved in the purine salvage pathway, acting in the generation of nucleosides that are able to cross plasma membrane via specialized transporters. Another important ecto-enzyme present in a vast number of pathogenic organisms is the ecto-phosphatase. These enzymes are able to hydrolyze extracellular phosphorylated substrates, releasing free inorganic phosphate that can be internalized by the cell, crossing the plasma membrane through a Pi-transporter. Ecto-phosphatases are also involved in the invasion and survival of parasite in the host cells. Several alternative functions have been suggested for these enzymes in parasites, such as participation in their proliferation, differentiation, nutrition and protection. In this context, the present chapter provides an overview of recent discoveries related to the occurrence of ecto-nucleotidase and ecto-phosphatase activities in Leishmania and Trypanosoma parasites.
The effect of the administration of commercial diets supplemented with 9 mg kg(-1) 3,5,3'-triiodo-l-thyronine (T(3)) or 10% (w/w) NaCl was evaluated on the ouabain-insensitive Na+-ATPase activity in rainbow trout gill microsomes. The trial, carried out following the seasonal trend from March to mid-May, included a treatment phase in freshwater and a subsequent transfer to brackish water (22 per thousand salinity) where trout were not treated. pH dependence, apparent Km values for Mg(2+) and Na+, and Hill coefficients evaluated throughout the trial for Na+-ATPase were generally not affected by the treatments and habitat change. In comparison with the control group, in both treated groups, Na+-ATPase activity was lower during the freshwater phase and higher after brackish-water transfer. As compared with untreated trout, gill (Na++K+)-ATPase activity during the freshwater phase was stimulated by NaCl treatment and also by T(3) treatment after transfer to brackish water. The results indicate that NaCl and T(3) administration act differently on the two ATPase activities involved in Na+ regulation and suggest a prevalent role of Na+-ATPase activity in hypoosmotic conditions.
Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na(+)-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2mM), a specific inhibitor of Na(+)-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na(+)-ATPase activity with IC(50)=18+/-5 microg mL(-1). This effect was shown to be reversible, dependent on the pH and Ca(2+). The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na(+)-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na(+)-ATPase and PKC activities.
Angiotensin-(1-7) (Ang-(1-7)) modulates the Na+-ATPase, but not the Na+,K+-ATPase activity present in pig kidney proximal tubules. The Na+-ATPase, insensitive to ouabain, but sensitive to furosemide, is stimulated by Ang-(1-7) (68% by 10(-9) M), in a dose-dependent manner. This effect is due to an increase in Vmax, while the apparent affinity of the enzyme for Na+ is not modified. Saralasin, a general angiotensin receptor antagonist, abolishes the stimulation, demonstrating that the Ang-(1-7) effect is mediated by receptor. The Ang-(1-7) stimulatory effect is not changed by either PD 123319, an AT2 receptor antagonist, or A779, an Ang-(1-7) receptor antagonist. On the other hand, increasing the concentration of the AT1 receptor antagonist losartan from 10(-11) to 10(-9) M, reverses the Ang(1-7) stimulation completely. A further increase to 10(-3) M losartan reverses the Na+-ATPase activity to a level similar to that obtained with Ang-(1-7) (10(-9) M) alone. The stimulatory effect of Ang-(1-7) at 10(-9) M is similar to the effect of angiotensin II (AG II) alone. However, when the two peptides are both present, Na+-ATPase activity is restored to control values. These data suggest that Ang-(1-7) selectively modulates the Na+-ATPase activity present in basolateral membranes of kidney proximal tubules through a losartan-sensitive receptor. This receptor is probably different from the receptor involved in the stimulation of the Na+-ATPase activity by angiotensin II.
In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.
The plasma membrane of cells contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using living cells. In this work we describe the ability of living promastigotes of Leishmania amazonensis to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (5.39 +/- 0.71 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 30.75 +/- 2.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.21 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.98 mM and free Mg(2+) did not increase the ecto-ATPase activity. In the pH range from 6.8 to 8.4, in which the cells were viable, the acid phosphatase activity decreased, while the Mg(2+)-dependent ATPase activity increased. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. A comparison between the Mg(2+)-dependent ATPase activity of virulent and avirulent promastigotes showed that avirulent promastigotes were less efficient than the virulent promastigotes in hydrolyzing ATP.
This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 +/- 13.9 nmol Pi/h x 10(7) cells). This ecto-enzyme was stimulated by ZnCl2, CaCl2 and MgCl2, was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. D-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity.
In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42±0.31 nmol Pi/h×108 cells). ATP hydrolysis was stimulated by MgCl2, and the Mg-dependent ecto-ATPase activity was 27.15±2.91 nmol Pi/h×108 cells. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2 or SrCl2. The apparent K
m for Mg-ATP2− was 0.61 mM, and free Mg2+ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4′.diisothiocyanostylbene 2′-2′-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg2+-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.
We have measured fluid secretion rate in Rhodnius prolixus upper Malpighian tubules (UMT) stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. Thirteen pharmacological agents were applied: ouabain, Bafilomycin A1, furosemide, bumetanide, DIOA, Probenecid, SITS, acetazolamide, amiloride, DPC, BaCl2, pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter-transporters and ion and water channels. The basic assumption is that water movement changes reflect changes in ion transport mechanisms, which we localize as follows: (i) At the basolateral cell membrane, fundamental are a Na+-K+-2Cl− cotransporter and a Cl−-HCO
3− exchanger; of intermediate importance are the Na+-K+-ATPase, Cl− channels and Rp-MIP water channels; K+ channels play a lesser role: (ii) At the apical cell membrane, most important are a K+-Cl− cotransport that is being located for the first time, a V-H+-ATPase; and a Na+-H+ exchanger; a urate-anion exchanger and K+ channels are less important, while Cl− channels are not important at all. A tentative model for the function of the UMT cell is presented.
In this study, the effect of toxaphene (camphechlor) on ATPase activity in the microsomal fraction of the Unio tumidus's digestive gland was determined. Toxaphene is a man-made mixture consisting of polychlorinated monoterpens, predominantly bornanes. This compound was primarily used as an insecticide, but in 1982 was officially banned because of its destructive effects on human and animal health. Toxaphene can be transported in the air at long distances and can persist in air, soil and water for years revealing acute and chronic toxicity towards aquatic organisms and wildlife, the increasing risk of cancer in both humans and animals. The microsomal fraction isolated from digestive glands was exposed to 1 x 10(-3) M, 1 x 10(-5) M and 1 x 10(-7) M of toxaphene. The obtained data showed that toxaphene induced a loss of ATPase activity in all used concentrations. The Lineweaver-Burk plots for microsomal Na+K+-ATPase in the presence or the absence of toxaphene as an inhibitor indicated a competitive type of inhibition.
Insects maintain haemolymph homeostasis under different environmental conditions by modulating the concentrations of Na+, K+ and Cl- ions. One group of proteins involved in ion transport across cell membranes consists of cation-chloride cotransporters that form a family of structurally similar proteins. Although much is known about these proteins in mammalian systems, our understanding of them in insects is lacking. The recent sequencing of two insect genomes, Drosophila and Anopheles, enabled us to identify globally members of the family of cation chloride cotransporters in these insects. Using RT-PCR we monitored the transcription of members of this family in development and in several tissues. Our analyses showed that transcription of these genes differ considerably from the ubiquitously and highly expressed CG5594 gene to the almost silent gene CG31547. Comparison of Drosophila CG12773 and its Aedes homologue AaeCG12773 showed that they have similar transcript expression profiles. Immunohistochemical analysis of AaeCG1277 gene expression revealed that it is highly expressed in the gut of larvae and female adults but not in Malpighian tubules. A more detailed analysis showed that this protein is localized predominantly in the basolateral membrane of these tissues. This expression pattern confirmed the results of RT-PCR analysis. We also created a mutant for one of the genes, CG10413, in Drosophila using P-element excision. Analysis of this mutant showed this protein does not appear to be essential for development.
When a mosquito takes in a blood meal that is twice its body weight, flying and easy maneuvering become a problem. To get rid of the extra water, the mosquito produces a copious flow of urine that begins even before the meal has been completed. How is this diuresis controlled?
The ribose-modified nucleotides 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP were used to probe the catalytic sites on soluble beef heart mitochondrial adenosine triphosphatase (F1). Both compounds were potent competitive inhibitors of ATP hydrolysis catalyzed by F1, Ki = 5.5 and 10 nM, respectively, and by submitochondrial particles, Ki (TNP-ATP) = 21 nM. Both compounds also were potent competitive inhibitors of ATP synthesis during oxidative phosphorylation (Ki = 1300 nM). Both analogs inhibited the 32Pi-ATP exchange reaction and the ATP-dependent reduction of NAD+ by succinate, catalyzed by submitochondrial particles. TNP-ATP and TNP-ADP were bound by F1. The presence of two binding sites on the enzyme for TNP-adenine nucleotides was determined by titrations of difference absorbance spectra, of the increase in fluorescence of the analog which occurred upon interaction with protein, and by titrations with the centrifuge column method using 32P-labeled TNP-adenine nucleotides. The first binding site bound the analogs with an affinity too high to be measured. The Kd for analog binding by the second site was 20 to 80 nM. In the presence of Mg2+, the 2 sites were filled with the TNP-ATP at a rate too rapid to be resolved by the procedure used. TNP-[gamma-32P]ATP was hydrolyzed by F1, Km = 0.2 microM, Vmax = 1.1 mol of 32Pi formed/mol of F1/s. It was shown, using the isotope trap technique as well as the inhibitor efrapeptin, that the 2 binding sites for TNP-ATP on F1 are hydrolytic sites.
The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K+ or Cl− or HCO
3−by Na+, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na+ by tetramethylammonium (TMA+) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10−4m, anthracene-9-COOH 5×10−4m, furosemide 5×10−4m or ethacrynic acid 5×10−5m, also abolished RVI. On the other hand, addition of either Ba2+ 10−3m, SITS 5× 10−4m, ouabain 10−3m or vanadate 10−3m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10−6m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na+, K+, Cl−, Ca2+ and HCO
3−transport pathways and a ouabain-insensitive ATPase stimulated by Na+.
This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.
Insect Malpighian tubules secrete fluid into the lumen as part of their function as excretory organs. The underlying ion transport is, when stimulated, faster than in any other known tissue. It is driven by the activity of an H+-transporting V-ATPase situated on the luminal cell membranes. This ATPase, together with cation/H+ antiporter(s), constitutes a common cation pump which can transport sodium ions, potassium ions or both. Treatments that selectively slow cation transport across the epithelium cause the secreted fluid to become alkaline, whereas those that selectively reduce the rate of anion passage lead to secretion of acid fluid.
The cell membranes of virtually all animal cells are highly permeable to water, and hence the cell volume is determined by the cellular content of osmotically active solutes and by the osmolality of the extracellular fluid. It should be noted, however, that in epithelial cells only the membrane on the serosal side is highly water permeable; the mucosal side membrane can be quite tight, as in the epithelial cells of late distal tubules and collecting ducts of the kidney in the absence of antidiuretic hormone.
The energy-dependent mechanisms that extrude Na+ from the cells of higher-order animals, against large electrochemical potential differences, are responsible for counterbalancing the colloid osmotic force of intracellular macromolecular anions, to which the cell is normally impermeable. In this chapter, the results of early experiments on rat kidney cortex slices are summarized that suggest the presence of an ouabain-insensitive sodium pump. The chapter also provides evidence that basolateral plasma membrane fractions from proximal tubular cells of the rat kidney have ouabain-insensitive Na+-ATPase with different kinetic characteristics from those of Na+,K'-ATPase. The chapter further presents the results of experiments indicating two different modes of Na+ uptake in inside-out basolateral plasma membrane vesicles associated with specific ATPase activities. The chapter concludes by providing the results of recent experiments dealing with the relationship between cell volume and the activity of ouabain-insensitive Na+-ATPase.
Single segments of rat nephron contain two distinct ouabain-insensitive, K-independent, Na-dependent ATPase activities: a Na-stimulated ATPase and a Na-inhibited ATPase. Na-inhibited ATPase activity is found in the proximal tubule and the thick ascending limb of Henle's loop but is absent in the collecting tubule whereas Na-stimulated ATPase is exclusively located in the proximal convoluted tubule. Na-inhibited ATPase, but not Na-stimulated ATPase, is totally abolished in the presence of 100 μM Ca2+. Conversely, Na-stimulated ATPase, but not Na-inhibited ATPase, is curtailed when nephron segments are preincubated at pH 7.2 whereas it is activated at pH 7.8. Finally, Na-stimulated ATPase displays an apparent Km for Na+ of ≈ 10 mM, and is dose-dependently inhibited by the diuretic triflocin (IC50 ≈ 6 · 10−6M).
Current information on the ionic basis of fluid secretion in Malpighian tubules is reviewed, with the emphasis on electrophysiological data, almost all of which has appeared in the last decade. Different electrical techniques are compared. Evidence for the various channels, carriers and pumps thought to be involved in the transport of K, Na, H and Cl ions is discussed. Few insect species have been studied in any detail, and major differences between them are apparent. Rapid advances are anticipated when synthetic diuretic peptides become available for electrophysiological studies.
1.1. Incubation of a microsomal fraction from the outer medulla of rabbit kidney with deoxycholate rapidly increases the specific activity of (ouabain sensitive; ATP phosphohydrolase, EC 3.6.1.3) from 45 to 270 μmoles Pi per mg protein per h if the conditions for incubation are optimal with respect to temperature, pH and concentrations of protein and detergent. A procedure for evaluation of the conditions for maximum activation by deoxycholate is described.2.2. Measurements of the surface tension show that the marked influence of changes in the pH on the activation by deoxycholate is due to changes in the capillary activity of deoxycholate. The optimum concentrations of deoxycholate, sodium dodecyl sulfate, and Lubrol-14 for activation of are different, but it is common for the three detergents that maximal activation is obtained when the critical micelle concentration is reached.3.3. Fractionation by zonal centrifugation shows that the remains associated with membranes after activation by deoxycholate, whereas inactive protein is removed and solubilized by the detergent. The treatment with deoxycholate reduces the content of Mg2+-ATPase (ouabain insensitive; ATP phosphohydrolase, EC 3.6.1.3) in the fractions which contain the .4.4. Tracer studies show that the activation of is not associated with binding of significant amounts of deoxycholate to the membranes. The activation does not change the molecular activity of .5.5. The data suggest that the activation of is due to exposure of latent enzyme sites in the preparation. The removal of protein may lead to opening of vesicular structures resulting in free access of substrate and activators to their respective sites on the membrane.
Thin ascending limb cells of Henle's loop from Wistar rats were studied with in vitro microperfusion and video-optical techniques to investigate their ability in regulating cell volume during osmotic shock and to identify mechanisms of ion transport involved in the process. These cells showed a clear volume regulatory decrease (VRD) response in hyposmotic medium, but no volume regulatory increase in hyperosmotic medium. The presence of barium in the bath abolished VRD. Removal of K+ from bath and perfusate also inhibited the VRD response. Reintroduction of K+ in hyposmotic conditions reestablished cell volume regulation. Introduction of anthracene-9-COOH to the basolateral medium blocked cell volume regulatory response. Cl- removal from perfusate and bath solutions also inhibited VRD, probably because of a significant intracellular Cl- depletion. Exposure of cells to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid in perfusate and bath solutions reduced significantly Ca2+ concentration and impaired VRD. Reintroduction of Ca2+ in hyposmotic conditions restored volume regulation. The presence of ouabain in basolateral medium also inhibited VRD. These data suggest that the following mechanisms in the basolateral membrane are involved in VRD response: K+ and Cl- conductive pathways, which might be Ca2+ dependent for activation, and an Na(+)-K(+)-adenosinetriphosphatase.
1. Two Na(+)-stimulated ATPase activities were determined in gill homogenates from squid, shrimp and teleost fish; in kidney slice homogenates from teleost fish, bullfrog, toad, iguana, chicken, duck, rat, pig and cow, as well as in homogenates from rat small intestinal cells, brain cortex and liver slices. The two Na(+)-stimulated ATPase activities, the Na- and the Na,K-ATPase, showed a different behavior toward K+ and ouabain. 2. The ouabain-insensitive, K(+)-independent, Na-ATPase activity for all the studied homogenates was completely inhibited by 2 mM furosemide. 3. An increase in cell volume of the kidney, brain cortex and liver slice preparations, as well as of the rat small intestinal cells, produced a concomitant increase of the ouabain-insensitive Na-ATPase.
Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.
A ouabain-insensitive, K+-independent, sodium pump, has been demonstrated in guinea-pig and rat kidney proximal tubular cells. This pump is thought to be distinct from the ouabain-sensitive Na+/K+ pump. We present evidence here indicating the modulation of the biochemical expression of the Na+ pump, i.e. the ouabain-insensitive Na+-ATPase, by the cell volume in rat kidney proximal tubular cells. Thus, basolateral plasma membranes from swollen cells show a ouabain-insensitive Na+-ATPase activity 10-times higher than that in membranes from control cells. If the swollen cells recover their volume, the activity decreases ten times to control values. The ouabain-sensitive Na+/K+-ATPase is not affected by changes in the cell volume.
Inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can accumulate Na+ actively in two ways. Mode 1, which is K+-independent, is ouabain-insensitive and is inhibited by furosemide and mode 2, which is K+-dependent, is inhibited by ouabain and is insensitive to furosemide. The presence of Mg2+ and ATP in the incubation medium is essential for both modes of Na+ uptake to proceed and in both cases, the nucleotide is hydrolyzed during the process. These results are consistent with the idea of the existence, in these membranes, of two Na+ pumps: one, which can work in the absence of K+ (Na+ pump) and another, which needs K+ to work (Na+ + K+ pump).
The ouabain-insensitive, active Na+ uptake of inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can be increased by the presence of micromolar concentrations of Ca2+ in the assay medium. The concomitant ATP hydrolysis associated with the Na+ uptake is also increased by the presence of Ca2+. The Na+ uptake and the concomitant ATP hydrolysis are inhibited by 2 mM furosemide. The effect of Ca2+ is not due to the activity of an Na+-Ca2+ exchanger. The present results are in accordance with our previous model (Proverbio, F., Proverbio, T. and Marín, R. (1982) Biochim. Biophys. Acta 688, 757-763) in which we proposed that Ca2+ seems to modulate the activity of the ouabain-insensitive Na+ pump, in two different ways: (1) in a strong association with the membranes in which Ca2+ (stable component) is essential for the pump activity and (2) in a weak association with the membranes in which Ca2+ (labile component) can be quickly and easily removed by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM. The Ka for Ca2+ (for the labile component) is around 5 microM. The Ca2+ modulation of the ouabain-insensitive Na+ pump is an indication that Ca2+ could regulate the magnitude of the Na+ extrusion accompanied by Cl- and water present in rat kidney proximal tubular cells.
The ouabain-insensitive, Mg2+-dependent, Na+-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca2+ to the assay medium. The Ca2+ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily 'deactivated' by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM; and as a stable component, which can be 'deactivated' by preincubating the membranes for periods of 3-4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca2+. The Ka of the system for Na+ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca2+ on the Na+-stimulated ATPase is on the Vmax, and not on the Ka of the system for Na+. The activating effect of Ca2+ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca2+ concentration may modulate the ouabain-insensitive, Na+-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na+ accompanied by Cl- and water that these cells show, and to which the Na+-ATPase has been associated as being responsible for the energy supply of this mode of Na+ extrusion.
Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.
ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).
Comparation of the enzymatic properties of the Na,K-ATPase α3β1 and α3β2 isoenzymes
- Blanco
Evidências para a presença da H+/K+-ATPase em túbulo de Malpighi de Rhodnius
- Caruso-Neves
Modulação da atividade da Ca2+-ATPase de homogenato de túbulo de Malpighi de Rhodnius durante choque hiperosmótico
- Caruso-Neves