RESUMEN
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 microM 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 approximately 10 mM, and is dose-dependently inhibited by the diuretic triflocin (IC50 approximately 6 x 10(-6) M).
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Nefronas/enzimología , Ouabaína/farmacología , Cloruro de Sodio/farmacología , Animales , Cloruro de Calcio/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Masculino , Colagenasa Microbiana/farmacología , Nefronas/citología , Nefronas/efectos de los fármacos , Ratas , Ratas Endogámicas , Cloruro de Sodio/administración & dosificaciónRESUMEN
Since it is now well established that the Na-H exchanger accounts for only part of tubular proton secretion, we attempted to characterize the molecular processes responsible for the remaining moiety. In particular, we evaluated the possible roles of proton pumps in urinary acidification. For this purpose, we characterized ATPase activities associated with the electrogenic H pump, on the one hand, and with H-K-ATPase, on the other. In order to circumvent the axial heterogeneity of nephron, this study was carried out on microdissected segments of nephron. The present report summarized experiments which aimed: (1) at characterizing H-ATPase and H-K-ATPase on kinetic and pharmacologic bases in the successive segments of mammalian nephrons; (2) at evaluating the ionic transport mediated by these two ATPases; and (3) at determining the factors which control the activity of these pumps.
Asunto(s)
Túbulos Renales/enzimología , Nefronas/enzimología , Adenosina Trifosfatasas/metabolismo , Animales , Transporte Biológico , Proteínas de Transporte de Catión , ATPasa Intercambiadora de Hidrógeno-Potásio , Iones , Masculino , Deficiencia de Potasio/enzimología , Ratas , Ratas Endogámicas , Distribución TisularRESUMEN
On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1). GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 micrograms protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 micrograms protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 +/- SEM; control: 23.8 +/- 1.8; GLP, 88 micrograms protein/ml: 8.2 +/- 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis.
Asunto(s)
Endotoxinas/fisiología , ATPasa Intercambiadora de Hidrógeno-Potásio/fisiología , Leptospira interrogans , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , ConejosRESUMEN
We report a technique of cephalic pancreatico-duodenectomy preserving the pylorus which differs from that initially devised by Longmire and Traverso. Restoration of digestive tract continuity consists successively of end-to-end duodeno-jejunal anastomosis, end-to-side choledoco-jejunal anastomosis and end-to-side pancreatico-jejunal anastomosis. The simple technique has the advantage of restoring a perfectly physiological circuit. It seems to produce satisfactory immediate and mid- or long-term results with improvements in the patients' comfort.
Asunto(s)
Duodeno/cirugía , Pancreatectomía/métodos , Humanos , Antro PilóricoAsunto(s)
Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Túbulos Renales/enzimología , Nefronas/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenilil Ciclasas/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Corteza Renal/enzimología , Médula Renal/enzimología , Leptospira interrogans , Masculino , Ratas , Ratas WistarRESUMEN
Intercalated cells of the distal segments of the mammalian nephron are able to reabsorb K through an active mechanism, particularly during K depletion. However, the molecular basis of this transport is unknown. Therefore, we attempted to determine whether a K-ATPase similar to K-H-ATPase described in gastric mucosa and colon might be present in segments of the distal nephron and thereby account for active K reabsorption. K-stimulated ATPase activity was detected in microdissected segments of rabbit nephron: its activity was proportional to the density of intercalated cells, since it was highest in the connecting tubule, intermediate in the cortical collecting tubule, lowest in the outer medullary collecting tubule, and was not detectable in all other nephron segments. K-ATPase had a high affinity for K (Km approximately equal to 0.2-0.4 mM), was inhibited by vanadate and omeprazole, and was insensitive to ouabain, indicating that it is different from Na+-K+-ATPase but similar to K-H-ATPase. In the rat kidney, K-ATPase was also detected in the collecting tubule and its activity was markedly increased (+100-200%) following K depletion. This stimulation occurred before morphological alterations and might therefore be a primary event responsible for K conservation during K depletion. In summary, these results demonstrate the presence of a vanadate-sensitive, ouabain-insensitive K-ATPase activity in distal nephron segments of mammalian tubules. It is suggested that K-ATPase activity originates in intercalated cells where it might account, at least in part, for K reabsorption.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Túbulos Renales Colectores/metabolismo , Túbulos Renales Distales/metabolismo , Túbulos Renales/metabolismo , Deficiencia de Potasio/enzimología , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Azidas/farmacología , Proteínas de Transporte de Catión , Masculino , Ouabaína/farmacología , Conejos , Ratas , ATPasa Intercambiadora de Sodio-Potasio , Especificidad por Sustrato , Factores de Tiempo , Vanadatos , Vanadio/farmacologíaRESUMEN
Besides its role in the control of the rate of functioning of each Na-K-ATPase unit (as a substrate of the enzyme), the intracellular sodium concentration also regulates the number of active Na-K-ATPase units, as previously described in cultured cells. To evaluate such a possibility in kidney epithelial cells, the intracellular concentration of sodium in rat cortical collecting tubules (CCT) maintained in vitro was altered by the use of the sodium ionophore nystatin. When CCT were preincubated for 2-3 h at 37 degrees C in the presence of nystatin, the enzymatic activity of Na-K-ATPase was markedly stimulated as compared to tubules preincubated without nystatin or in the presence of the ionophore but in the absence of extracellular sodium. Although nystatin increased both Na-K-ATPase activity and [3H]ouabain specific binding in CCT, its action was independent of de novo synthesis of the pump since neither actinomycin D nor cycloheximide abolished it. It is suggested that increasing the sodium concentration in CCT cells induces the recruitment of a latent pool of Na-K-ATPase units. The size of this latent pool of enzyme is under the control of corticosteroids as it is markedly decreased in CCT from adrenalectomized rats.
Asunto(s)
Corticoesteroides/fisiología , Túbulos Renales Colectores/metabolismo , Túbulos Renales/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Adrenalectomía , Animales , Cicloheximida/farmacología , Dactinomicina/farmacología , Epitelio/metabolismo , Ionomicina/farmacología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Nistatina/farmacología , Ouabaína/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The aim of this study was to develop an in vitro system in which we could study the causal relationship between short-term stimulation of Na+,K+-ATPase in the collecting tubule by aldosterone on the one hand and protein synthesis and changes in intracellular Na+ concentration on the other hand. Previous in vivo studies suggested that triiodothyronine might facilitate aldosterone-induced stimulation of Na+,K+-ATPase. Results show that when segments of cortical collecting tubules microdissected from collagenase-treated kidneys of adrenalectomized rats were incubated for 3 hr in the presence of either 10(-8) M aldosterone or 10(-8) M triiodothyronine alone Na+,K+-ATPase activity was not altered, whereas the addition of both hormones markedly stimulated the activity and the number of catalytic sites of Na+,K+-ATPase. This stimulation was abolished by actinomycin D and cycloheximide, whereas it was not altered in the absence of extracellular sodium or in the presence of the luminal Na+-channel blocker amiloride. Thus, triiodothyronine facilitates the in vitro induction of Na+,K+-ATPase synthesis by aldosterone. Aldosterone action on Na+,K+-ATPase is independent of Na+ availability.
Asunto(s)
Aldosterona/farmacología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Triyodotironina/farmacología , Adrenalectomía , Amilorida/farmacología , Animales , Inducción Enzimática/efectos de los fármacos , Técnicas In Vitro , Masculino , Ouabaína/metabolismo , Ratas , Sodio/farmacologíaRESUMEN
An N-ethyl-maleimide (NEM)-sensitive ATPase that displays the properties of an electrogenic proton pump has been described in the different segments of the rat nephron where it mediates part of the active tubular proton secretion. Because corticosteroids are known to control kidney acidification, we evaluated whether or not NEM-sensitive ATPase is a target of corticosteroids in some nephron segments. For this purpose we measured NEM-sensitive ATPase activity in the different segments of nephron microdissected from normal and adrenalectomized rats. Results indicate that within 1 wk after adrenalectomy NEM-sensitive ATPase activity was markedly decreased in both cortical and outer medullary portions of the collecting tubule (cortex, from 398 +/- 12 (+/-SE) to 145 +/- 20; outer medulla, from 293 +/- 21 to 112 +/- 14 pmol X mm-1 X h-1); however, it was not altered in any other segment of the nephron. These results demonstrate that kidney NEM-sensitive ATPase is under the control of corticosteroids and suggest that mineralocorticoids rather than glucocorticoids are involved in this regulation that specifically occurs in mineralocorticoid-sensitive nephron segments. This paper also describes a new computerized method for the automatic determination of the length of single nephron segments.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Corticoesteroides/fisiología , Adrenalectomía , Nefronas/enzimología , Equilibrio Ácido-Base , Acidosis/fisiopatología , Animales , Etilmaleimida/farmacología , Nefronas/anatomía & histología , RatasRESUMEN
Because previous studies indicated that in the collecting tubule, N-ethylmaleimide (NEM)-sensitive ATPase, the biochemical equivalent of the proton pump, is controlled by mineralocorticoids in the long term, the present study was designed to investigate whether such control also exists in the short term. Therefore we investigated the in vivo and in vitro effects of aldosterone on the enzyme activity in cortical and outer medullary collecting tubules (CCT and MCT, respectively) from adrenalectomized rats. Administration of aldosterone (10 micrograms/kg body wt) markedly stimulated NEM-sensitive ATPase activity in the CCT and MCT within 3 h. Similarly, incubating CCT or MCT for 3 h in the presence of 10(-8) M aldosterone enhanced NEM-sensitive ATPase activity up to values similar to those previously measured in the corresponding nephron segments of normal rats. In vitro stimulation of NEM-sensitive ATPase was dose dependent in regard to aldosterone (apparent affinity constant approximately 10(-9) M), appeared after a 30-min lag period, and reached its maximum after 2-2.5 h. Finally, actinomycin D and cycloheximide totally abolished the in vitro action of aldosterone, demonstrating the involvement of protein synthesis in this process.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Aldosterona/farmacología , Etilmaleimida/farmacología , Túbulos Renales Colectores/enzimología , Túbulos Renales/enzimología , Adrenalectomía , Animales , Cicloheximida/farmacología , Dactinomicina/farmacología , Corteza Renal/enzimología , Médula Renal/enzimología , Cinética , Masculino , Ratas , Ratas Endogámicas , Triyodotironina/farmacologíaRESUMEN
Rat collecting ducts exhibit type I or type III K(+)-ATPase activities when animals are fed a normal (NK) or a K(+)-depleted diet (LK). This study aimed at determining functionally the cell origin of these two K(+)-ATPases. For this purpose, we searched for an effect on K(+)-ATPases of hormones that trigger cAMP production in a cell-specific fashion. The effects of 1-deamino-8-D-arginine vasopressin (dD-AVP), calcitonin, and isoproterenol in principal cells, alpha-intercalated cells, and beta-intercalated cells of cortical collecting duct (CCD), respectively, and of dD-AVP and glucagon in principal and alpha-intercalated cells of outer medullary collecting duct (OMCD), respectively, were examined. In CCDs, K(+)-ATPase was stimulated by calcitonin and isoproterenol in NK rats (type I K(+)-ATPase) and by dD-AVP in LK rats (type III K(+)-ATPase). In OMCDs, dD-AVP and glucagon stimulated type III but not type I K(+)-ATPase. These hormone effects were mimicked by the cAMP-permeant analog dibutyryl-cAMP. In conclusion, in NK rats, cAMP stimulates type I K(+)-ATPase activity in alpha- and beta-intercalated CCD cells, whereas in LK rats it stimulates type III K(+)-ATPase in principal cells of both CCD and OMCD and in OMCD intercalated cells.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Inhibidores Enzimáticos/farmacología , Hormonas/farmacología , Imidazoles/farmacología , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/enzimología , Animales , Proteínas de Transporte de Catión , Desamino Arginina Vasopresina/farmacología , Glucagón/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Deficiencia de Potasio/enzimología , Ratas , Ratas Sprague-Dawley , Rubidio/metabolismoRESUMEN
The rat nephron displays two ouabain-sensitive K-ATPases: one, which is present in proximal tubules and thick ascending limbs of normal rats, is specifically activated by K+ and is down-regulated by K+ depletion, whereas the other one appears in collecting ducts of K+-depleted rats and is activated by either Na+ or K+. To determine which of these two ATPases is similar to colonic-type H,K-ATPase, we quantitated by reverse transcriptase-polymerase chain reaction (RT-PCR) the mRNAs encoding the colonic H,K-ATPase alpha subunit in microdissected nephron segments. In normal rats, statistically significant amounts of colonic H,K-ATPase mRNAs were detected exclusively in cortical thick ascending limbs and cortical collecting ducts (200-500 copies/mm). Because these levels of expression were low (1-1.2 copies/target cell), they probably have no physiological relevance. In rats fed a K+-depleted diet for 2 weeks, expression of colonic H,K-ATPase was markedly enhanced in cortical and medullary collecting ducts (5000-12,000 copies/mm or 30-40 copies per cell), whereas it remained low in all other nephron segments. Thus, colonic H,K-ATPase alpha subunit is specifically expressed in cortical and outer medullary collecting ducts of K+-depleted rats where it likely accounts for the ouabain-sensitive K-ATPase activity.
Asunto(s)
Colon/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/biosíntesis , Nefronas/enzimología , Potasio/fisiología , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Colon/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , Masculino , Datos de Secuencia Molecular , Mutación , Nefronas/efectos de los fármacos , Ouabaína/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/química , Ratas , Ratas Sprague-DawleyRESUMEN
Adrenal insufficiency is associated with an impairment of kidney diluting and concentrating ability, defects that may result from alterations of vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The purpose of this study were 1) to localize the sites of decreased vasopressin-stimulated adenylate cyclase (AC) activity along the nephron of adrenalectomized rats; 2) to determine whether the response of AC to other hormones is altered by adrenalectomy; 3) to evaluate whether changes in AC are due to the deficiency in mineralocorticoids and/or glucocorticoids; and 4) to characterize the mechanism of action of corticosteroids on the AC system. Results indicate that adrenalectomy reduced AC stimulation by vasopressin, glucagon, and calcitonin in the thick ascending limb, whereas only the response to vasopressin decreased in the collecting tubule. Glucocorticoid administration curtailed adrenalectomy-induced alterations of AC in the thick ascending limb, whereas that in the collecting tubule was prevented by mineralocorticoids. Adrenalectomy did not alter forskolin-stimulated AC, whereas it decreased responses to aluminum fluoride and cholera toxin. Finally, alterations of fluoride- and cholera toxin-stimulated AC were prevented by glucocorticoid and mineralocorticoid repletion in the thick ascending limb and collecting tubule, respectively.
Asunto(s)
Adenilil Ciclasas/metabolismo , Glucocorticoides/fisiología , Mineralocorticoides/fisiología , Nefronas/enzimología , Corticoesteroides/farmacología , Adrenalectomía , Animales , Fenómenos Biomecánicos , AMP Cíclico/metabolismo , Corteza Renal , Médula Renal , Túbulos Renales Colectores/enzimología , Asa de la Nefrona/enzimología , RatasRESUMEN
The sensitivity of Na(+)-K(+)-ATPase to Na was determined in single segments of rabbit nephron isolated by microdissection. In the cortical collecting tubule (CCT), Na(+)-K(+)-ATPase was threefold more sensitive to Na (apparent K0.5 approximately 3 mM) than in proximal convoluted tubule and cortical thick ascending limb (apparent K0.5 approximately 10 mM). Furthermore, increasing K concentration from 5 to greater than 100 mM markedly reduced the affinity of the pump for Na in all three nephron segments. In fact, the main shift in Na affinity occurred when K changed from 100 to 120 mM; in the CCT, increasing K concentration from 100 to 120 mM while maintaining Na concentration at 10 mM reduced Na(+)-K(+)-ATPase activity by greater than 35%. These findings confirm that, in kidney cells as in other cells, intracellular Na limits the rate of Na(+)-K(+)-ATPase. Thus any alteration of intracellular Na concentration modifies the pump activity in a way that contributes to the restoration of intracellular Na homeostasis. This adaptive property is particularly efficient in the collecting tubule in which the apparent K0.5 of the pump for Na is close to normal intracellular Na concentration. Furthermore, changes in intracellular K concentration, which usually accompany those of Na so as to maintain the total cation concentration constant, potentiate the regulatory role of Na through modifications of its affinity for the pump.
Asunto(s)
Nefronas/metabolismo , Potasio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Animales , Túbulos Renales/metabolismo , Túbulos Renales Colectores/metabolismo , Asa de la Nefrona/metabolismo , Masculino , Nefronas/enzimología , Concentración Osmolar , ConejosRESUMEN
This study is aimed both at characterizing an ATPase activity in rat kidney equivalent to the proton pump described in bovine kidney medulla and at localizing this enzyme along the nephron. Membrane fractions isolated from kidney homogenates by differential and density gradient centrifugations were enriched 7-fold in ATPase activity sensitive to N-ethylmaleimide (NEM). These fractions also displayed ATP-dependent proton transport. ATPase activity and proton transport in vesicles had similar pharmacological properties as both were insensitive to vanadate and ouabain and had similar sensitivities toward NEM (apparent Ki = 20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 50 microM). Proton transport was dependent on chloride availability as chloride addition to the extravesicular medium stimulated proton transport in a dose-dependent fashion (apparent K 1/2 = 7 mM). NEM-sensitive ATPase activity displaying similar pharmacological properties as proton transport in vesicles was also found in single segments of nephron. It was insensitive to vanadate and ouabain, was inhibited by similar concentrations of NEM (apparent Ki = 15-20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 30 microM), and is therefore likely to be a proton pump. NEM-sensitive ATPase was localized in all the segments of the rat nephron; its activity was highest in proximal convoluted tubules; intermediate in proximal straight tubules, thick ascending limbs, and cortical collecting tubules; and lowest in outer medullary collecting tubules.
Asunto(s)
Etilmaleimida/farmacología , Riñón/enzimología , Nefronas/enzimología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Animales , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Cloruros/farmacología , Ácido Desoxicólico/farmacología , Diciclohexilcarbodiimida/farmacología , Riñón/efectos de los fármacos , Masculino , Nefronas/efectos de los fármacos , Ouabaína/farmacología , Ratas , Ratas Endogámicas , Factores de Tiempo , Valinomicina/farmacología , Vanadatos , Vanadio/farmacologíaRESUMEN
A quantitative assay based on the method of reverse transcription and polymerase chain reaction (RT-PCR) was developed to study the expression of calcitonin (CT) receptors in microdissected rat nephron segments. Steady-state mRNA levels of two CT-receptor spliced variants (CT1a and CT1b) were measured using a mutant cRNA as internal standard. CT1a, but not the CT1b isoform, was detected in the kidney cortex, outer medulla, and papilla. Among the tested segments, predominant expression of CT1a mRNA was found in the cortical thick ascending limb of Henle's loop (754 +/- 87 mRNA molecules/mm tubular length; n = 8). Lower expression levels were measured in the medullary thick ascending limb (460 +/- 62 molecules/mm tubule length; n = 7) and in the cortical collecting duct (327 +/- 61 molecules/mm tubule length; n = 6). A weak expression was also detected in the outer medullary collecting duct and the glomerulus. No expression was found in the proximal convoluted tubule, pars recta, and thin descending and thin ascending limb of Henle's loop. We conclude that only the CT1a-receptor mRNA is present in the rat kidney, with a significant level of expression in the cortical and medullary thick ascending limb and in the cortical collecting duct.
Asunto(s)
Nefronas/metabolismo , ARN Mensajero/metabolismo , Receptores de Calcitonina/metabolismo , Animales , Secuencia de Bases , Isomerismo , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Receptores de Calcitonina/genética , Transcripción GenéticaRESUMEN
Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long-lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.
Asunto(s)
Mutación , Oxidorreductasas/genética , Sordariales/genética , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Complejo IV de Transporte de Electrones/genética , Fertilidad , Mitocondrias/enzimología , Proteínas Mitocondriales , Consumo de Oxígeno/genética , Proteínas de Plantas , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismo , Eliminación de Secuencia , Sordariales/enzimología , Sordariales/fisiologíaRESUMEN
This study was designed to correlate morphological alterations induced in rat collecting tubule by potassium depletion with changes in the activity of enzymatic markers of the cell basolateral membrane. Results show the following responses. 1) Potassium depletion induced a huge and progressive hypertrophy of the outer medullary collecting tubule (MCT). Hypertrophy was paralleled by enhancements of vasopressin- and forskolin-dependent adenylate cyclase (AC) activities. Glucagon-sensitive AC was also increased, but with a different kinetics, whereas isoproterenol-dependent AC was only modestly stimulated. 2) In cortical (CCT) and papillary collecting tubules, AC response to hormones did not change. The concentrating defect of K-deprived rats, therefore, does not appear to result from an intrinsically defective adenylate cyclase system in any portion of the collecting tubule. Decreased AC response of the medullary thick ascending limb to vasopressin and glucagon, observed after 3-5 wk of K depletion, might account, at least in part, for reduced hypertonicity of medullary tissue. 3) Na+-K+-ATPase activity fell in CCT, probably in relation to decreased K secretion. Conversely, in MCT, Na+-K+-ATPase rose much more than tubular volume. The physiological significance of this latter observation remains to be established.
Asunto(s)
Adenilil Ciclasas/metabolismo , Túbulos Renales Colectores/metabolismo , Túbulos Renales/metabolismo , Deficiencia de Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Arginina Vasopresina/farmacología , Calcitonina/farmacología , Glucagón/farmacología , Túbulos Renales Colectores/patología , Deficiencia de Potasio/patología , Ratas , Factores de TiempoRESUMEN
To evaluate the involvement of K(+)-ATPase activity in K+ transport in the terminal segments of the rat nephron, we searched for the existence of a component of Rb+ uptake into microdissected segments of collecting tubule associated with the activity of this ATPase. Results indicated that K(+)-ATPase is stimulated by K+ and by Rb+ in a similar fashion and that it is specifically inhibited by the imidazopyridine Sch 28080 (apparent affinity approximately 5 x 10(-7) M). In both cortical and outer medullary collecting tubules (CCT and MCT) of normal rats, 10(-4) M Sch 28080 significantly inhibited the initial rate of Rb+ uptake. Sch 28080-sensitive Rb+ uptake in these two nephron segments was not altered by ouabain, as K(+)-ATPase activity. Finally, both K(+)-ATPase activity and Sch 28080-sensitive Rb+ uptake were increased by similar factors in the CCT and MCT of rats fed a K(+)-depleted diet for 3 days. In these two nephron segments, the apparent stoichiometry of K(+)-ATPase was 1 Rb+:1 ATP. These results demonstrate that K(+)-ATPase reflects the activity of a K+ pump that is pharmacologically similar to the gastric H(+)-K+ pump.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Túbulos Renales Colectores/fisiología , Rubidio/farmacocinética , Animales , Antiulcerosos/farmacología , Transporte Biológico/fisiología , Proteínas de Transporte de Catión , Imidazoles/farmacología , Túbulos Renales Colectores/metabolismo , Masculino , Potasio/fisiología , Ratas , Ratas Endogámicas , Rubidio/metabolismoRESUMEN
An N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) displaying the kinetic and pharmacological properties of an electrogenic proton pump has been described in the different segments of rat nephron, where it mediates part of the active tubular proton secretion. This study was therefore designed to evaluate whether changes in urinary acidification observed during metabolic acidosis or alkalosis were associated with alterations of the activity of tubular NEM-sensitive ATPase, and if so, to localize the nephron segments responsible for these changes. Within 1 wk after the onset of ammonium chloride treatment, rats developed a metabolic acidosis, and NEM-sensitive ATPase activity was markedly increased in the medullary thick ascending limb of Henle's loop and outer medullary collecting tubule, and slightly increased in the cortical collecting tubule. Conversely, treatment with sodium bicarbonate induced a metabolic alkalosis that was accompanied by decreased NEM-sensitive ATPase activity in medullary thick ascending limb and outer medullary collecting tubule. NEM-sensitive ATPase activity was not altered in any other nephron segment tested in alkalotic and acidotic rats, i.e., the proximal tubule and the cortical thick ascending limb of Henle's loop. Changes qualitatively similar were observed as soon as 3 h after the onset of NaHCO3 or NH4Cl-loading. In the medullary collecting tubule, alterations of NEM-sensitive ATPase activity are in part due to hyperaldosteronism observed in both acidotic and alkalotic rats.(ABSTRACT TRUNCATED AT 250 WORDS)