RESUMO
Disfunction of proximal tubules (PT) in cadmium (Cd) nephrotoxicity in mammals results from the diminished functional capacity of brush-border membrane (BBM) caused by (a) direct inhibition of BBM transporters by Cd, (b) shortening and loss of microvilli, and (c) loss of specific BBM transporters. The loss of transporters may partially result from impaired intracellular vesicle recycling due to loss or/and inhibition of vacuolar H+-ATPase in the PT cell organelles. Cytoskeleton plays an important role in vesicle-mediated recycling and processing of BBM transporters in PT cells. Experiments in vitro have indicated that Cd may affect the state of polymerization of some cytoskeletal proteins. In this work we studied the in vivo effect of CdCl2-treatment in rats (2 mg Cd/kg b. m., s.c., daily for 14 days) upon abundance and arrangement of actin filaments, actin-bundling protein villin, and microtubules (MT) in PT cells. Cd-treatment elicited a dramatic accumulation of Cd in the kidney cortex (200 microg/g tissue wet mass after 14 days) and a strongly increased abundance of metallothionein in PT cells. As revealed by immunocytochemistry in tissue cryosections, the staining intensity of actin and villin in PT cells of Cd-treated rats was generally decreased, without a marked change in their intracellular distribution, whereas MT became largely irregular, diminished in most cells, and lost in many cells. However, the immunoblots revealed an increased content of villin and alpha-tubulin in cortical tissue homogenates from Cd-treated rats, thus indicating an impaired bundling of actin and greatly depolymerized MT in cells intoxicated with Cd. The partial loss of apical actin and villin in PT cells of Cd-treated rats may reflect (or cause) shortening and loss of microvilli, whereas derangement and depolymerization of MT may contribute to the impairment of intracellular recycling of BBM proteins, and lead to the loss of BBM transporters.
Assuntos
Intoxicação por Cádmio/metabolismo , Proteínas do Citoesqueleto/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Actinas/metabolismo , Animais , Cloreto de Cádmio/farmacocinética , Cloreto de Cádmio/toxicidade , Proteínas de Transporte/metabolismo , Imuno-Histoquímica , Córtex Renal/metabolismo , Fígado/metabolismo , Masculino , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Ratos , Ratos Wistar , Tubulina (Proteína)/metabolismoRESUMO
By breeding selection for the extreme values of platelet serotonin level (PSL), two sublines of Wistar-derived rats, with constitutionally high or low PSL and platelet serotonin uptake (PSU), have been developed. Searching for the basis of these differences, we performed quantitative western blot analysis of serotonin transporter (5HTt) in platelet membranes isolated from both rat sublines. A polyclonal anti-5HTt antibody labeled a single, 5HTt-related 94 kDa protein band in platelet membranes, with significantly stronger intensity in membranes from rats that exhibited a high PSL. We conclude that the inherited differences in PSL and PSU in rats, following breeding selection, are determined by the level of 5HTt expression in platelet membranes.
Assuntos
Plaquetas/metabolismo , Proteínas de Transporte/sangue , Proteínas de Transporte/genética , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Serotonina/sangue , Animais , Transporte Biológico , Western Blotting , Proteínas de Transporte/isolamento & purificação , Membrana Celular/metabolismo , Cinética , Masculino , Glicoproteínas de Membrana/isolamento & purificação , Ratos , Ratos Wistar , Proteínas da Membrana Plasmática de Transporte de SerotoninaRESUMO
Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.
Assuntos
Genitália Masculina/enzimologia , ATPases Translocadoras de Prótons/análise , ATPases Vacuolares Próton-Translocadoras , Animais , Western Blotting , Citoplasma/enzimologia , Epididimo/enzimologia , Células Epiteliais/enzimologia , Imunofluorescência , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Masculino , Próstata/enzimologia , Ratos , Ratos Wistar , Glândulas Seminais/enzimologia , Trocadores de Sódio-Hidrogênio/análise , Distribuição Tecidual , Ducto Deferente/enzimologiaRESUMO
Poisoning of experimental animals with cadmium (Cd), mercury (Hg), lead (Pb) or cis-diamminedichloroplatinum (cis-Pt) causes shortening and focal loss of microvilli in proximal tubule (PT) cells, thus indicating that the reduced reabsorptive surface due to damaged integrity of brush-border membrane (BBM) may contribute to the reabsorptive and secretory defects in these toxic states. In addition, in in vitro studies with isolated renal cortical BBM vesicles (BBMV), heavy metals (HM) inhibit transport of various compounds, and these data were interpreted as being a result of a direct inhibition of the respective membrane transporters. In this work we used a DeltapH-driven acridine orange fluorescence quench assay to test if various divalent cations affect in vitro the integrity of BBMV and basolateral membrane vesicles (BLMV) isolated from the rat renal cortex. In Cd-treated BBMV we found that: (a) the integrity of vesicles decreased with increasing concentrations of Cd; and (b) the loss of sealed vesicles was high at 37 degrees C, intermediate at 25 degrees C, and very low at 0 degrees C. The loss of sealed BBMV was caused also by Hg, Cu, Pb and Zn (Hg>>>Cu=Cd>Pb=Zn). Cis-Pt, Al, Fe, Ba, Mg and Mn had no effect. BLMV were damaged by HM with an efficiency Hg>>>Cd=Pb=Cu, whereas other divalent cations, including Zn, were ineffective. An SH-group protector, dithiothreitol, prevented the loss of sealed vesicles in some (Hg, Pb, Cu) but not in all (Cd, Zn) cases. We conclude that the nephrotoxic HM directly damage the integrity of PT cell plasma membranes; this may cause shortening and loss of microvilli and basolateral invaginations in HM-treated experimental animals in vivo. The data also indicate that caution should be taken when effects of HM on various transports are studied in isolated membrane vesicles in vitro; an impaired transport may result from the loss of vesicle integrity, and not necessarily from the direct inhibition of a transporter.
Assuntos
Membrana Celular/efeitos dos fármacos , Vesículas Citoplasmáticas/efeitos dos fármacos , Córtex Renal/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Metais Pesados/toxicidade , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Fluorometria , Córtex Renal/citologia , Córtex Renal/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Ratos , Ratos EndogâmicosRESUMO
In rats, an acidic luminal pH maintains sperm quiescence during storage in the epididymis. We recently showed that vacuolar H(+)ATPase-rich cells in the epididymis and vas deferens are involved in the acidification of these segments. Treatment of rats with cadmium (Cd) leads to alkalinization of this fluid by an unknown mechanism. Because Cd may affect H(+)ATPase function, we examined 1) the in vivo effect of Cd poisoning on H(+)ATPase-rich cell morphology and on the abundance and distribution of the 31-kDa H(+)ATPase subunit in cells along the rat epididymis, and 2) the in vitro effect of Cd on H(+)ATPase activity and function in the isolated vas deferens. Immunofluorescence and immunoblotting data from rats treated with Cd for 14-15 days (2 mg Cd/kg body mass/day) showed that 1) H(+)ATPase-positive cells regressed to a prepubertal phenotype, and 2) H(+)ATPase was lost from the apical pole of the cell and was redistributed into an intracellular compartment. In experiments in vitro, Cd inhibited bafilomycin-sensitive ATPase activity in isolated total cell membranes and, as measured using a proton-selective extracellular microelectrode, inhibited proton secretion in isolated vas deferens. We conclude that alkalinization of the tubule fluid in the epididymis and vas deferens of Cd-treated rats may result from the loss of functional H(+)ATPase enzyme in the cell apical domain as well as from a direct inhibition of H(+)ATPase function by Cd.
Assuntos
Cádmio/farmacologia , Inibidores Enzimáticos/farmacologia , Epididimo/enzimologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Vacúolos/enzimologia , Animais , Western Blotting , Imunofluorescência , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Masculino , ATPases Translocadoras de Prótons/análise , ATPases Translocadoras de Prótons/metabolismo , Prótons , Ratos , Ratos Wistar , Maturidade Sexual , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismoRESUMO
The Na/K-ATPase plays a fundamental role in the physiology of various mammalian cells. In the kidney, previous immunocytochemical studies have localized this protein to the basolateral membrane in different tubule segments. However, intercalated cells (IC) of the collecting duct (CD) in rat and mouse were unlabeled with anti-Na/K-ATPase antibodies. An antigen retrieval technique has been recently described in which tissue sections are pretreated with sodium dodecyl sulfate before immunostaining. This procedure was used to reexamine the presence of Na/K-ATPase in IC along the rat nephron using monoclonal antibodies against the Na/K-ATPase alpha-subunit. Subtypes of IC along the nephron were identified by their distinctive staining with polyclonal and monoclonal antibodies to the 31-kD vacuolar H+ -ATPase subunit, whereas principal cells (PC) were labeled with a polyclonal antibody to the water channel aquaporin-4 (AQP-4). In PC, the Na/K-ATPase and AQP-4 staining colocalized basolaterally. In contrast to previous reports, we found that IC of all types showed basolateral labeling with the anti-Na/K-ATPase antibody. The staining was quantified by fluorescence image analysis. It was weak to moderate in IC of cortical and outer medullary collecting ducts and most intense in IC of the initial inner medullary collecting duct. IC in the initial inner medulla showed a staining intensity that was equivalent or stronger to that in adjacent principal cells. Models of ion transport at the cellular and epithelial level in rat kidney, therefore, must take into account the potential role of a basolateral Na/K-ATPase in intercalated cell function.
Assuntos
Néfrons/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Anticorpos Monoclonais , Aquaporina 4 , Aquaporinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica/métodos , Néfrons/citologia , Néfrons/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/fisiologiaRESUMO
The mechanism of cadmium (Cd)-induced damage in the mammalian proximal tubule that is manifested by defects in reabsorption of various compounds, is poorly understood. A vacuolar H(+)-ATPase (V-ATPase) in proximal tubule (PT) brush border and intracellular vesicles may be affected by Cd, and this may influence intracellular vesicle trafficking and reabsorption of the filtered proteins. We studied the effects of Cd on V-ATPase and endocytosis in rat renal PT in vivo and on acidification mechanisms in isolated renal cortical organelles in vitro. The V-ATPase activity in brush border membrane (BBM) from Cd-intoxicated rats was 40% lower compared to that in control animals. Immunofluorescence studies in cortical tissue sections and Western blot studies in BBM from Cd-treated rats showed a strongly decreased abundance of the 31 kDa and 70 kDa V-ATPase subunits. Functional studies in vivo showed a dramatically diminished endocytosis of fluorescein-labeled dextran in PT cells from Cd-treated animals, whereas morphological studies revealed a loss of endocytic invaginations and subapical vesicles in the same cells. In studies in vitro, Cd inhibited V-ATPase activity in a concentration- and time-dependent manner in both BBM and endocytic vesicles, whereas in endocytic vesicles, Cd inhibited ATP-driven intravesicular acidification and accelerated the dissipation of transmembrane pH gradients. We conclude that Cd may impair acidification in cell organelles by (a) causing a loss of V-ATPase protein in their limiting membranes, (b) inhibiting the intrinsic V-ATPase activity, and (c) dissipating the transmembrane pH gradient. This may inhibit endocytosis of filtered proteins and impair vesicle-mediated recycling of some membrane transporters, thus contributing to the loss of reabsorptive capacity of the PT.
Assuntos
Cádmio/farmacologia , Endocitose/efeitos dos fármacos , Córtex Renal/enzimologia , Córtex Renal/fisiologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Vacúolos/enzimologia , Equilíbrio Ácido-Base , Animais , Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Rim/efeitos dos fármacos , Rim/fisiopatologia , Córtex Renal/ultraestrutura , Microscopia Eletrônica , Ratos , Ratos WistarRESUMO
Using antibodies against the 31-kD and 70-kD subunits of vacuolar type H+-ATPase (V-ATPase) and light microscopic immunocytochemistry, we have demonstrated the presence of this V-ATPase in rat submandibular gland. We have also investigated the adaptive changes of this transporter during acid-base disturbances such as acute and chronic metabolic acidosis or alkalosis. Our results show intracellularly distributed V-ATPase in striated, granular, and main excretory duct cells in controls, but no V-ATPase immunoreaction in acinar cells. Both acute and chronic metabolic acidosis caused a shift in V-ATPase away from diffuse distribution towards apical localization in striated and granular duct cells, suggesting that a V-ATPase could be involved in the regulation of acid-base homeostasis. In contrast, during acidosis the main excretory duct cells showed no changes in the V-ATPase distribution compared to controls. With acute and chronic metabolic alkalosis, no changes in the V-ATPase distribution occurred. (J Histochem Cytochem 46:91-100, 1998)
Assuntos
Desequilíbrio Ácido-Base , Adaptação Fisiológica , ATPases Translocadoras de Prótons/metabolismo , Glândula Submandibular/enzimologia , ATPases Vacuolares Próton-Translocadoras , Desequilíbrio Ácido-Base/induzido quimicamente , Acidose/induzido quimicamente , Acidose/enzimologia , Adaptação Fisiológica/efeitos dos fármacos , Alcalose/induzido quimicamente , Alcalose/enzimologia , Cloreto de Amônio/farmacologia , Animais , Western Blotting , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar , Ductos Salivares/citologia , Ductos Salivares/efeitos dos fármacos , Ductos Salivares/enzimologia , Bicarbonato de Sódio/farmacologia , Glândula Submandibular/citologia , Glândula Submandibular/efeitos dos fármacosRESUMO
Rat small intestinal brush-border membrane (BBM) vesicles were solubilized to test for the presence of an intravesicular H+-ATPase. Several detergents that in rat renal BBM vesicles revealed a bafilomycin-sensitive H+-ATPase failed to expose a similar ATPase in small intestinal vesicles. Antibodies against the 31-kDa and 70-kDa H+-ATPase subunits labelled respective antigens in the BBM of renal proximal tubules, but not in the BBM of enterocytes. The results demonstrate that a bafilomycin-sensitive, vacuolar-type H+-ATPase is absent from the BBM of enterocytes and, hence, cannot contribute to H+ secretion.
Assuntos
Intestino Delgado/enzimologia , Macrolídeos , ATPases Translocadoras de Prótons/metabolismo , Vacúolos/enzimologia , Animais , Antibacterianos/farmacologia , Detergentes/farmacologia , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Microvilosidades/enzimologia , Octoxinol/farmacologia , Ratos , Ratos WistarRESUMO
The cellular mechanisms of cadmium (Cd) nephrotoxicity are poorly understood. In this study we investigated the cellular causes of the Cd-induced phosphaturia in the rat. Compared to controls, Cd-treated rats (2 mg Cd/kg body weight, s.c. for 14 days) showed a marked polyuria, proteinuria and phosphaturia. As studied by the rapid filtration technique in isolated cortical brush-border membrane vesicles (BBMV), Na+-gradient-driven uptake of phosphate ([32Pi]) and of [3H] glucose were markedly decreased in Cd-treated rats, whereas uptake of sulphate ([35S]) remained unchanged. By Western blotting of BBMV proteins and by indirect immunocytochemistry in 4-micron thick frozen fixed kidney sections, using an antibody against the type II Na/Pi-cotransporter (NaPi-2), we found a diminished expression of this protein in the brush-border membrane from Cd-treated rats. How ever, the expression of the water channel aquaporin 1, estimated from the specific antibody staining in brush-border membranes, remained unchanged by Cd. Northern blot analysis showed a strong reduction of 2.7 kb NaPi-2-related mRNA in Cd-affected kidneys. Our data indicate that: (1) Cd may reduce reabsorption of Pi in proximal tubules by affecting the expression of the functional Na/Pi-cotransporters in the luminal membrane, and (2) Cd effects on brush-border transporters are selective.
Assuntos
Aquaporinas , Cádmio/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Canais Iônicos/metabolismo , Córtex Renal/metabolismo , Simportadores , Animais , Aquaporina 1 , Northern Blotting , Western Blotting , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/metabolismo , Córtex Renal/enzimologia , Microvilosidades/enzimologia , Microvilosidades/metabolismo , Ratos , Ratos Wistar , Cotransportador de Sódio-Sulfato , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIRESUMO
The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations.