RESUMO
The Na(+)- and Cl(-)-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodium-myo-inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver.
Assuntos
Encéfalo/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Rim/fisiologia , Fígado/fisiologia , Animais , Anticorpos/farmacologia , Membrana Celular/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA/imunologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Células HEK293 , Hepatócitos/fisiologia , Humanos , Medula Renal/fisiologia , Túbulos Renais Coletores/fisiologia , Fígado/citologia , Alça do Néfron/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Pressão Osmótica/fisiologia , RNA Mensageiro/metabolismo , Coelhos , Cloreto de Sódio/farmacologiaRESUMO
Ten normal cheek teeth (CT) were extracted at post mortem from donkeys that died or were euthanased for humane reasons. Decalcified histology was performed on three sections (sub-occlusal, mid-tooth and pre-apical) of each tooth, and undecalcified histology undertaken on sub-occlusal sections of the same teeth. The normal histological anatomy of primary, regular and irregular secondary dentine was found to be similar to that of the horse, with no tertiary dentine present. Undecalcified histology demonstrated the normal enamel histology, including the presence of enamel spindles. Scanning electron microscopy was performed on mid-tooth sections of five maxillary CT, five mandibular CT and two incisors. The ultrastructural anatomy of primary and secondary dentine, and equine enamel types-1, -2 and -3 (as described in horses) were identified in donkey teeth. Histological and ultrastructural donkey dental anatomy was found to be very similar to equine dental anatomy with only a few quantitative differences observed.
Assuntos
Odontologia/veterinária , Equidae , Microscopia Eletrônica de Varredura/veterinária , Dente , Animais , Odontologia/métodos , Incisivo/anatomia & histologia , Incisivo/patologia , Incisivo/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Dente/anatomia & histologia , Dente/patologia , Dente/ultraestruturaRESUMO
Post-mortem examination of 19 donkey skulls showed that donkeys have a greater degree of anisognathia (27% width difference between upper and lower jaws) compared to horses (23%). Teeth (n=108) were collected from 14 skulls and examined grossly and by computed axial tomography (CAT). A greater degree of peripheral enamel infolding was found in mandibular cheek teeth (CT) compared to maxillary CT (P<0.001). A significant increase in peripheral cementum from the apical region to the clinical crown was demonstrated in all CT (P<0.0001). All donkey CT had at least five pulp cavities with six pulp cavities present in the 06s and 11s. A new endodontic numbering system for equid CT has been proposed. A greater occlusal depth of secondary dentine (mm) was present in older donkeys (>16 years) than in the younger (<15 years) donkeys studied. Based on gross and CAT examinations, donkey dental anatomy was shown to be largely similar to that described in horses.
Assuntos
Odontologia/veterinária , Equidae , Radiografia Dentária/veterinária , Tomografia Computadorizada por Raios X/veterinária , Doenças Dentárias/veterinária , Dente , Envelhecimento , Animais , Cemento Dentário/anatomia & histologia , Cemento Dentário/diagnóstico por imagem , Polpa Dentária/diagnóstico por imagem , Odontologia/métodos , Radiografia Dentária/instrumentação , Radiografia Dentária/métodos , Tomografia Computadorizada por Raios X/métodos , Dente/anatomia & histologia , Dente/diagnóstico por imagem , Doenças Dentárias/diagnóstico , Doenças Dentárias/diagnóstico por imagemRESUMO
The major renal adaptive changes in response to selective dietary phosphate restriction are a marked reduction in urinary excretion of phosphate and an increased urinary excretion of calcium; at the cellular level, there is selective increase in renal cortical brush border membrane phosphate uptake and increase in specific activity of alkaline phosphatase. In the present study we examined whether these functional and biochemical adaptive changes could be blocked by drugs known to inhibit protein synthesis. Administration of actinomycin D or cycloheximide to rats switched from a diet with normal phosphate content (0.7%) to a diet with low (0.07%) phosphate content either completely (actinomycin D) or partially (cycloheximide) prevented the expected decrease in urinary excretion of phosphate and increase in the urinary excretion of calcium. The specific activity of alkaline phosphatase measured in crude membrane fraction (washed 100,000 g pellet) from renal cortical homogenate in animals fed a low phosphate diet and treated with actinomycin D or with cycloheximide was significantly lower than in control animals also on a low phosphate diet receiving placebo; but there were no differences between treated and untreated animals in the activities of two other brush border enzymes, gamma-glutamyltransferase and leucine aminopeptidase. Actinomycin D administered to rats maintained on a normal phosphate diet throughout the course of the experiment caused an increase in the urinary excretion of phosphate on the last (6th) day of the experiment but did not change urinary excretion of calcium. In acute clearance experiments, infusion of actinomycin D to rats adapted to a low phosphate diet did not increase fractional excretion of phosphate. In separate experiments, using the same dietary protocol as above, brush border membrane fraction (vesicles) was prepared from renal cortex of rats sacrificed at the end of the experiment. In this preparation Na(+)-dependent (32)Pi and d-[(3)H]glucose uptake and activities of brush border enzymes membrane were determined. Brush border membrane vesicles prepared from rats fed a low phosphate diet showed significantly higher Na(+)-dependent (32)Pi uptake compared with rats fed a normal phosphate diet. This increase in (32)Pi uptake was completely prevented when rats on a low phosphate diet were simultaneously treated with actinomycin D. These differences were specific for (32)Pi transport as no differences were observed in d-[(3)H]glucose uptake among the three groups. There was a positive correlation (r = 0.82, P < 0.01) between (32)Pi uptake and specific activity of alkaline phosphatase measured in aliquots of the same brush border membranes, whereas no such correlation was observed with two other brush border membrane enzymes gamma-glutamyltransferase and leucine aminopeptidase. These observations show that actinomycin D prevents both the functional and cellular renal adaptive changes induced by a low phosphate diet. Taken together, these observations suggest that renal adaptation to a low phosphate diet could be prevented by inhibition of de novo protein synthesis.
Assuntos
Dactinomicina/farmacologia , Dieta , Rim/fisiopatologia , Fosfatos/deficiência , Animais , Cálcio/fisiologia , Cicloeximida/farmacologia , Depressão Química , Glucose/metabolismo , Córtex Renal/metabolismo , Masculino , Microvilosidades/metabolismo , Fosfatos/administração & dosagem , Fosfatos/fisiologia , Ratos , Sódio/metabolismoRESUMO
In these experiments we investigated whether NAD could serve as an intracellular modulator of the brush border membrane (BBM) transport of inorganic phosphate (Pi). NAD, both oxidized (NAD+) and reduced (NADH) form, inhibited the Na+-dependent uptake of 32Pi in the concentration range of 10-300 microM NAD when added in vitro to BBM vesicles isolated from rat kidney cortex, but did not inhibit BBM uptake of D-[3H]glucose or BBM uptake of 22Na+. Neither nicotinamide (NiAm) nor adenosine alone influenced BBM uptake of 32Pi. NAD had a similar relative effect (percent inhibition) in BBM from rats stabilized on low Pi diet (0.07% Pi), high Pi diet (1.2% Pi), or normal Pi diet (0.7% Pi). Subsequently, we examined the renal effects of changing the tissue NAD level in vivo. Rats stabilized on low Pi diet were injected intraperitoneally with NiAm (0.25-1.0 g/kg body wt); urinary excretions of Pi (UPiV), of fluid, and of other solutes were measured before and after NiAm injection, then renal cortical tissue nucleotide content was determined, and a BBM fraction was isolated for transport measurements. In BBM from NiAm-treated rats, the Na+-dependent uptake of 32Pi was decreased, but BBM uptake of D-[3H]glucose and BBM uptake of 22Na+ were not changed. NiAm injection elicited an increase in NAD+ (maximum change, 290%), a lesser increase in NADH (maximum change, +45%), but no change in the content of ATP or cyclic AMP in the renal cortex. Na+-dependent BBM uptake of 32Pi ws inversely correlated with NAD+ content in renal cortex (r = -0.77 +/- 0.1; P less than 0.001) and with UPiV (r = -0.67 +/- 0.13; P less than 0.01). NAD+ in renal cortex was positively correlated with UPiV (r = 0.88 +/- 0.05; P less than 0.001). Injection of NiAm elicited a marked increase in UPiV, but no change in excretions of creatinine or K+, or in urine flow; excretion of Na+ and Ca declined. NiAm injection caused similar renal responses, in normal and in thyroparathyroidectomized rats, as well as in rats on normal Pi diet and low Pi diet. We conclude that NAD can serve as an intracellular modulator (inhibitor) of Na+-dependent transport of Pi across the renal luminal BBM and across the proximal tubular wall by its direct interaction with BBM. We propose that at least some hormonal and/or metabolic stimuli elicit phosphaturia by increasing NAD+ in cytoplasm of proximal tubular cells.
Assuntos
Córtex Renal/metabolismo , NAD/fisiologia , Fosfatos/metabolismo , Adenosina/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Sistema Livre de Células , Dieta , Glucose/metabolismo , Túbulos Renais Proximais/metabolismo , Microvilosidades/metabolismo , Niacinamida/farmacologia , Ratos , Sódio/metabolismoRESUMO
Changes in the kinetics of sodium gradient-dependent brush border Pi transport in response to dietary phosphorus deprivation were analysed using initial rate conditions. In rats adapted to low phosphorus diet the apparent Vmax, determined from a double-reciprocal plot, was increased 2-fold but the apparent Km was not different compared to control rats fed normal phosphorus diet. In contrast when renal adaptation to low phosphorus diet was reversed by fasting the apparent Vmax was not significantly different but the apparent Km was increased 5-fold. The results suggest that regulation of renal Pi transport in vivo may occur not only through changes in the apparent Vmax of the brush border Pi transport system but also, in certain circumstances, through changes in the apparent Km.
Assuntos
Jejum , Córtex Renal/ultraestrutura , Fosfatos/metabolismo , Fósforo/administração & dosagem , Animais , Transporte Biológico Ativo , Cinética , Microvilosidades/metabolismo , RatosRESUMO
Accumulation of osmolytes by renal cells is due in part to increased uptake via specific transporters. These include amino acid transport system A and the betaine/GABA transporter (BGT1). Transport changes have been characterized using intact cells which makes the intracellular mechanisms difficult to determine. In this study the hypertonic upregulation of system A and BGT1 was studied directly at the membrane level in Madin-Darby canine kidney (MDCK) cells. Both system A and BGT1 transport systems were detected in an isolated membrane fraction containing plasma membranes. System A transport was increased in membranes prepared from cells after 6 h hypertonic stress (449 mosmol/kg) but BGT1 activity was minimal and not different from isotonic controls. The increase in system A was blocked by inhibitors of RNA and protein synthesis. BGT1 transport was induced in membranes prepared after 24 h hypertonicity. At this time system A activity in the membrane fraction remained increased, unlike the downregulation observed in intact MDCK cells. We conclude that differential upregulation of system A and BGT1 by hypertonic stress is due to intrinsic changes in these transporters at the membrane level. In contrast, the downregulation of system A in intact cells when hypertonicity is prolonged for 24 h is likely due to the action of an intracellular repressor that is not present in the isolated membranes.
Assuntos
Betaína/metabolismo , Estresse Fisiológico/metabolismo , Aminoacilação de RNA de Transferência , Ácido gama-Aminobutírico/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cães , Proteínas da Membrana Plasmática de Transporte de GABA , Líquido Intracelular/fisiologia , Rim , Solução Salina Hipertônica , Aminoacilação de RNA de Transferência/efeitos dos fármacosRESUMO
In the presence of NAD+, renal brush-border membranes are mono-ADP-ribosylated by an endogenous ADP-ribosyltransferase. The reaction is inhibited by arginine methyl ester and is markedly stimulated by EDTA. Stimulation by EDTA is likely due, at least in part, to EDTA preventing the destruction of intact NAD+ by other enzymes in the brush-border membrane.
Assuntos
Ácido Edético/farmacologia , Córtex Renal/ultraestrutura , Nucleotidiltransferases/metabolismo , ADP Ribose Transferases , Animais , Guanilil Imidodifosfato/farmacologia , Córtex Renal/efeitos dos fármacos , Microvilosidades/efeitos dos fármacos , Microvilosidades/enzimologia , NAD/metabolismo , Poli(ADP-Ribose) Polimerases , Ratos , Temperatura , Fatores de TempoRESUMO
Deprivation of dietary phosphate leads to a decrease in urinary excretion of ammonium in rats which may be mediated through an alteration in ammonium transport in the proximal tubule. In the present study the OK renal epithelial cell line, a model for the proximal tubule, was used to determine if NH4+ transport was changed during acute phosphate deprivation. The intracellular pH after perfusion with NH4Cl solution was used for calculation of intracellular NH4+ concentration. Intracellular [NH4+] increased linearly during the first 2 min of acidification with NH4Cl. NH4+ transport, defined by the initial rate of the increase in intracellular [NH4+], was decreased by 32% (P < 0.005) in phosphate-deprived cells. This transport process was inhibited by barium chloride, but not by DIDS, amiloride or ouabain, suggesting that the NH4+ transport pathway may utilize K+ channels. Acute phosphate deprivation may inhibit NH4+ transport in OK cells by decreasing membrane K+ permeability.
Assuntos
Amônia/metabolismo , Túbulos Renais Proximais/metabolismo , Fosfatos/deficiência , Cloreto de Amônio/metabolismo , Animais , Bicarbonatos/metabolismo , Transporte Biológico , Linhagem Celular , Permeabilidade da Membrana Celular , Concentração de Íons de Hidrogênio , GambásRESUMO
In an attempt to identify the renal Na+/Pi cotransporter, Xenopus laevis oocytes were used to express mRNA isolated from the renal cortex of rat kidney. Na(+)-dependent uptake of Pi in oocytes, injected with mRNA, resulted in an increase of 2-4-fold as compared to oocytes injected with water. Both the new expressed and endogenous Na(+)-dependent Pi uptake activity were inhibited with 2 mM phosphonoformic acid (PFA). Expression of Pi uptake into oocytes was dose-dependent with the amount of mRNA injected. When mRNA was fractionated on a sucrose gradient, a mRNA fraction of 2.5 kilobases expressed the Na+/Pi cotransport activity in oocytes. This fraction resulted in a 6-fold stimulation of Na(+)-dependent Pi transport when compared to oocytes injected with water. The Km and Vmax for Na(+)-dependent Pi uptake were 0.18 mM and 118 pmol/oocyte per 30 min, respectively.
Assuntos
Proteínas de Transporte/biossíntese , Rim/metabolismo , Simportadores , Animais , Transporte Biológico , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Clonagem Molecular , Foscarnet , Cinética , Oócitos/metabolismo , Ácido Fosfonoacéticos/análogos & derivados , Ácido Fosfonoacéticos/farmacologia , RNA Mensageiro/metabolismo , Ratos , Proteínas Cotransportadoras de Sódio-Fosfato , Xenopus laevisRESUMO
When grown in monolayer culture, OK cells display endocytic uptake of soluble fluid-phase markers such as lucifer yellow (LY) and horseradish peroxidase (HRP). The response of this process to metabolic inhibitors was characterized in the present study. Inhibition of cell metabolism by cyanide produced a decrease in cell ATP content which was accompanied by a decrease in uptake of both LY and HRP, confirming the energy-dependence of fluid-phase endocytosis in OK cells. Use of iodoacetate also decreased cell ATP content but its action on endocytosis was unexpected. Cell uptake of HRP was decreased by iodoacetate, similar to the effect of cyanide, but there was a marked increase in LY uptake. Additional studies showed that cyanide did not change intracellular Na+ or intracellular K+ and did not interfere with the Na(+)-dependency of Pi uptake. In contrast, iodoacetate produced a marked increase in Na+, a decrease in K+, and abolished the Na(+)-dependency of Pi transport. The latter was due primarily to a 10-fold increase in Na(+)-independent uptake of Pi. These findings suggest, indirectly, that plasma membrane permeability to Na+, K+, Pi, and small molecules such as LY, may be increased by iodoacetate, possibly through its action as an alkylating agent. This mechanism may allow increased cell uptake of LY through a non-endocytic pathway, and may mask the inhibitory action of iodoacetate on endocytic uptake of LY. These additional effects complicate the use of iodoacetate to interrupt endocytosis.
Assuntos
Endocitose/efeitos dos fármacos , Iodoacetatos/farmacologia , Rim/fisiologia , Animais , Linhagem Celular , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Corantes Fluorescentes , Peroxidase do Rábano Silvestre/metabolismo , Ácido Iodoacético , Isoquinolinas , Cinética , Gambás , Fosfatos/metabolismo , Potássio/metabolismo , Cianeto de Potássio/farmacologia , Sódio/metabolismoRESUMO
1. Diazotized 2,6-diiodosulfanilic acid (DDISA) appears to have properties suitable to serve as an artificial, non-penetrating label of cell surface membranes. Therefore, the conditions for selective labeling of cell surface membranes as compared to intracellular proteins as well as a method for its chemical determination were explored in the present study. 2. DDISA reacts with alpha-naphthol at neutral pH to produce a compound (1-hydroxy-4-(2,6-diiodo-4-sulfo-1-phenylazo-(naphthylene)), DSPN) with a characteristic spectrum in the visible range (Amax 430 nm). The absorbance of the reaction product, DSPN, is linearly proportional to the concentration of DDISA and can be used as a method for the colorimetric determination of DDISA. Reaction of DDISA with a molar excess of alpha-naphthol was also used as a method for inactivating unreacted DDISA to terminate labeling prior to cell fractionation. 3. [125I]DDISA reacts avidly with a variety of basic, neutral and acidic proteins as well as with cell membranes to form an acid-stable covalent azo linkage. 4. Effectiveness of labeling of the surface membrane of intact erythrocytes after incubation with [125I]DDISA was assessed by th ratio of 125I incorporated into membrane proteins compared to intracellular proteins. When intact erythrocytes were exposed to [125I]DDISA, the optimal labeling of membranes occurred at 37 degrees C after 20 min of incubation time and at a concentration of 10(-4) M [125I]DDISA in the incubation media. Under these conditions the ratio of the specific activity (cpm 125I/mg protein) of the membrane fraction to the specific activity of the soluble protein fraction (membrane/supernatant ratio) was greater than 500. When incubations were conducted at 4 degrees C this ratio was less than 50. However, when osmotically lysed erythrocytes were incubated with [125I]DDISA the majority of the label reacted with the soluble protein fraction resulting in a membrane/supernatant ratio of 0.14. 5. The results thus suggest that [125I]DDISA used under the appropriate incubation conditions, including the inactivation and removal of [125I]DDISA by washing with alpha-naphthol, can serve as a highly selective membrane label with minimal incorporation into intracellular soluble proteins. The general applicability of this method for other cell types remains to be explored.
Assuntos
Membrana Eritrocítica/análise , Eritrócitos/análise , Proteínas de Membrana/sangue , Compostos de Diazônio , Histonas/análise , Humanos , Radioisótopos do Iodo , Marcação por Isótopo , Proteínas/análise , Ácidos Sulfanílicos/análogos & derivadosRESUMO
Cholera toxin was used to enhance ADP-ribosylation of rabbit renal brush-border membranes. Treatment of brush-border membrane sheets with cholera toxin in the presence of NAD resulted in a specific inhibition of the initial phase of Na+-dependent Pi uptake, compared to controls incubated with NAD alone. The Pi uptake was determined after conversion of the membrane sheets to vesicles. The equilibrium uptake of Pi, the Na+-independent uptake of Pi, the Na+-dependent uptake of L-proline and the activities of several brush-border membrane enzymes were not changed. The inhibition of Pi transport was dependent on the presence of both NAD and cholera toxin. Incubation of membrane sheets with [3H]NAD produced acid-stable binding of radioactivity to the membranes and the binding was increased 5-fold by the presence of cholera toxin. The use of [32P]NAD and autoradiography confirmed that the bound radioactivity was associated with several different membrane proteins, and that cholera toxin increased binding to these proteins including three that were not labelled in the absence of the toxin. The specific inhibitory action of cholera toxin on Na+/Pi cotransport is probably mediated by ADP-ribosylation of membrane proteins, suggesting that the Pi transport system can be regulated by ADP-ribosylation, at least in vitro.
Assuntos
Toxina da Cólera/farmacologia , Rim/metabolismo , Fosfatos/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Fracionamento Celular , Técnicas In Vitro , Rim/enzimologia , Potenciais da Membrana , Proteínas de Membrana/análise , Microscopia Eletrônica , Microvilosidades/enzimologia , Microvilosidades/metabolismo , Peso Molecular , Prolina/metabolismo , Coelhos , Sódio/fisiologiaRESUMO
Nicotinamide, like parathyroid hormone, is a rapidly acting specific inhibitor of Na+-dependent transport of phosphate (Pi) across the brush-border membrane of the proximal tubule of the mammalian kidney. Pretreatment of rats with colchicine (0.7 mg/kg body weight) for 1 h led to a significantly diminished phosphaturic response to parathyroid hormone (synthetic 1-34 fragment, 4 micrograms/kg). In contrast, the same dose of colchicine had no effect on the renal response to nicotinamide (1.0 g/kg), measured both as the change in urinary Pi excretion and as Na+-dependent Pi uptake by isolated brush-border membrane vesicles. These data suggest indirectly that the intracellular mechanism that mediates the inhibitory effects of nicotinamide on renal Pi transport does not require intact microtubules.
Assuntos
Colchicina/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Niacinamida/farmacologia , Hormônio Paratireóideo/antagonistas & inibidores , Fragmentos de Peptídeos/antagonistas & inibidores , Fosfatos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Ratos , Ratos Endogâmicos , Sódio/metabolismo , TeriparatidaRESUMO
Parathyroid hormone (PTH) inhibits Na(+)-dependent Pi uptake in renal epithelial cells from opossum kidney (OK). This requires an intact endocytic pathway, suggesting that one action of PTH may be to promote endocytic removal of Na+/Pi cotransporters from the cell membrane. The present study tested if PTH, at a dose that inhibited membrane Pi transport, also produced an increase in endocytic activity. Pi transport was measured in isolated plasma membrane vesicles. Endocytosis was measured by allowing cells to take up horseradish peroxidase (HRP) followed by assay of triton-sensitive (latent) HRP activity in subcellular fractions isolated by density gradient centrifugation. Incubation of OK cells with 10(-7) M PTH for 3 h decreased Na+/Pi cotransport by membrane vesicles to 328 +/- 54 pmol/mg/min compared to 448 +/- 67 pmol/mg/min (mean +/- S.E., P < 0.03) in controls. Latent HRP content of endosomal fractions was dependent on the time and temperature used to load cells with HRP and on the concentration of HRP. However, incubation of OK cells with 10(-7) M PTH for either 1 or 3 h produced no change in latent HRP activity. Thus the action of PTH on the Na+/Pi cotransporter in the plasma membrane of OK cells does not require a change in the rate of endocytosis.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Rim/metabolismo , Hormônio Paratireóideo/farmacologia , Fosfatos/metabolismo , Simportadores , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Endocitose , Peroxidase do Rábano Silvestre , Rim/efeitos dos fármacos , Cinética , Gambás , Proteínas Cotransportadoras de Sódio-FosfatoRESUMO
We tested the hypothesis that NAD plays a role in the cellular mechanism of action of parathyroid hormone (PTH) on phosphate transport. PTH significantly increased urinary cyclic AMP and phosphate excretions in thyroparathyroidectomized rats. The NAD+/NADH ratio, but not total NAD content, in renal cortical tissue was significantly higher in rats infused with PTH than in controls. The results demonstrate that the phosphaturic effect of PTH is associated with a shift in the renal cortical NADH to NAD+ and provides evidence for a role for NAD+ in the cellular regulation of phosphate transport.
Assuntos
Córtex Renal/metabolismo , NAD/metabolismo , Hormônio Paratireóideo/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , AMP Cíclico/urina , Córtex Renal/efeitos dos fármacos , Masculino , Oxirredução , Glândulas Paratireoides/fisiologia , Fosfatos/metabolismo , Fosfatos/urina , Ratos , TireoidectomiaRESUMO
The wealth of new information on BBM transport of Pi which has accumulated in recent years gives an indication of the importance and intellectual challenge that the mechanism of this process poses to investigators. In this brief reflection on the field, we have tried to draw attention to some general principles and features which may be helpful as working hypotheses in the development of the field. To date, a disproportionate amount of effort may have been spent on deciphering putative intracellular regulatory mechanisms, without knowing some essential fundamental properties of the Na+-Pi-COT. We suggest that a major effort should be exerted towards elucidating biogenesis of the Na+-Pi-COT, the possible existence of a membrane cycling mechanism, and a refined analysis of the Na+-Pi-COT in specific subsegments of proximal tubules. Advances in these areas together with studies of both the rapid and long-term adaptive regulation of Pi transport are needed, given the central role of the kidney in total body Pi homeostasis both in health and disease.
Assuntos
Túbulos Renais Proximais/metabolismo , Fosfatos/metabolismo , Adaptação Fisiológica , Animais , Transporte Biológico , Proteínas de Transporte/metabolismo , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Microvilosidades/metabolismo , NAD/metabolismo , Hormônio Paratireóideo/fisiologia , Sódio/metabolismo , Fatores de TempoRESUMO
The aims of the study were to determine whether 8-bromo-cyclic AMP (8BcAMP) in vivo mimics the inhibitory action of parathyroid hormone (PTH) on phosphate transport across the brush border membrane (BBM) of the renal proximal tubule, and to examine whether changes in BBM transport are accompanied by changes in the rate of renal gluconeogenesis. Thyroparathyroidectomized dogs were anesthetized and equilibrated, and control urine collections were obtained prior to removing the left kidney. Subsequent intravenous infusion of 8BcAMP at 50 mg/hr for 2 hr increased fractional excretion of phosphate from 4 +/- 1 (controls) to 29 +/- 4% (P less than 0.001) without changing glomerular filtration. In BBM vesicles isolated from the renal cortex, the initial Na+-dependent transport of phosphate was decreased from 747 +/- 135 (controls) to 564 +/- 126 pmoles per mg per 0.25 min after 8BcAMP (P less than 0.025), but Na+-independent phosphate uptake and Na+-dependent L-proline uptake were not changed significantly. Renal gluconeogenesis in the same animals was increased from 2.5 +/- 0.3 (controls) to 5.3 +/- 0.5 mumoles glucose per g tissue per hr after infusion of 8BcAMP (P less than 0.001). Infusion of PTH, like 8BcAMP, inhibited BBM phosphate transport and stimulated renal gluconeogenesis. We conclude that the inhibitory action of cyclic AMP and PTH on BBM phosphate transport is accompanied by stimulation of gluconeogenesis which suggests, indirectly, that changes in gluconeogenesis may be part of the intracellular mechanism for regulating BBM phosphate uptake in response to certain stimuli.
Assuntos
Membrana Celular/metabolismo , AMP Cíclico/análogos & derivados , Gluconeogênese/efeitos dos fármacos , Córtex Renal/metabolismo , Microvilosidades/metabolismo , Hormônio Paratireóideo/farmacologia , Fosfatos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica , Animais , Transporte Biológico/efeitos dos fármacos , AMP Cíclico/farmacologia , Cães , Feminino , Córtex Renal/efeitos dos fármacos , Cinética , Microvilosidades/efeitos dos fármacos , Glândulas Paratireoides/fisiologia , Prolina/metabolismo , TireoidectomiaRESUMO
Available information on the bioeffects of ESWL is insufficient to characterize the tissue injury induced by shock waves. The cellular mechanisms have not been elucidated, nor are there enough data to establish objective criteria for treatment.
Assuntos
Litotripsia/efeitos adversos , Cálculos Urinários/terapia , Animais , Células Cultivadas , Edema/etiologia , Gastroenteropatias/etiologia , Hemorragia/etiologia , Humanos , Rim/lesões , Rim/patologia , Nefropatias/etiologiaRESUMO
Transmission electron microscopy has been used to study the ultrastructure of rat jejunum, ileum and caecum after dietary supplementation with 10% (w/w) gum arabic, 7% (w/w) gum karaya, 4% (w/w) gum tragacanth for 45 days. Scrutiny of 65 micrographs, undertaken independently by 2 specialists, revealed no abnormalities in any of the organelles in any of the micrographs. Twelve representative micrographs are reproduced here.