共查询到20条相似文献,搜索用时 15 毫秒
1.
V M Bresler S N Val'ter V V Isaev-Ivanov E N Kazbekov A P Kle?ner 《Tsitologiia》1987,29(10):1177-1184
The kinetics of the transport of 3H-para-aminohippuric acid (PAH) and the influence of the temperature on the initial rate of transport were studied on the vesicles of a purified fraction of the apical membrane isolated from cells of kidney proximal tubules. The PAH transport is accomplished owing to the facilitate diffusion mechanism. The apparent Michaelis constant at 36 degrees C was equal to 7.0 + 1.0 mM, the maximum rate was 15 nmol/min on 1 mg of protein, the inhibition constant for the PAH transport by probenecid being 0.5 mM. At 22 degrees C the apparent Michaelis constant was drastically increased. When the temperature dependence of the initial rate of PAH transport into vesicles was replotted in the form of the Arrhenius plot, there was a turning-point of the line at 28-30 degrees C. The same turning-point is shown on the Arrhenius plot for temperature dependence of alkaline phosphatase activity (a marker enzyme for the apical membrane). The electron paramagnetic resonance spectra analysis of 5-doxylstearate-labeled apical membrane preparation reveals a thermotropic transition near 21-29 degrees C. It is concluded that the function of the carrier and the activity of alkaline phosphatase depend on the phasic state of membrane lipids; the normal function of membrane proteins is possible under the liquid-crystalline state of the lipid bilayer. 相似文献
2.
A A Nikiforov 《Tsitologiia》1985,27(7):834-837
Cadmium ions (10(-5)-10(-3) M) stimulate Na-dependent transport of a weak organic acid, fluorescein, into the proximal tubules of surviving frog kidney. Their stimulatory action ceases with increasing the duration of incubation to 45-60 minutes (stimulation does not disappear after introducing acetate into the incubating medium), in the presence of amiloride in the tubular lumen or in the absence of Na+ from the medium. The data obtained in the present work coincide with the previously reported evidence of the influence of Cd2+ on the Na-independent fluorescein transport into the proximal tubules of rat kidney. They are in good accordance with the suggestion that the effect of Cd2+ of the weak organic acid transport is mediated through an acceleration of the active reabsorption of Na+ with the accompanying activation of Na,K-ATPase. 相似文献
3.
A.A. Nikiforov 《生物化学与生物物理学报:生物膜》1982,686(1):36-46
The effect of acetate on active fluorescein transport in intact proximal tubules of surviving frog kidney was studied. When the kidneys were incubated in a 120 mM Na+ medium, 10 mM acetate stimulated fluorescein uptake in the tubules. The stimulation was more pronounced if the kidneys had been previously preincubated for 3 h in the substrate-free solution. Lowering of the Na+ concentration in the bathing medium to 10 mM resulted in the disappearance of the acetate effect. Preincubation of the kidneys with acetate at 2–4°C gave rise to stimulation of the fluorescein transport only in the 120 mM Na+ acetate-free medium. The acetate effect on the fluorescein uptake was partially prevented by ouabain. The stimulation of the uptake by acetate in the 120 mM Na+ medium correlated with an increase in the extent of reduction of pyridine nucleotides in the tubules. The pyridine nucleotides were reduced more markedly after incubation of the kidneys in the 10 mM Na+ medium, when acetate had no effect on the fluorescein transport. In both the 120 mM and the 10 mM Na+ media, the cold preincubation of the kidneys with 2.5 mM ADP or 2.5 mM ATP resulted in only slight stimulation of the fluorescein uptake. But in both media the uptake was significantly enhanced after cold preincubation of the kidneys with 2 mM NADH. After the cold precincubation with ADP, stimulation of the fluorescein transport by acetate was observed in the case of the 10 mM Na+ medium also. The absence of any stimulatory effect of acetate on the organic acid transport in the 10 mM Na+ medium is explained as the result of the transformation of mitochondria in the tubular cells into the inactive state 4 due to a decrease in the intracellular ADP level. Reducing equivalents are supposed to take part in energization and/or regulation of transport processes in plasma membranes of the renal proximal tubules. 相似文献
4.
Calcium dependence of the stimulating action of cadmium on organic acid transport in the frog kidney
A A Nikiforov 《Tsitologiia》1985,27(8):887-894
The stimulatory effect of cadmium ions on the Na-dependent fluorescein transport into the frog renal proximal tubules ceased with decreasing Ca++ concentration in solution on both the sides of the cell layer down to micromolar level. The decrease in Ca++ concentration per se stimulated fluorescein uptake during short-term incubations. A further diminution of Ca++ concentration in the tubular lumen with the aid of EGTA resulted in a sharp inhibition of the organic acid transport. Amiloride, which prevented the stimulatory effect of cadmium, inhibited the fluorescein transport at both millimolar and micromolar levels of Ca++ concentration, but it failed to affect the transport process after introducing EGTA into the tubular lumen. The results are discussed within the frames of a model regarding extracellular Ca++ as an allosteric inhibitor, and intracellular Ca++ as an allosteric activator of sodium channels in the apical membrane. Cd++ is assumed to compete with Ca++ for binding to centers of the allosteric inhibition, thereby accelerating the sodium ion flux across the cells of the proximal tubules. 相似文献
5.
The embryonic kidneys of larval aquatic vertebrates such as fish and frogs serve as excellent model systems for exploring the early development of nephric organs. These experimental systems can easily be manipulated by microsurgery, microinjection, genetics, or combinations of these approaches. However, little is known about how physiologically similar these simple kidneys are to the more complex mammalian adult kidneys. In addition, almost nothing is known about proximo-distal patterning of nephrons in any organism. In order begin to explore the physiological specialization of the pronephric tubules along the proximo-distal axis, a combination of uptake assays using fluorescently tagged proteins, LDL particles and dextrans, and an informatics-targeted in situ screen for transport proteins have been performed on embryos of the frog, Xenopus laevis. Genes identified to be expressed within unique subdomains of the pronephric tubules include an ABC transporter, two amino acid cotransporters, two sodium bicarbonate cotransporters, a novel sodium glucose cotransporter, a sodium potassium chloride cotransporter (NKCC2), a sodium chloride organic solute cotransporter (ROSIT), and a zinc transporter. A novel combination of colorimetric and fluorescent whole-mount in situ hybridization (FCIS) was used to precisely map the expression domain of each gene within the pronephros. These data indicate specialized physiological function and define multiple novel segments of the pronephric tubules, which contain at least six distinct transport domains. Uptake studies identified functional transport domains and also demonstrated that early glomeral leakage can allow visualization of protein movement into the pronephric tubules and thus establish a system for investigating experimentally induced proteinuria and glomerulonephritis. 相似文献
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7.
E K Park S K Hong J Goldinger G Andres B Noble 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1985,180(1):174-184
Organic ion transport across the basolateral membrane of proximal tubules was measured by means of the tissue slice technique in each of the four different stages of Heymann nephritis. Impairment of both organic anion and cation transport was detected early in Stage 2, and became more severe in Stage 3 of Heymann nephritis. The decreased transport function was associated with extensive damage to proximal tubule cells, including loss of brush border microvilli and basal infoldings. Despite these abnormalities of structure and function, oxygen consumption of proximal tubule cells remained essentially normal. Partial recovery of organic cation transport was noted late in Heymann nephritis (Stage 4). Recovery of the cation transport function was associated with a partial restoration of brush border microvilli and basal infoldings to proximal tubule cells. However, organic anion transport remained depressed throughout the entire course of disease. Impairment of organic ion transport in rats with Heymann nephritis appeared to result from damage to basolateral membrane transport elements rather than general deterioration of the metabolic machinery of proximal tubule cells. Decreased organic cation transport appeared to be the consequence of a reduction in the number of carrier sites, a phenomenon that could have resulted from decreased membrane surface area. However, the depression of organic anion transport was associated with decreased substrate affinity of the anion carrier, indicating that qualitative, rather than quantitative changes, were primarily responsible for that defect. Specific antibody-mediated damage to the anion transport elements in basolateral membranes of proximal tubules is postulated to occur in Heymann nephritis. 相似文献
8.
We have shown previously that OK cells recover from an acid load in a medium nominally CO2-free by extruding H via a Na/H exchanger and a passive H-conductive pathway. In this work, the regulation of cell pH (pHi) was studied after addition or withdrawal of CO2/HCO3 (5% CO2, 95 mM HCO3, pH = 8) using the fluoroprobe BCECF. In the presence of Na and amiloride to inhibit Na/H exchange, the recovery of pHi after CO2 entry and CO2 exit were found to depend in part on HCO3 entry and exit, respectively. Efflux of H per se also contributed to restoring pHi after CO2 addition, whereas H influx may have played a smaller role to normalize pHi after CO2 removal. DIDS, 0.5 mM, significantly inhibited both recovery phases of pHi. Removal of Na failed to inhibit the recovery of pHi after CO2 addition and removal. Cl removal also failed to inhibit pHi recovery after CO2 removal. Cell depolarization in the presence of Na moderately stimulated the pHi recovery rate after CO2 addition whereas it markedly inhibited the normalization of pHi after CO2 removal. Cell depolarization in the absence of sodium had only a slight effect to increase pHi recovery after CO2 addition but markedly prevented the pHi recovery after CO2 removal. These results indicate that OK cells lack Na or Cl-dependent HCO3 transport systems. The OK cell possesses a novel stilbene-sensitive electrogenic HCO3 transport system that is involved in the regulation of cell pH. 相似文献
9.
R W Chesney I Zelikovic S Dabbagh A Friedman S Lippincott 《The Journal of experimental zoology》1988,248(1):25-32
This study focuses on the maturation of the renal beta-amino acid transport system and uses dietary manipulation as a probe. The epithelial surface of the renal proximal tubule is responsible for the conservation of ions and organic solutes including beta-amino acids. This beta-amino acid transport system is stimulated during periods of reduced dietary intake and permits increased excretion following dietary excess. We have examined transport of the sulfur-containing beta-amino acid, taurine, as a measure of this renal adaptive response to fluctuations in dietary sulfur amino acid intake and as a substrate for the beta-amino acid transport system. A precession of taurine uptake values by brush border membrane vesicles (BBMV) prepared from nursing rats from youngest to oldest was evident. However, these membranes demonstrate the full renal adaptive response to altered sulfur amino acid intake after the first week of life. This adaptive response is expressed at the brush border surface by transport changes in both directions ("up regulation" and "down regulation"), through changes in the initial rate (15 sec) of Na+-taurine cotransport. No alterations in the lipid microenvironment of the membrane, as detected by altered membrane fluidity, were uncovered. Although vesicles from 7-day-old pups demonstrate adaptation and accumulate taurine to a limited extent, the accumulation of Na+, which energizes uptake, may be altered, thereby preventing full expression of the adaptive response and of transport capacity at this age. 相似文献
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12.
The kinetics of 14C-para-aminohippuric acid (PAH) transport in the vesicles and the influence of the temperature on the initial rate of this transport were studied using a purified fraction of the apical membrane isolated from the kidney cortex of the Campbell strain rats with an autosomic recessive gene. The transport was brought about owing to the facilitate diffusion mechanism. At 36 degrees C the apparent Michaelis constant was equal to 29 mM, the maximum rate--62 nmol/min on 1 mg of protein, the inhibition constant for the PAH-transport by probenecid--1.5 mM. The temperature dependence of the initial rate of PAH-transport in vesicles and that of the rate of substrate splitting by alkaline phosphatase show the break point on the Arrhenius plot at 36 degrees C-38 degrees C. The analysis of electron magnetic resonance reveals the thermotropic transition at temperatures near 30 degrees-35 degrees C. Therefore the affinity of the carrier to its substrates in vesicles of the Campbell strain rats is strongly reduced and the lipid layer is more viscous than in the normal rats. We decide therefore that the mutation taking place in the Campbell strain leads to pleotropic membrane reconstructions in different organs (eye, kidney). The discovery of such a mutation is of considerable biological interest and promotes bases for development of the membrane biochemical genetics. 相似文献
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14.
Summary We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (V
bl) in rabbit proximal convoluted tubule (PCT. Sl segment). The present study is aimed at understanding the origin of the membrane repolarization following the intial effect of addition of luminal cotransported solutes. Luminal addition of 10–15mMl-alanine produced a rapid and highly significant depolarization ofV
bl (20.3±1.1 mV,n=15) which was transient and associated with an increase in the fractional K+ conductance of the basolateral membrane (t
K) from 8 to 29% (P<0.01,n=6). Despite the significant increase int
K, the repolarization was only slightly reduced by the presence of basolateral Ba2+ (2mM,n=6) or quinine (0.5 mM,n=5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4isothiocyamostilbene-2,2-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pH
i
) determined with the fluorescent dye 2, 7-bis-2-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF), averaged 7.39±0.02 in control solution (n=9) and increased to 7.50±0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16±0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K+ conductance but also through a cellular acidification reducing the basolateral HCO
3
–
exit through the electrogenic Na-3(HCO3) cotransport mechanism. 相似文献
15.
The proximal cecum of 5- and 13-wk-old chickens takes up L-proline (Pro) by two saturable pathways that differ in their requirement of Na+. The kinetic properties of Pro influx in cecal segments incubated in Na(+)-free conditions, have been studied and the effect of HgCl2 on the uptake process has been tested. Experiments were carried out using an in vitro everted-sleeve method. Kinetic parameters were estimated by nonlinear regression analysis. One min Pro fluxes in tissues incubated in presence of HgCl2 fit a straight line, indicating that a Na-independent saturable component was inhibited. Estimated Kd* values are the same in 5- and 13-wk-old chickens, 0.023 +/- 0.001 and 0.027 +/- 0.001 microliters.mg-1 x min-1, respectively. HgCl2-sensitive fluxes fit a Michaelis hyperbola, with similar Km* values, 4.85 +/- 1.86 (5-wk) and 9.47 +/- 3.0 (13-wk) mmol/l. However, Vmax* in 5-wk chickens (0.662 +/- 0.053 nmol.mg-1 x min-1) is higher than in 13-wk birds (0.420 +/- 0.039), in accordance with previous results. The present data give further support to the existence of a Na-independent L-proline carrier in the chicken proximal cecum which is inhibitable by HgCl2. 相似文献
16.
The molecular and cellular physiology of basolateral organic anion transport in mammalian renal tubules 总被引:1,自引:0,他引:1
Basolateral transport of organic anions (OAs) into mammalian renal proximal tubule cells is a tertiary active transport process. The final step in this process involves movement of OA into the cells against its electrochemical gradient in exchange for alpha-ketoglutarate (alphaKG) moving down its electrochemical gradient. Two homologous transport proteins (OAT1 and OAT3) that function as basolateral OA/alphaKG exchangers have been cloned and sequenced. We are in the process of determining the functional distribution and regulation of OAT1 and OAT3 in renal tubules. We are using rabbit OAT1 (rbOAT1) and OAT3 (rbOAT3) expressed in heterologous cell systems to determine substrate specificity and putative regulatory steps and isolated rabbit proximal renal tubule segments to determine functional distribution and physiological regulation of these transporters within their native epithelium. Rabbit OAT1 and OAT3 differ distinctly in substrate specificity. For example, rbOAT1 has a high affinity for the classical renal OA transport substrate, p-aminohippurate (PAH), whereas rbOAT3 has no affinity for PAH. In contrast, rbOAT3 has a high affinity for estrone sulfate (ES), whereas rbOAT1 has only a very slight affinity for ES. Both rbOAT1 and rbOAT3 appear to have about the same affinity for fluorescein (FL). These differences and similarities in substrate affinities make it possible to functionally map transporters along the renal tubules. Initial data indicate that OAT1 predominates in S2 segments of the rabbit proximal tubules, but studies of other segments are just beginning. Transport of a given substrate in any tubule segment depends on both the affinity of each transporter which can accept that substrate as well as the level of expression of each of those processes in that particular tubule segment. Basolateral PAH transport (presumably OAT1 activity) appears to be down-regulated by activation of protein kinase C (PKC) and up-regulated via mitogen-activated protein kinase (MAPK) through phospholipase A(2) (PLA(2)), prostaglandin E(2) (PGE(2)), cyclic AMP, and protein kinase A (PKA) activation. 相似文献
17.
Properties and regulation of organic cation transport in freshly isolated human proximal tubules. 总被引:4,自引:0,他引:4
G Pietig T Mehrens J R Hirsch I Cetinkaya H Piechota E Schlatter 《The Journal of biological chemistry》2001,276(36):33741-33746
The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases. 相似文献
18.
With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na+-flow in the opposite direction was studied. It was shown that the complete removal of Na+ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na+ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10?5 M), a specific inhibitor of (Na+, K+)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na+ should be present in proximal tubules lumen.Thus, the various effects on the Na+ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport () with unchanged . It is suggested that the system of Na+ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na+ concentration which may be higher than the summary Na+ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na+-dependent organic acid transport. So, there is a double dependence of the active transport system on Na+, and the stages where Na+ is needed are: (1) the formation of a carrier-substrate-Na+ complex and (2) the production of substantial membrane-side Na+ gradient at the expense of Na+ extrusion from the tubules. 相似文献
19.
Harold Koenig Alfred Goldstone Chung Y. Lu 《Biochemical and biophysical research communications》1982,106(2):346-353
Testosterone induced a rapid (<1 min) stimulation of endocytosis, amino acid and hexose transport, measured by the temperature-sensitive uptake of HRP, 14C-AIB and 3H-DG, in mouse kidney cortex slices. The hormonal increment in uptake persisted for at least 60–120 min, showed time-, energy-, and Na+-dependence, and varied with substrate and testosterone concentration. Testosterone was maximally effective at 10?8 to 10?7 M. Peroxidase histochemistry indicated that the hormonal increase in HRP uptake is restricted to proximal tubules. Testosterone was more effective than DHT, whereas cyproterone acetate, androsterone and dexamethasone had little or no stimulating effect on this uptake. Kidney slices from androgen-insensitive mice did not respond to testosterone. The rapid increase in endocytosis, amino acid and hexose transport may represent a direct, receptor-mediated response of the surface membrane of target cells to testosterone. 相似文献
20.
N Burgmeier R Zawislak F V Defeudis C Bollack J J Helwig 《European journal of biochemistry》1985,151(2):361-364
4-Aminobutyric acid (GABA) synthesis was examined in purified glomeruli and tubules of rat kidney cortex that were incubated in the presence of [2,3-3H2]glutamate. The GABA that was formed was separated from glutamate using anion-exchange resin, and identified by means of an automatic amino acid analyser. In the renal cortex only the tubules were able to form GABA (35.0 nmol mg-1 h-1); the remaining GABA synthesis found in the glomerular preparations can most probably be attributed to a contamination by cortical tubules (9%), as shown by determination of a known tubular marker enzyme (L-gamma-glutamyltransferase). Hydroxylamine (1 mM) and ethanolamine-O-sulfate (10 mM), well-known inhibitors of cerebral GABA formation and GABA catabolism respectively, inhibited renal tubular GABA formation at 100% and 44% respectively. 相似文献