首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
A Cl/HCO3 exchanger mediates HCO3 extrusion across rat jejunal basolateral membrane. Previous studies demonstrated that anion antiport activity is positively affected by Na, but evidence was given that this cation is not translocated by the carrier protein. Basolateral membranes isolated from rat jejunum were used to give more insight on Na effect. Uptake studies, performed together with vesicle sidedness determinations, indicated that the greatest stimulation of Cl-dependent HCO3 uptake occurs when Na is present at both vesicle surfaces. The kinetic dependence of Cl/HCO3 exchange on equal intra- and extravesicular Na concentration showed a hyperbolic relationship, and the calculated kinetic parameters were V max=0.153 ± 0.006 nmol mg protein-1 sec-1, K m =23.0 Mm. Ion replacement studies indicated that Na can be partially substituted only by Li and not by other monovalent cations. Results of this study suggest that Na could act as a nonessential activator of the Cl/HCO3 exchanger. A possible role of the Na-sensitive modifier site in the physiology of jejunal enterocyte is suggested.  相似文献   

2.
Summary Basolateral membrane vesicles isolated from rat jejunal enterocyte and well purified from brush border contamination were tested to examine Cl and HCO3 movements. Uptake experiments provided no evidence for a coupling between Na and HCO3 fluxes; K–HCO3 and K–Cl cotransports also could be excluded. Transport studies revealed the presence of a Cl/HCO3 exchanger accepting other anions and inhibitable by the disulfonic stilbenes SITS and DIDS. We can exclude that the evidenced HCO3-dependent Cl uptake is due to brush border contamination, since in jejunal brush border membranes this mechanism, if present, has a very low transport rate. Besides the Cl/HCO3 antiporter, a Cl-conductive pathway seems to exist in jejunal basolateral membranes.  相似文献   

3.
The cellular model of short chain fatty acid stimulation of electroneutral Na-Cl absorption in large intestine proposes that SCFA, following its uptake across the apical membrane, recycles and is coupled to functional Na-H and Cl-short chain fatty acid exchanges. To establish the presence of a Cl-butyrate exchange (used as a model short chain fatty acid), studies of 36Cl and 14C-butyrate uptake across apical membrane vesicles of rat distal colon were performed. An outward butyrate-gradient stimulated transient accumulation of 36Cl uptake that was not inhibited by pH clamping with valinomycin (a K ionophore) and FCCP (a proton ionophore). Outward butyrate-gradient-stimulated 36Cl uptake was inhibited by 4,4-diisothiocyanatostilbene2,2-disulfonic acid (DIDS) with a half-maximal inhibitory concentration (IC50) of 68.4 m, and was saturated by both increasing extravesicular Cl concentration (K m for Cl of 26.8 ±3.4 mm and a V max of 12.4±0.6 nmol/mg protein·9 sec) and increasing intravesicular butyrate concentration (K m for butyrate of 5.9 mm and a V max for Cl of 5.9 nmol/mg protein · 9 sec). 36Cl uptake was also stimulated by outward gradients of other short chain fatty acids (e.g., propionate, acetate and formate). In contrast, an outward Cl gradient failed to enhance 14C-butyrate uptake. Extravesicular Cl more than extravesicular butyrate enhanced 36Cl efflux from apical membrane vesicles. These studies provide compelling evidence for the presence of an electroneutral, pH-activated, Cl-butyrate exchange which in concert with Na-H exchange is the mechanism by which butyrate stimulates electroneutral Na-Cl absorption.Abbreviations used AMV apical membrane vesicles - BLMV basolateral membrane vesicles - DIDS 4,4-diisothiocyanatostilbene 2,2-disulfonic acid - FCCP carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - MES 1-[N-morpholino]ethanesulfonic acid - NMG N-memyl-d-glucamine - SCF Ashort chain fatty acid This study was supported in part by a Public Health Service research Grant (DK 14669) provided by the National Institute of Diabetes, Digestive and Kidney Diseases. Ms. Mary Guidone provided excellent secretarial assistance.  相似文献   

4.
Summary The fluorescence enhancement of 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. Dix and A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (K d =3.8 m) and rat (3.2 m); 100 m dihydro-4,4-diisothiocyano-2,2-disulfonic stilbene (H2DIDS) blocked >95% of binding. H2DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDSK d doubled with 10mm SO4, 50mm HCO3 and 100mm Cl, but was not altered by Na or pH (6–8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO4, HCO3 and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used35SO4 uptake to characterize several modes of rabbit BLM anion transport: H/SO4 and Na/SO4 cotransport, and Cl/SO4 countertransport. Each transport process was electroneutral and was inhibited by H2DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparentK t 's for DBDS (3–20 m) were similar toK d for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measureable optically by a fluorescent stilbene.  相似文献   

5.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K m of 39.2 ± 4.8 mm and a J max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997  相似文献   

6.
Summary The effect of bicarbonate (HCO3) on fluid absorption by guinea pig gallbladder was investigatedin vitro. Stimulation of fluid absorption was concentration dependent resulting in a fourfold increase in transport over the range 1 to 50mm. Phosphate, Tris, glycodiazine and glutamine buffers failed to substitutte for HCO3 in stimulating absorption. Unidirectional22Na fluxes were measured across short-circuited sheets of guinea pig and rabbit gallbladders mounted in Ussing-type chambers. In both species the net Na flux was unaffected by serosal HCO3 alone but was stimulated by addition of HCO3 to the mucosal bathing solution. Transepithelial electrical potential difference in rabbit gallbladder was about 1.4 mV (lumen positive) when HCO3 was present in the mucosal or in both compartments. This fell to 0.2 mV under HCO3-free conditions or when HCO3 was present only in the serosal solution. The respective values for guinea pig gallbladder were –1.6 and –0.6 mV (lumen negative). HCO3 stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 7.8, an effect resulting mainly from a reduction inJ mis Na . Tris buffer (25mm) inhibited HCO3-dependent fluid absorption in this species completely at pH 8.5 and partially at 7.5. These results indicate that HCO3 stimulates gallbladder transport in both species by an action from the mucosal side. This effect cannot be attributed to simple buffering of H+ but may be explained by the participation of HCO3 in the maintenance of intracellular H+ for a Na/H-exchange.  相似文献   

7.
The first step of riboflavin absorption was studied by determining the uptake of the vitamin by rat small intestinal brush border membrane vesicles. Vesicles were incubated at 25°C in the presence of [3H]-riboflavin at concentrations within the physiological intraluminal range for rat. The time course of [3H]-riboflavin uptake was unaffected by Na+ or K+ gradients. The 5 sec uptake rate plotted as a function of the initial concentration of [3H]-riboflavin in the medium (0.125 to 7.5 m) revealed the presence of a dual mechanism, with a saturable component (apparent kinetic constants: 0.12 m for K m and 0.36 pmol · mg-1 protein · 5 sec-1 for J max) prevailing at low concentrations (<2 m), and a nonsaturable component prevailing at higher concentrations. The presence of a carrier-mediated system for riboflavin was validated by counter-transport experiments. At equilibrium, uptake was almost completely accounted for by membrane binding, whereas at earlier times the transport component accounted for about 30% of total uptake. The plot of [3H]-riboflavin binding at equilibrium as a function of its concentration in the medium was quite similar to that of the 5 sec uptake rate in both intact and osmotically shocked vesicles and demonstrated the occurrence of a saturable component: binding constants were 0.07 (K d) in m), 0.54 (B max in pmol · mg-1 protein), and 0.11 (K d), 1.13 (B max, respectively, indicating the existence of specific riboflavin binding sites. The specificity of riboflavin binding to the membrane was confirmed by preliminary studies with structural analogues. Specific binding could represent the first step of a specific riboflavin entry mechanism in enterocytes.This research was supported by grants from Italian MPI 60% (1989, 1992) and CNR n. 90, 02467 CT 04. We wish to express our gratitude to Prof. E. Perucca (Department of Internal Medicine, Clinical Pharmacology Unit, University of Pavia) for revising the English, and to Mrs. M. Agrati Greco and Mrs. P. Vai Gatti for secretarial assistance and excellent typing.  相似文献   

8.
Relationships between short-circuit current (I sc), cell Cl and the mechanism(s) of Cl accumulation in toad bladder epithelial cells were investigated. In serosal Cl-free gluconate Ringer, 80% of the cell Cl (measured by x-ray microanalysis) was lost over 30–60 min with an associated decrease in cell water content. Concomitantly, I sc fell to 20% of its initial value within 10 min but then recovered to 45% of its initial value despite continued Cl loss. With the reintroduction of Cl, cell Cl and I sc both recovered within 10 min. Serosal SITS (4 acetamido-4-isothiocyano-stilbene-2,2-disulfonate; 0.5 mm) plus bumetanide (0.1 mm), did not prevent the fall in I sc or the loss of cell Cl in gluconate medium, although they did inhibit subsequent recovery of I sc in this medium. They also prevented the recovery of I sc in Cl medium but not the reaccumulation of Cl by the cells. Although SITS and bumetanide did not prevent the loss or recovery of Cl, they modified the pattern of the ion changes. In their absence, changes in cellular Cl were twice that of the changes in measured cellular cations implicating basolateral Cl/HCO3 exchange in Cl movement. With SITS plus bumetanide present, changes of similar magnitude in Cl were associated with equivalent changes in cation, consistent with the inhibition of Cl/ HCO3 exchange.This work was supported by a grant from the Medical Research Council of New Zealand. Purchase of equipment was made possible through grants from the Medical Research Council of New Zealand, the Medical Distribution Committee of the Lottery Board, the University Grants Committee, the Telford Trust, the New Zealand Neurological Foundation and the National Heart Foundation. The expert technical assistance of S. Zellhuber-McMillan is gratefully acknowledged.  相似文献   

9.
Summary Models for active Cl transport across epithelia are often assumed to be universal although they are based on detailed studies of a relatively small number of epithelia from vertebrate animals. Epithelial Cl transport is also important in many invertebrates, but little is known regarding its cellular mechanisms. We used short-circuit current, tracer fluxes and ion substitutions to investigate the basic properties of Cl absorption by locust hindgut, an epithelium which is ideally suited for transport studies. Serosal addition of 1mm adenosine 35-cyclic monophosphate (cAMP), a known stimulant of Cl transport in this tissue, increased short-circuit current (I sc) and net reabsorptive36Cl flux (J net Cl ) by 1000%. Cl absorption did not exhibit an exchange diffusion component and was highly selective over all anions tested except Br. Several predictions of Na- and HCO3-coupled models for Cl transport were tested: Cl-dependentI sc was not affected by sodium removal (<0.05mm) during the first 75 min. Also, a large stimulation ofJ net Cl was elicited by cAMP when recta were bathed for 6 hr in nominally Na-free saline (<0.001 to 0.2mm) and there was no correlation between Cl transport rate and the presence of micromolar quantities of Na contamination. Increased unidirectional influx of36Cl into rectal tissue during cAMP-stimulation was not accompanied by a comparable uptake of22Na.J net Cl was independent of exogenous CO2 and HCO3, but was strongly dependent on the presence of K. These results suggest that the major fraction of Cl transport across this insect epithelium occurs by an unusual K-dependent mechanism that does not directly require Na or HCO3.  相似文献   

10.
Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (I SC), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on I SC when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent I SC response to calcium ionophores. Carbachol (100 m) transiently elevated intracellular free calcium ([Ca2+] i ) by 3-fold in confluent cells cultured on glass coverslips with a time course resembling the I sc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (–54 mV) with pipette solution containing 150 mm KCl (37°C). This rectification persisted when patches were bathed in symmetrical 150 mm KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mm barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated I SC was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues.We thank Dr. Marcel Crest (Laboratoire de Neurobiologie, CNRS, Marseille) for providing a sample of kaliotoxin. This work was supported by the Canadian Cystic Fibrosis Foundation and the Respiratory Health Network of Centres of Excellence. J.W.H. is a Chercheur-Boursier of the Fonds de la recherche en santé du Québec.  相似文献   

11.
Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO3 gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO3 gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound3[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK D of 3.5×10–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.  相似文献   

12.
We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140 mm) NaHCO3. Experimental data suggest that NaHCO3 secretion occurs in three steps: (i) accumulation of HCO 3 across the basolateral membrane of the duct cell by Na(HCO3) n cotransporters, Na+/H+ exchangers and proton pumps; (ii) secretion of HCO 3 across the luminal membrane on Cl/HCO 3 antiporters operating in parallel with Cl channels; and (iii) diffusion of Na+ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that this mechanism for HCO 3 secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO 3-rich fluid, it can only raise the luminal HCO 3 concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO3 by the model it is necessary to: (i) reduce the conductive Cl permeability and increase the conductive HCO 3 permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl/HCO 3 antiporters. Under these conditions most of the HCO 3 is secreted via a conductive pathway. Based on our data, we propose that HCO 3 secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO 3 concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO 3 to ∼140 mm). Received: 15 November 1999/Revised: 29 March 2000  相似文献   

13.
The jejunal basolateral Cl/HCO3 exchanger is modulated by two Na-dependent regulatory sites located on the inner and outer membrane surfaces. The aim of this work was to focus on the interaction between the anion exchanger and intracellular or extracellular sodium. Uptake studies, performed using basolateral membrane vesicles, provided kinetic parameters as a function of outside or inside Na concentration. The intracellular Na-sensitive modifier site seems to be primarily involved in the modulation of the Cl/HCO3 exchanger.  相似文献   

14.
Electrogenic 2 Na+/1 H+ exchange in crustanceans   总被引:4,自引:0,他引:4  
Summary Hepatopancreatic brush border membrane vesicles of the freshwater prawn,Macrobrachium rosenbergii and the marine lobster,Homarus americanus exhibited22Na uptake which was Cl-independent, amiloride sensitive, and stimulated by a transmembrane H gradient (H i >H o ). Sodium influx by vesicles of both species were sigmoidal functions of [Na] o , yielding Hill coefficients that were not significantly different (P>0.5) than 2.0. Estimations of half-saturation constants (K Na) were 82.2mm (prawn) and 280.1mm (lobster), suggesting a possible adaptation of this transporter to environmental salinity.Trans-stimulation andcis-inhibition experiments involving variable [H] suggested that the exchangers in both species possessed single internal cation binding sites (pK 6.5–6.7) and two external cation binding sites (prawn, pK 4.0 and 5.7; lobster pK 3.5 and 6.1). Similarcis inhibition studies using amiloride as a competitive inhibitor of Na uptake supported the occurrence of dual external sites (prawn,K i 50 and 1520 m; lobsterK i 9 and 340 m). Electrogenic Na/H exchange by vesicles from both crustaceans was demonstrated using equilibrium shift experiments where a transmembrane potential was used as the only driving force for the transport event. Transport stoichiometries of the antiporters were determined using Static Head analysis where driving forces for cation transfer were balanced using a 101 Na gradient, a 1001 H gradient, and a stoichiometry of 2.0. These electrogenic 2 Na/1 H exchangers appear thermodynamically capable of generating sufficient gastric acidification for organismic digestive activities.  相似文献   

15.
Summary The electrical responses induced by external applications of neutral amino acids were used to determine whether different carriers are expressed in the membrane of embryonic epithelial cells ofXenopus laevis. Competition experiments were performed under voltage-clamp conditions at constant membrane potential.Gly,l-Ala,l-Pro,l-Ser,l-Asn andl-Gln generate electrical responses with similar apparent kinetic constants and compete for the same carrier. They are [Na] o and voltage-dependent, insensitive to variations in [Cl] o and [HCO3] o , inhibited by pH o changes, by amiloride and, for a large fraction of the current, by MeAIB. The increase in [K] o at constant and negative membrane potential reduces the response, whereas lowering [K] o augments it. l-Leu,l-Phe andl-Pro appear to compete for another carrier. They generate electrogenic responses insensitive to amiloride and MeAIB, as well as to alterations of membrane potential, [Na] o and [K] o . Lowering [Cl] o decreases their size, whereas increasing [HCO3] o at neutral pH o increases it.It is concluded that at least two and possibly three transport systems (A, ASC and L) are expressed in the membrane of the embryonic cells studied. An unexpected electrogenic character of the L system is revealed by the present study and seems to be indirectly linked to the transport function. l-Pro seems to be transported by system A or ASC in the presence of Na and by system L in the absence of Na. MeAIB induces an inward current.  相似文献   

16.
Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J Cl) was increased significantly from 0.32±0.12 nmol · sec–1 · mg protein–1 (mean±sem) to 0.50±0.07 nmol · sec–1 · mg protein–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ Cl from a control value of 0.37±0.03 nmol · sec–1 · mg protein–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ Cl significantly (J Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H2DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H2DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J Br andJ 1, respectively) were not significantly different fromJ Cl.J Cl was a saturable function of Cl concentration with apparentK d of 24mm and apparentV max of 0.54 nmol · sec–1 · mg protein–1. Measurement of the temperature dependence ofJ Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.  相似文献   

17.
Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK m approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK d 0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK i values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK d 1.05±0.13nM and apparentK i values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM r on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.  相似文献   

18.
Summary Intracellular C1, K and Na activities (a Cl i ,a k i anda Na i ) and transmucosal membrane potential (E m) in epithelial cells ofNecturus gallbladder were measured at different external Na concentrations ([Na]o), with liquid ion-exchanger and conventional microelectrodes. Bladders were mounted in a divided chamber at 23°C between identical HCO3-free Ringer solutions containing 5mm K. The pH was 7.2. Tris was substituted for Na. Measurements were made under steady-state conditions as determined by the constancy of the transepithelial potential difference. Both,a Cl i anda Na i increased in a saturable fashion with [Na]o.E m did not change significantly. Average values (±sem) under normal conditions ([Na]o=100mm) fora Cl i ,a Na i andE m were 16.8±0.8mm (n=9), 9.7±0.6mm (n=10) and –52.6±0.6 mV (n=26), respectively. In Na-free mediaa Cl i declined to its equilibrium value.a K i (96±2mm;n=7) did not change when [Na]o was varied between 100 and 10mm but decreased to 80±3mm (n=4) in Na-free media.Transmembrane electrochemical potential differences, , for Cl and Na were calculated at four different [Na]o levels. A highly significant linear relation between and was found, indicating that Cl and Na transport are energetically linked. The results support the view that the energy necessary for intracellular Cl accumulation is derived from the simultaneous dissipation of the chemical potential gradient of Na across the apical membrane and that the coupled entry mechanism is electroneutral.  相似文献   

19.
Summary Gallbladder fluid and electrolyte transport was investigatedin vitro. In guinea pig gallbladder, equimolar substitution of acetate, propionate, butyrate or valerate for HCO3 was increasingly effective in stimulating fluid absorption. The stimulatory potency of these compounds was a function of their chloroform water partition coefficients. The stimulatory effects of the isomers isobutyrate and isovalerate were less than predicted from their partition coefficients. Acidification of the gallbladder lumen, however, was strictly dependent on the partition coefficients for all of the above fatty acids. Unidirectional22Na fluxes were measured in rabbit and guinea pig gallbladders under short-circuit conditions. In the presence of butyrate stimulation of net Na flux was due entirely to an increase in the mucosal-to-serosal Na flux. Stimulation by butyrate was abolished by its omission from the mucosal bathing solution. The transepithelial electrical potential difference in both rabbit and guinea pig gallbladder became more lumen positive following mucosal but not serosal addition of butyrate. Net14C-butyrate fluxes were too small to account for stimulation of Na absorption in either species. Butyrate stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 8.1. Tris buffer (25mm) partially inhibited butyrate-dependent gallbladder fluid absorption by rabbit and guinea pig at pH 6.4 and 7.0, respectively, and completely at pH 8.4. These results reveal a marked similarity between butyrate and HCO3 stimulation of gallbladder NaCl and fluid absorption. The results are best explained by a double ion-exchange model, in which butyrate (HCO3) in the mucosal solution acts to maintain the intracellular supply of H+ and butyrate (HCO3) for countertransport of Na and Cl, respectively.  相似文献   

20.
Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin=6.0, pHout=8.0)22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin=pHout=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K 1 =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulatedp-nitrophenyl phosphatase. In addition22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号