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1.
In human keratinocytes, mediated transport of Cl- was found to occur mainly by two mechanisms: an anion exchange and an electrically conductive pathway. The contribution of the anion exchange, which accounted for about 50% of overall Cl- efflux, was assessed either by its sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and by means of Cl- substitution experiments. The anion exchange exhibited a saturation behaviour over the range 10-135 mM Cl-; Cl- was more efficient than HCO3-, Br- and NO3- in increasing Cl- efflux rate, whereas SO4(2-) and I- inhibited Cl- efflux. The electrically conductive Cl- pathway, which accounted for about 40% of total Cl- efflux, was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and was at least partially sensitive to variation of the plasma membrane potential. The Cl- channel was insensitive to elevation in the intracellular concentration of either cyclic AMP and calcium ions. Indomethacin, an inhibitor of the cyclooxygenase, failed to reduce Cl- efflux, whereas nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase, induced 50% inhibition of Cl- efflux. These results support the conclusion that endogenous production of lipoxygenase-derived arachidonic acid metabolite(s) might be responsible for high basal Cl- permeability in human keratinocytes. 相似文献
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B M King T Rozaklis J J Hopwood G S Harper 《Biochemical medicine and metabolic biology》1992,47(3):260-264
The glycoconjugate component of cystic fibrosis (CF) epithelial secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation (delta F508/delta F508) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [35S]SO4(2-) uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts. 相似文献
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Altered intestinal chloride transport in cystic fibrosis 总被引:18,自引:0,他引:18
H M Berschneider M R Knowles R G Azizkhan R C Boucher N A Tobey R C Orlando D W Powell 《FASEB journal》1988,2(10):2625-2629
Sodium ion and chloride transport was studied in vitro in small intestinal and colonic tissue from patients with cystic fibrosis (CF) and from non-CF control subjects matched as to age and sex. Normal histological appearance and substantial response to mucosal glucose (5 mM, ileum) or mucosal amiloride (10(-5) M, colon) indicated normal tissue viability in both control and CF tissues. Electroneutral NaCl absorption was demonstrated in the small intestine of control subjects and CF patients. Small intestinal and colonic tissues of control subjects responded to four secretagogues (theophylline, 5 mM; prostaglandin E2, 10(-6) M; calcium ionophore (A23187), 10(-5) M; bethanechol, 5 x 10(-5) M), with electrogenic chloride secretion. The tissues of CF patients, however, did not respond to any of the test secretagogues. These studies demonstrate that an abnormality in chloride transport is present in the small intestinal and colonic epithelia of CF patients. Unlike airway epithelia, which secrete chloride in response to Ca ionophore, the intestinal epithelia of CF patients do not respond to either cAMP- or Ca-mediated secretagogues. This abnormality in intestinal electrolyte transport may play a role in the pathogenesis of meconium impactions in CF patients. 相似文献
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Putrescine transport was examined in normal and cystic fibrosis fibroblasts. No differences were observed in accumulation pattern, kinetics of uptake, or efflux between CF and normal cells. In both growing and growth-arrested CF and normal fibroblasts, exogenously supplied putrescine remained unchanged for at least 60 min. Some differences were observed in the response of CF and normal cells to environmental (media) changes.This research was supported by a grant from the Cystic Fibrosis Foundation and by a grant from the National Institutes of Health, Training Grant (GM01316 11 GNC). 相似文献
5.
Jerome L. Sullivan JoAnn C. Kelly William B. Roess A. Gib DeBusk 《Biochemical genetics》1977,15(11-12):1125-1132
Initial rate kinetics of methionine transport, time course of accumulation of methionine, and efflux of accumulated methionine were studied in three normal and four CF human diploid fibroblast strains. The range of apparent Km's was 12.7-32.1 micrometer for the CF strains and 18.3-39.2 micrometer for the normal strains. The range of apparent Vmax's was 6.69-9.22 nmole mg-1 min-1 for the CF strains and 5.59-7.87 nmole mg-1 min-1 for the normal strains. The patterns of accumulation and efflux are quite similar in all the strains studied except for WI-38, which showed somewhat higher efflux and lower accumulation than for others. There was no significant difference in the kinetic parameters of methionine transport between CF and normal skin fibroblasts, and methionine transport will not serve as a marker for cystic fibrosis in cultured fibroblasts. 相似文献
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A biochemical link is proposed between recent observations on defective regulation of Cl– transport in CF respiratory epithelial cells and studies showing altered biological activity of calmodulin in exocrine glands from CF patients. A consensus is emerging that defective -adrenergic secretory responsiveness in CF cells is caused by a defect in a regulator protein at a site distal to cyclic AMP formation. Our results indicate that this protein might be a specific calmodulin acceptor protein which modifies the activity of calmodulin in epithelial cells. Alteration in Ca2+/calmodulin dependent regulation of Cl– transport and protein secretion could explain (i) alterations in Ca2+ homeostasis seen in CF, (ii) defective -adrenergic responses of CF cells, and (iii) the observed inability of cyclic AMP (acting via its specific protein kinase, A-kinase) to open apical membrane Cl– channels in CF epithelial cells. Most of the physiological abnormalities of CF including elevated sweat electrolytes and hyperviscous mucus can be explained on this basis.Abbreviations -adrenergic agonist acting at its receptor cAMP
cyclic AMP
- PDE
phosphodiesterase
- CaM
calmodulin
- Pase
phosphatase 相似文献
9.
Chloride permeability in 9 cystic fibrosis- and 11 normal-skin fibroblast lines has been investigated. Chloride efflux, under steady-state conditions, involves two intracellular compartments characterized by slow- and fast-rate constants of efflux. We show here that the fast rate constant in cystic fibrosis cells is reduced by 25% in comparison with controls. The data presented support recent studies indicating that isolated sweat glands and respiratory epithelia of patients suffering from cystic fibrosis have an unusual low permeability to chloride ions compared to control epithelia. It is concluded that variation in chloride transport can successfully be studied in cultured fibroblasts, which are not directly involved in the pathology of the disease. 相似文献
10.
It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In the present study, the effect of elevation of intracellular concentration of cyclic AMP and calcium on the efflux of Cl- from human fibroblasts has been investigated. Cyclic AMP analogs (8-bromo cAMP and dibutyryl cAMP) and a beta agonist (isoproterenol) induced only a weak stimulation (5-10%) of Cl- efflux. Conversely, elevation of cytoplasmic calcium concentration produced by addition of the Ca2+ ionophore A23187 in the efflux medium, did not affect Cl- efflux. Our data indicate that the response of Cl- efflux to elevation of cAMP and calcium is similar in normal and cystic fibrosis fibroblasts. Exposure to hypotonic medium induced a significant stimulation of Cl- efflux in fibroblasts from both normal and cystic fibrosis individuals. Substitution of Cl- in the medium by gluconate and the subsequent addition of furosemide did not inhibit the effect of hypotonicity, indicating the involvement of a conductive pathway for Cl- transport, which was insensitive to oligomicin C. 相似文献
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C M Liedtke 《FASEB journal》1992,6(12):3076-3084
The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main-stem bronchi), bronchioles, and alveoli. Na+ absorption occurs in all three pulmonary segments but by different transporters: apical Na+ channels in large airways and bronchioles; Na+/H+ exchange and Na+ channels in adult alveoli. The Na+ channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na+ channels. Fetal alveoli display spontaneous Cl- secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl- channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated Cl- secretion. Electroneutral carriers, such as NaCl(K) cotransport, Cl-/HCO3- exchange, and Na+/HCO3- exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl- conductance and increased Na+ conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation. 相似文献
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Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts. 总被引:7,自引:0,他引:7
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The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60 min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells. 相似文献
16.
B Sarkadi D Bauzon W R Huckle H S Earp A Berry H Suchindran E M Price J C Olson R C Boucher G A Scarborough 《The Journal of biological chemistry》1992,267(3):2087-2095
Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells. 相似文献
17.
Membrane function was examined in cultured cells from cystic fibrosis patients by assaying several enzymes on intact skin fibroblasts attached to culture dishes. This technique required few cells and minimized disruption of cellular organization. Comparison of enzyme activities of intact and broken cells showed that 12% of total glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was measurable using intact cells, while all adenosine monophosphatase was measurable using intact cells. Alkaline paranitrophenylphosphatase activity was divided between the cell surface and interior. Substrate competition experiments indicated that substrate specificities for adenosine monophosphatase and paranitrophenylphosphatase activities were different. Adenosine monophosphatase activities of 2 control and 2 cystic fibrosis strains fluctuated similarly during the cell culture cycle. The apparent Km values relative to adenosine monophosphate were similar in all strains. A chromatographic fraction of serum from a cystic fibrosis patient that was inhibitory to oyster ciliary activity had no effect on adenosine monophosphatase activity of normal fibroblasts. Furthermore, fractions of media from cystic fibrosis homozygote and heterozygote fibroblast cultures were not inhibitory to adenosine monophosphatase activities of intact normal fibroblasts or of part iculate fractions prepared from them. In light of previous studies that showed that factors from cystic fibrosis serum of culture medium disrupted specific membrane activities, it is proposed that the cystic fibrosis factor interacts with the plasma membrane, interfering most conspicuously with the protein functions that are sensitive to changes in their membrane environment. 相似文献
18.
B S Danes 《Texas reports on biology and medicine》1976,34(1):135-150
Observations on the characteristics of the cell with the cystic fibrosis (CF) genotype in culture are reviewed. Although numerous and diverse abnormalities have been described, none were specific for the CF gene. The relevance of each of these abnormalities to the clinical syndrome known as CF is discussed, emphasizing that the value thus far of such cell culture research has been to learn how the CF gene influences cellular function. 相似文献
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The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP. Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells. 相似文献