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1.
Comparison of locomotion, chemotaxis and adhesiveness of rabbit neutrophils from blood and peritoneal exudates 总被引:2,自引:0,他引:2
We compared the spontaneous behaviour (motility, adhesiveness, locomotion) and the chemotactic responses of exudate and blood-borne neutrophils. Directional locomotion of exudate neutrophils in 2% HSA-Gey's towards exudate fluid was not significantly changed, the response to activated autologous plasma diminished, and that to f-Met-Leu-Phe (10(-9) M) increased in comparison with blood-borne cells. The spontaneous behaviour of exudate cells in 2% HSA-Gey's (no gradient) differed markedly from that of blood-borne cells. In tissue culture medium (2% HSA-Gey's) exudate cells showed heightened motility in suspension and greater adhesiveness to glass substrata. These differences were eliminated by culturing the cells in their physiological media (i.e. plasma or exudate fluid). In contrast to blood-borne cells, exudate neutrophils tended to aggregate spontaneously. There was no correlation between neutrophil aggregation and adhesion to glass substrata of exudate cells in exudate fluid. 相似文献
2.
T H Fischer 《The Biochemical journal》1983,211(3):771-774
To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy. 相似文献
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Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min). Stimulation of Group B proteins was observed by fMet-Leu-Phe (ED50 0.15 nM, 1 min), leukotriene B4 (ED50 0.15 nM, 1 min), phorbol 12-myristate 13-acetate (PMA) (ED50 25 ng/ml, 10 min) and partially by ionophore A23187 (1 microM, 1 min). Pretreatment of the cell with the protein kinase inhibitor H-7 (25 microM, 5 min) and PMA (0.1 microgram/ml, 3 min) partially inhibited the fMet-Leu-Phe effect. However, pretreatment of the cells with quin 2/AM (20 microM, 10 min) completely inhibited the fMet-Leu-Phe effect. The results indicate that rapid regulation of tyrosine phosphorylation is an early event occurring in stimulated neutrophils. Furthermore the effect of fMet-Leu-Phe on tyrosine phosphorylation may require Ca2+ mobilization and may partially require the activity of H-7-sensitive protein kinases. 相似文献
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P H Naccache H J Showell E L Becker R I Sha'afi 《Biochemical and biophysical research communications》1979,87(1):292-299
We have found that arachidonic acid rapidly and selectively induces the release of lysosomal enzymes from cytochalasin B treated rabbit peritoneal neutrophils. 5, 8, 11, 14-eicosatetraynoic acid inhibits the arachidonate induced release with an apparent KD of 1.5 × 10?6M. 5,8,11,14-eicosatetraynoic acid (2.5 × 10?5M also inhibits the chemotactic factors and the A23187 induced release in the presence of cytochalasin B but does not affect the degranulation induced by A23187 alone. These observations strongly suggest a role for arachidonate metabolites in rabbit neutrophil physiology. 相似文献
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The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump 总被引:2,自引:0,他引:2
Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites. 相似文献
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Three phosphorylated reaction intermediates (EP) of Na,K-ATPase, and ADP-sensitive K+-insensitive EP (E1P), an ADP- and K+-sensitive EP (E*P), and a K+-sensitive ADP-insensitive EP (E2P), have been discovered at present. By using Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme, we found in this study that E*P existed even in the presence of K+ on both sides of the PL and that there was a sidedness difference in K+ sites between E*P and E2P. Cytoplasmic K+ (K+cyt) accelerated the conversion of E*P to E2P but did not dephosphorylate the E2P. Although the extracellular K+ accelerated the dephosphorylation of E2P, it did not interact with E*P directly. This K+cyt effect was also verified by the activation of Na+-pump in the Na+-K+ exchange mode. In the presence of K+cyt, both the ATP hydrolysis and Na+ uptake rates of the PL containing K+ inside vesicles increased sigmoidally with the concentrations of ATP and cytoplasmic Na+ (Na+cyt). However, in the absence of K+cyt, these Na+-pump reactions in PL containing K+ inside vesicles had only a hyperbolic curve. These results imply that the E*P to E2P conversion is one of the rate-limiting steps of the Na+-pump in the presence of a high concentration of ATP and that K+cyt may control this reaction step by enhancing the conversion rate of E*P to E2P. 相似文献
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The objective of the present investigation was to characterize the ATP-dependent Na+-Na+ exchange, with respect to cation sensitivity on the two aspects of the Na+/K+-pump protein. In order to accomplish this, we used Na+/K+-ATPase reconstituted with known orientation in the proteoliposomes. Activation by cytoplasmic Na+ shows cooperative interaction between three sites. The apparent intrinsic site constants displayed transmembrane dependence on the extracellular Na+ concentration. However, the apparent K0.5 for cytoplasmic Na+ is independent of the extracellular Na+ concentration. The activation by extracellular Na+ at a fixed cytoplasmic Na+ concentration is biphasic with a component which saturates at a concentration of about 1-2 mM extracellular Na+, a plateau phase up to 20 mM, and another component which tends to saturate at about 80 mM followed by a slight deactivation at higher concentrations of Na+. The apparent K0.5 value for extracellular Na+ is also found to be independent of the Na+ concentration on the opposite side of the membrane. The activation by extracellular Na+ can be explained by the negative cooperativity in the binding of extracellular Na+, but positive cooperativity in the rate of dephosphorylation of enzyme species with one and three sodium ions bound extracellularly. Na+ bound to E2-PNa has a transmembrane effect on the cooperativity between binding of cytoplasmic Na+, and E2-PNa2 does not dephosphorylate. K0.5/Vm for cytoplasmic as well as for extracellular Na+ decreases with an increase in the trans Na+ concentration in the non-saturating concentration range. The experiments indicate that at a step in the reaction simultaneous binding of extracellular and cytoplasmic Na+ occurs. 相似文献
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A chemotactic peptide CHO.Met.Leu.Phe.OH has been synthesized classically using the mixed anhydride procedure. The formyl group was introduced by coupling formic acid in the presence of dicyclohexylcarbodiimide to the partially protected triptide. The final product was obtained by treatment of the intermediate CHO.Met.Leu.Phe.OBzl with hydrogen fluoride. The ED50 of the peptide in the Boyden chamber assay was 7 x 10(-11) M; in the lysozyme release assay 2.4 x 10(-10) M and in the beta-glucuronidase release assay 2.6 x 10(-10) M. In a radioreceptor assay the ID50 of the peptide was 3.3 x 10(-10) M. 相似文献
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Regional distribution of the Na+ and K+ currents around the crystalline lens of rabbit 总被引:3,自引:0,他引:3
Early studies described asymmetricalelectrical properties across the ocular lens in theanterior-to-posterior direction. More recent results obtained with avibrating probe indicated that currents around the lens surface are notuniform by showing an outwardly directed K+ efflux at thelens equator and Na+ influx at the poles. The latterstudies have been used to support theoretical models for fluidrecirculation within the avascular lens. However, the existence of anonuniform current distribution in the lens epithelium from theanterior pole to the equator has never been confirmed. The present workdeveloped a modified short-circuiting technique to examine the netflows of Na+ and K+ across arbitrarily definedlens surface regions. Results indicate that passive inflows ofNa+ occur at both the anterior polar region and posteriorlens surface, consistent with suggestions derived from the vibratingprobe data, whereas K+ efflux plus theNa+-K+ pump-generated current comprise thecurrents at the equatorial surface and an area anterior to it.Furthermore, Na+-K+ pump activity was absent atthe posterior surface and its polar region in all lenses examined, aswell as from the anterior polar region in most lenses. The latterunexpected observation suggests that the monolayered epithelium, whichis confined to the anterior surface of the lens, does not express anactive Na+-K+ pump at its anterior-most aspect.Nevertheless, this report represents the first independent confirmationthat positive currents leave the lens around the equator and reenteracross the polar and posterior surfaces. 相似文献
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K+Nutrition and Na+Toxicity: The Basis of Cellular K+/Na+Ratios 总被引:38,自引:0,他引:38
The capacity of plants to maintain a high cytosolic K+/Na+ratiois likely to be one of the key determinants of plant salt tolerance.Important progress has been made in recent years regarding theidentification and characterization of genes and transportersthat contribute to the cytosolic K+/Na+ratio. For K+uptake,K+efflux and K+translocation to the shoot, genes have been isolatedthat encode K+uptake and K+release ion channels and K+carriersthat are coupled to either a H+or Na+gradient. Although thepicture is less clear for the movement of Na+, one pathway,in the form of non-selective ion channels, is likely to playa role in Na+uptake, whereas Na+efflux and compartmentationare likely to be mediated by H+-coupled antiport. In addition,several proteins have been characterized that play prominentroles in the regulation of K+and/or Na+fluxes. In this BotanicalBriefing we will discuss the functions and interactions of thesegenes and transporters in the broader context of K+nutritionand Na+toxicity. Copyright 1999 Annals of Botany Company Salinty, K+/N+ratio, transporter, membrane. 相似文献
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The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (beta- and gamma-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished approximately 79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (less than 0.06 mM) and stage-related differences in blastocoele volume. Analysis of beta- and gamma-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P less than 0.001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst. 相似文献
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Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes 总被引:2,自引:0,他引:2
Hansen PS Buhagiar KA Kong BY Clarke RJ Gray DF Rasmussen HH 《American journal of physiology. Cell physiology》2002,283(5):C1511-C1521
To examine effects of cytosolicNa+, K+, and Cs+ on the voltagedependence of the Na+-K+ pump, we measuredNa+-K+ pump current (Ip)of ventricular myocytes voltage-clamped at potentials(Vm) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na+ concentration ([Na]pip)of 80 mM and a K+ concentration from 0 to 80 mM or withsolutions containing Na+ in concentrations from 0.1 to 100 mM and K+ in a concentration of either 0 or 80 mM. When[Na]pip was 80 mM, K+ in pipette solutionshad a voltage-dependent inhibitory effect on Ipand induced a negative slope of theIp-Vm relationship. Cs+ in pipette solutions had an effect onIp qualitatively similar to that ofK+. Increases in Ip with increasesin [Na]pip were voltage dependent. The dielectriccoefficient derived from[Na]pip-Ip relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K+ and 0.06 when pipette solutions were K+ free. 相似文献
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J N Karli G A Karikas P K Hatzipavlou G M Levis S N Moulopoulos 《Life sciences》1979,24(20):1869-1875
Lysophosphatidylcholine (LPC) added at 30 μM or 10 μM to sarcolemma (SL) membranes of rabbit or dog causes about 50% inhibition of the Mg++ dependent Na+ + K+ stimulated ATPase activity. Higher concentration (100 μM) of free fatty acid (FFA) is needed to produce the same inhibition. Lysophosphatidyl ethanolamine, phosphorylcholine, phosphorylethanolamine also inhibit the enzyme activity. The inhibition of LPC is competitive for the Na+ site of the enzyme and it also affects the K+ site. The inhibitory effect of LPC is not additive to that produced by FFA or ouabain, on the contrary at low concentrations of these inhibitors the inhibition of LPC is reversed. The possible effects of increased levels of blood or tissue LPC on heart cell functions related to the Na+ + K+ ATPase activity are discussed. The possible interference of LPC with cardiac glycoside action is also discussed. 相似文献
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The ATP hydrolysis dependent Na+-Na+ exchange of reconstituted shark (Na+ + K+)-ATPase is electrogenic with a transport stoichiometry as for the Na+-K+ exchange, suggesting that translocation of extracellular Na+ is taking place via the same route as extracellular K+. The preparation thus offers an opportunity to compare the sided action of Na+ and K+ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K+. With Na+ but no K+ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K+ in concentrations of 50 microM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K+ also gives biphasic ATP-activation curves, however, only when the K+ concentration is 50 mM or higher (Na+ + K+ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E2(Na2+) and E2(K2+), respectively, and in which the dephosphorylation of E2-P is rate limiting in the presence of Na+ (but no K+) extracellular, whereas in the presence of extracellular K+ it is the deocclusion of E2(K2+) which is rate limiting. 相似文献
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