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1.
The effect of the immunosuppressive drug cyclosporin A (CsA) on the K (Rb) influx, intracellular K and Na contents, and on the major parameters of lymphocyte activation have been investigated in human peripheral blood lymphocytes activated by phytohemagglutinin (PHA). CsA suppressed protein, RNA, DNA syntheses and cell proliferation by 49.8 +/- 4.3, 67.6 +/- 10.1, 60.4 +/- 5.3 and 60.0 +/- 5.1%, respectively (n = 10) within 48 h. It also inhibited the late long-term Na+,K+ pump activation, as determined from the ouabain-sensitive Rb uptake, and prevented the increase in the intracellular K content at the late stages of G0/G1/S progression. Cyclosporin A did not affect the early transient pump activation, the dynamics, of ouabain-resistant influxes and the intracellular Na content in PHA-activated lymphocytes. When added 1 h after PHA, CsA neither affected the activation of the pump-mediated Rb influxes nor the increase in the intracellular K content. It is concluded that in activated human lymphocytes, the long-term activation of Na+,K+ pump associated with the mitogen-induced blast transformation, as well as the late increase in K content depend on the T-cell growth factor interleukin-2. 相似文献
2.
Gusev GP Ivanova TI 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2003,173(5):429-435
The present study was designed to evaluate the role of protein phosphatases in regulation of sodium transport in the marsh frog erythrocytes using 22Na as a tracer. For this purpose the cells were treated with several known inhibitors of protein phosphatases. In standard isotonic medium, exposure of the cells to 10 mmol l(-1) NaF, 20 nmol l(-1) calyculin A or 0.1 mmol l(-1) cantharidin resulted in a significant (1.7-fold) increase in unidirectional ouabain-insensitive Na+ influx. The Na+ influx in frog red cells was progressively activated as the medium osmolality was increased by addition of 100, 200 or 300 mmol l(-1) sucrose to standard isotonic medium. The stimulatory effect of protein phosphatase blockers on Na+ influx was much higher in hypertonic medium containing 100 or 200 mmol l(-1) sucrose than that in isotonic medium. Stimulation of Na+ transport enhanced with increasing concentrations of calyculin A, and half-maximal activation (EC50) was obtained at 16 nmol l(-1). However, Na+ influx induced by strong hypertonic treatment (+300 mmol l(-1) sucrose) was not altered further in the presence of protein phosphatase inhibitors. The changes in Na+ influx evoked by protein phosphatase inhibitors and hypertonic treatment were associated with a rise in the intracellular Na+, but not K+, content. Enhancement in Na+ influx after addition of protein phosphatase blockers to cell suspension in isotonic or hypertonic media was almost completely inhibited by Na+/H+ exchange inhibitors, amiloride and ethyl-isopropyl-amiloride. The basal Na+ influx in frog erythrocytes in isotonic medium was relatively low (1.7 mmol/l cells/h) and not affected by 1 mmol l(-1) amiloride. Thus, the data obtained clearly indicate that Na+/H+ exchanger in the marsh frog red blood cells is under tight regulatory control, in all likelihood via protein phosphatases of types PP-1 and PP-2A. 相似文献
3.
Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. I. Sodium fluxes 总被引:4,自引:0,他引:4
L Simchowitz 《The Journal of biological chemistry》1985,260(24):13237-13247
The nature of Na+ fluxes in resting and in chemotactic factor-activated human neutrophils was investigated. In resting cells, ouabain-insensitive unidirectional 22Na+ in- and effluxes represented passive electrodiffusional fluxes through ion channels: they were nonsaturable and voltage-dependent (PNa = 4.3 X 10(-9) cm/s). Amiloride (1 mM) had little effect on resting 22Na+ influx (approximately 0.8 meq/liter X min), thereby suggesting a minor contribution of Na+/H+ exchange and a lack of amiloride-sensitive Na+ channels. When neutrophils were exposed to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM), 22Na+ influx was stimulated approximately 30-fold (initial rate approximately 22 meq/liter X min). The FMLP-induced 22Na+ influx was saturable with respect to external Na+ (Km 26-35 mM, Vmax approximately 28 meq/liter X min), was electroneutral, and could be competitively inhibited by amiloride (Ki 10.6 microM). From a resting value of approximately 30 meq/liter of cell water, internal Na+ in FMLP-stimulated cells rose exponentially to reach a concentration of approximately 60 meq/liter by 10-15 min. This uptake was blocked by amiloride. FMLP also stimulated the efflux of 22Na+ which followed a single exponential time course (rate coefficient approximately 0.16 min-1). The FMLP-induced 22Na+ fluxes were similar to those observed with 10 microM monensin, a known Na+/H+ exchanging ionophore. The data indicate that FMLP activates an otherwise quiescent, amiloride-sensitive Na+/H+ exchange. Furthermore, all of the FMLP-induced 22Na+ fluxes can be satisfactorily accounted for by transport through the exchanger, leaving little room for an appreciable increase in Na+ conductance. 相似文献
4.
Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters 总被引:6,自引:0,他引:6
The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent. 相似文献
5.
Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes 总被引:8,自引:0,他引:8
L Simchowitz 《The Journal of biological chemistry》1985,260(24):13248-13255
The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+. 相似文献
6.
The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 X 10(3) +/- 12 X 10(3)/cell in resting to 60 X 10(3) +/- 27 X 10(3)/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: there was an increase in the Vmax of ouabain binding, without variation in the Km; the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane. 相似文献
7.
Chemotactic factor-induced generation of superoxide radicals by human neutrophils: evidence for the role of sodium. 总被引:16,自引:0,他引:16
The role of sodium ion in superoxide (O2-) generation by human peripheral neutrophils was investigated. Cells were activated by exposure to the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and O2- release was assessed by ferricytochrome c reduction after 5 min of incubation at 37 degrees C in the presence of FMLP 4 X 10(-8) M. In the absence of monovalent cations (isotonic glucose), negligible O2- generation occurred. There was a progressive increase in the magnitude of FMLP-induced O2- generation with increasing Na+ concentration up to 90 mM, where the response was noted to plateau. Varying the K+ concentration (1 to 10 mM) had no effect on the amount of O2- produced in the presence of Na+ 140 mM. FMLP also stimulated 22Na+ and 48Ca2+ uptake by the cells in a dose- and time-dependent fashion. FMLP-induced 22Na+ uptake appeared to be independent of the external Ca2+ concentration ( to 4 mM). In contrast, there was a progressive decrease in themagnitude of the FMLP-induced increase in 45Ca2+ uptake as the Na+ concentration was reduced by replacement with choline+ or glucose. These studies support a requirement for Na+ in FMLP-induced O2- generation and suggest that a Na+ influx may underlie the nature of this requirement. The data are also consistent with the hypothesis that a Na+ influx may precede the Ca2+ influx in the FMLP-induced activation sequence. 相似文献
8.
The guanosine 3',5'-cyclic monophosphate (cGMP) level in the mouse splenic lymphocytes was increased about 2- to 3-fold by concanavalin A. This increase was completely dependent on the presence of Ca2+ in the medium. Homogenates of mouse splenic lymphocytes contained significant guanylate cyclase [EC 4.6.1.2] activity in both the 105,000 X g (60 min) particulate and supernatant fractions and both fractions required Mn2+ for full activity. Calcium ion (3mM) activated soluble guanylate cyclase 3-fold at a relatively low concentration of Mn2+ (less than 1mM) but inhibited the particulate enzyme slightly at all Mn2+ concentrations tested. Concanavalin A itself did not stimulate either fraction of guanylate cyclase. Thus these results suggest that elevation of the cGMP level in lymphocytes by concanavalin A might be brought about by stimulation of Ca2+ uptake and activation of soluble guanylate cyclase by the latter. 相似文献
9.
At concentrations as low as 10(-7) M, the cardiotonic glycosteroid ouabain, a specific inhibitor of the membrane Na+, K+-ATPase, is known to inhibit in vitro human lymphocyte proliferation produced in mixed lymphocyte cultures or induced by various stimulating agents (PHA, Con A, PWM, soluble antigens), while mouse lymphocyte proliferation is unaffected at this concentration. Ouabain inhibits most of proliferative response parameters at all stages of the transformation. This observation prompted us to suggest that ouabain could also act through inhibition of interleukin production which is known to occur during the first hours after T-cell stimulation in the presence of monocytes. In order to check the possible influence of ouabain on interleukin production, conditioned media from stimulated human mononuclear cells, prepared in the presence or in the absence of inhibitor, were tested for their ability to promote a mouse thymocyte response to PHA. Instead of the expected inhibition, we found that ouabain, even at high concentrations (2 X 10(-6) M) enhanced the stimulatory effect and/or the production of murine thymocyte activating factor(s). Moreover conditioned media from serum-free cultures of unstimulated human mononuclear cells exposed for 24 hr to low ouabain concentrations (10(-8) to 10(-7) M) showed a high activating effect on the response of murine thymocytes to PHA. This soluble factor produced upon ouabain treatment is produced by adherent cells and appears to be functionally similar to interleukin 1. 相似文献
10.
In order to establish a standard for sodium influx in erythrocytes for the black population, 22Na+ uptake was measured in 29 normotensive black volunteers. Nineteen males and 10 females during the follicular phase of the menstrual cycle were studied utilizing the procedure of Gambhir et al. (1). In the males, cell concentrations ranging from 0.64 to 2.0 X 10(9)/ml showed an influx of 0.42 to 1.34% of the total 22Na+ added, and in the females, using the same erythrocyte concentrations, the 22Na+ influx ranged from 0.37 to 1.1%; these differences were not significant. Intraassay variation of the 22Na+ data was 4.8%. Interassay variations have been explained elsewhere (1). These data provided a range of observed values for 22Na+ uptake in erythrocytes from a subpopulation of normotensive black males and females for comparison with hypertensive patients. 相似文献
11.
Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism. 相似文献
12.
Sacher A Nelson N Ogi JT Wright EM Loo DD Eskandari S 《The Journal of membrane biology》2002,190(1):57-73
We expressed mouse gamma-aminobutyric acid (GABA) transporter (mGAT3) in Xenopus laevis oocytes and examined its steady-state and presteady-state kinetics and turnover rate by using tracer flux and electrophysiological methods. In oocytes expressing mGAT3, GABA evoked a Na+-dependent and Cl(-)-facilitated inward current. The dependence on Na+ was absolute, whereas that for Cl(-) was not. At a membrane potential of -50 mV, the half-maximal concentrations for Na+, Cl(-), and GABA were 14 mM, 5 mM, and 3 microM. The Hill coefficient for GABA activation and Cl(-) enhancement of the inward current was 1, and that for Na+ activation was > or =2. The GABA-evoked inward current was directly proportional to GABA influx (2.2 +/- 0.1 charges/GABA) into cells, indicating that under these conditions, there is tight ion/GABA coupling in the transport cycle. In response to step changes in the membrane voltage and in the absence of GABA, mGAT3 exhibited presteady-state current transients (charge movements). The charge-voltage (Q-V) relation was fitted with a single Boltzmann function. The voltage at half-maximal charge (V(0.5)) was +25 mV, and the effective valence of the moveable charge (zdelta) was 1.6. In contrast to the ON transients, which relaxed with a time constant of < or =30 msec, the OFF transients had a time constant of 1.1 sec. Reduction in external Na+ ([Na+]o) and Cl(-) ([Cl(-)]o) concentrations shifted the Q-V relationship to negative membrane potentials. At zero [Na+]o (106 mM Cl(-)), no mGAT3-mediated transients were observed, and at zero [Cl(-)]o (100 mM Na+), the charge movements decreased to approximately 30% of the maximal charge (Q(max)). GABA led to the elimination of charge movements. The half-maximal concentrations for Na+ activation, Cl(-) enhancement, and GABA elimination of the charge movements were 48 mM, 19 mM, and 5 mM, respectively. Q(max) and I(max) obtained in the same cells yielded the mGAT3 turnover rate, 1.7 sec(-1) at -50 mV. The low turnover rate of mGAT3 may be due to the slow return of the empty transporter from the internal to the external membrane surface. 相似文献
13.
Mineralocorticoid-specificity of aldosterone-induced protein synthesis in giant-toad (Bufo marinus) urinary bladders. 总被引:1,自引:1,他引:0
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We have identified a group of proteins (Mr approximately 70000-80000; pI approximately 5.8-6.4) in giant-toad (Bufo marinus) urinary-bladder epithelial cells whose synthesis appears to be related to aldosterone-stimulated Na+ transport. To define this relationship further, we examined whether submaximal natriferic concentrations of aldosterone induced these proteins and whether spironolactone (a specific mineralocorticoid antagonist in renal epithelia) inhibited their synthesis. Short-circuit current was used to measure Na+ transport and epithelial-cell protein synthesis was detected with high-resolution two-dimensional polyacrylamide-gel electrophoresis and autoradiography. Submaximal natriferic concentrations of aldosterone (1.4 X 10(-8) M) induced the same proteins as maximal concentrations of the hormone (1.4 X 10(-7) M). In contrast, in previous experiments, similar proteins were not induced by subnatriferic concentrations (5.0 X 10(-8) M) of cortisol, a glucocorticoid. A spironolactone/aldosterone molar ratio of 2000:1 was required to inhibit aldosterone-stimulated Na+ transport completely; ratios of 200:1 and 500:1 produced partial inhibition. Concentrations of spironolactone that abolished aldosterone-stimulated Na+ transport also inhibited aldosterone-induced protein synthesis. We conclude that the synthesis of the proteins we have identified is specifically related to activation of the mineralocorticoid pathway. 相似文献
14.
Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells 总被引:2,自引:0,他引:2
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Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
15.
The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells. 相似文献
16.
Role of the Na+/K+/Cl- transporter in the positive inotropic effect of ouabain in cardiac myocytes 总被引:1,自引:0,他引:1
R Panet R Fixler D Snyder S Raz H Atlan Y Eilam Y Hasin 《Journal of cellular physiology》1990,145(1):24-29
In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain. 相似文献
17.
Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells 总被引:2,自引:0,他引:2
Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle. 相似文献
18.
Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells 总被引:1,自引:0,他引:1
Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells. 相似文献
19.
36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed-nephrectomized rats with plasma Cl concentrations between 14 and 120 mumol X ml-1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood-to-brain transfer constants for Cl, kCl, ranged from 1.8 X 10(-5) S-1 at the parietal cortex to 3.8 X 10(-5) S-1 at the thalamus-hypothalamus. kCl fell by 42-62% when mean plasma [Cl] was elevated from 16 to 114 mumol X ml-1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis-Menten saturation kinetics, where, for the parietal cortex, Km = 43 mumol X ml-1 and Vmax = 2.5 X 10(-3) mumol X S-1 X g-1, and for CSF Km = 68 mumol X ml-1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration-dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain. 相似文献
20.
A R Borgatti G Trigari A Pagliarani V Ventrella 《Comparative biochemistry and physiology. A, Comparative physiology》1985,81(1):127-135
Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested. 相似文献