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1.
《Comparative biochemistry and physiology. A, Comparative physiology》1992,101(4):763-766
- 1.1. Unidirectional Na+ influx in lamprey red blood cells was determined using 22Na as a tracer.
- 2.2. Total Na+ uptake and amiloride-inhibitable Na+ influx increased in a saturable fashion as a function of external Na+ concentration (Nae).
- 3.3. At 141 mM Nae, the average value of net Na+ influx was 13 ± 1.1 and the amiloride-sensitive Na+ influx was 5.3±1.1 mmol/l cells per hr (±SE).
- 4.4. The amiloride-sensitive component of Na+ influx was significantly activated by 10−5 M isoproterenol, by 2 × 10−5 M DNP, and by cell shrinkage.
- 5.5. Furosemide (1 mM) had no effect on the Na+ transport in red cells.
- 6.6. The residual amiloride-insensitive component of Na+ transport was a linear function of Nae in the range of 5–141 mM. This transport seems to be accounted for by simple diffusion.
2.
Our preliminary studies have shown that the Na,K-pump in frog erythrocytes is activated by isoproterenol (ISP), phosphodiesterase
blocker (3-isobutyl-methylxantine, IBMX), and by iodoacetate (MIA). The aim of the present study was to determine a mechanism
responsible for the effect of MIA on the Na,K-pump activity in frog red blood cells as well as the role of G proteins and
intracellular messengers in modulation of active K+ transport induced by ISP. An additive stimulation of active K+ (86Rb) transport in frog erythrocytes was found after exposure of the cells to MIA in a combination with ISP or IBMX. The treatment
of the red blood cells with 1 mM MIA for 1 or 2 h was associated with a significant decrease in intracellular Na+ concentration, on average, by 13 and 20%, respectively, suggesting a direct action of MIA on the Na,K-pump. Incubation of
cells in the presence of dibutyryl-cAMP (1 mM) or adenylate cyclase activator forskolin (0.1 mM) caused stimulation of the
active K+ influx by 21.8 and 27.9%, respectively. AlF
4
-
and cholera toxin able to increase cell cAMP levels via G protein interactions had no effect on the total and IPS-induced
K+ influx in frog erythrocytes. The treatment of the red blood cells with sodium nitroprusside that increases cGMP concentration
in cells also had no effect on the K+ influx. The stimulatory influence of ISP on the Na,K-pump was reduced with increase of the intracellular Na+ concentration. ISP increased affinity of the Na,K-pump to Na+ (the Mihaelis constant KM = 34.4 ± 5.1 in control and 25.3 ± 2.8 mM in the presence of ISP,p < 0.01), but did not change maximal velocity (8.1 ± 0.6 and 7.7 ± 0.3 mmol/1/h in the control and ISP-treated cells, respectively).
The results obtained indicate the presence of several different signal pathways involved in regulation of the Na,K-pump activity
in frog erythrocytes. 相似文献
3.
N. I. Agalakova A. V. Lapin G. P. Gusev 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1997,167(8):576-581
This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content
of frog erythrocytes. To eliminate K-C1 cotransport, the frog erythrocytes were incubated in nitrate media at 20 °C. Incubation
of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K+ influx measured as 86Rb uptake for 60 min. The Na+-K+-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose
or adenosine. Unexpectedly, the treatment of red cells with 1–2 mM glycolytic blocker iodoacetate produced a 2-fold increase
in the ouabain-sensitive K+ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells
for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K+ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation
with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K+ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast,
0.01–0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative
phosphorylation were without effect on K+ influx into frog erythrocytes. These results indicate that about one-half of the Na+ -K+-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate “membrane-associated” ATP
pool.
Accepted: 12 July 1997 相似文献
4.
Transport of Na+ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope 22
22Na. Treatment of erythrocytes with -adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na+ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na+ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of 22
22Na accumulation and intracellular Na+ concentration. These changes of the Na+ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na+/H+ exchanger. Hence, PMA produced activation of Na+/H+ exchange in frog erythrocytes. The unidirectional Na+ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na+ influx (IC50) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells. 相似文献
5.
Mitchel L. Villereal 《Journal of cellular physiology》1981,107(3):359-369
Human fibroblasts that have been serum deprived for 4 hours have a digitoxin-insensitive Na influx of 9.5 ± 1.0 (n = 4) μmol/g prot/min which is not significantly different from the influx of 9.4 ± 0.6 (n = 3) μmol/g prot/min measured in cells arrested in the G1/G0 state by serum-deprivation for a period of four days. The Na influx in serum-deprived cells is rapidly stimulated (within one minute) simply by assaying the cells in medium containing 10% fetal bovine serum (FBS). The digitoxin-insensitive Na influx for cells in the presence of 10% FBS is 22.9 ± 1.1 (n = 6) μmol/g prot/min. the stimulation of Na influx in serumdeprived cells can also be achieved by the addition of the purified mitogen, epidermal growth factor (EGF). Addition of EGF to serum-deprived cells gives a maximal stimulation of Na influx of approximately 1.6-fold, with the concentration for half-maximal stimulation being 7.5 ng/ml. The stimulation of Na influx results from the activation of an amiloride-sensitive pathway, which appears to be minimally active in serum-deprived cells. Kinetic analysis of Na influx experiments in the presence of 10% FBS and varying concentrations of amiloride indicate that at infinite concentrations of amiloride the Na flux would be reduced to 8.9 μmol/g prot/min, which is comparable to the level of Na flux measured in serum-deprived cells in the presence of 5 mM amiloride. Thus, amiloride can totally inhibit the serum-stimulated component of Na influx while inhibiting less than 10% of the Na influx in serum-deprived cells. The Na influx in serum-deprived cells can also be stimulated 2.5-fold by preincubating cells in the presence of the Ca+ ionophore A23187 to elevate the intracellular Ca content. This stimulation of Na influx by intracellular Ca+2 can be virtually eliminated by adding 1 mM amiloride. 相似文献
6.
Jin-Lin Zhang Anne M. Wetson Suo-Min Wang Ali R. Gurmani Ai-Ke Bao Chun-Mei Wang 《Biological trace element research》2011,139(1):108-117
Salinity is an increasing problem for agricultural production worldwide. The result of low-affinity Na+ uptake is toxic to the cytoplasm of most crop plants. Nevertheless, the pathways for this low-affinity Na+ uptake are still uncertain. In this work we used 22Na+ isotope tracing technology to investigate factors associated with determination of root 22Na+ influx in the salt accumulation halophyte Suaeda maritima. We found that a 2 min of exposure to the 22Na+ labeled uptake solution was optimal for determining 22Na+ influx into excised roots of S. maritima and that 7 min of blotting is suitable in 22Na+ influx experiments. 22Na+ influx did not increase linearly with the increasing external Na+ concentration, in the range tested, of 2 to 300 mM NaCl. But root 22Na+ influx and root Na+ concentration were well correlated. 22Na+ influx into excised roots of S. maritima was not, however, well correlated with the plant size. All the above results indicated further that this 22Na+ isotope influx procedure is a good method for quantify Na+ uptake rate by the roots of the salt accumulation halophyte. 相似文献
7.
In the present investigation, intracellular sodium ([Na+]i) levels were determined in GH4C1 cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na+]i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na+. Resting [Na+]i in GH4C1 cells was 26 ± 6.2 mM (mean ± SD). In cells incubated in Na+-buffer [Na+]i decreased to 3 ± 3.6 mM. If Na+/K+ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na+]i increased to 47 ± 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetete, or thapsigargin had no effect on [Na+]i. Incubating the cells in Ca2+-buffer rapidly increased [Na+]i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca2+, nimodipine, or Ni2+ to these cells immediately decreased [Na+]i, whereas Bay K 8644 enhanced the influx of Na+. In cells where [Na+]i was increased the TRH-induced increase in intracellular free calcium ([Ca2+]i) was decreased compared with control cells. Our results suggest that Na+ enters the cells via Ca2+ channels, and [Na+]i may attenuate TRH-induced changes in [Ca2+]i in GH4C1 cells. © 1993 Wiley-Liss, Inc. 相似文献
8.
The fluxes of 22Na+ and 86Rb+ in Arbacia sperm and oocytes were studied in order to determine how these cells carry out cation exchange with the sea environment. The uptake of these ions by serum followed a pattern of early rapid influx (initial 0.5 min) and subsequent efflux (1–3 min) followed by a gradual uptake (after 3 min). Neither the uptake nor the efflux of these cations by Arbacia sperm were affected by ouabain, suggesting that influx and efflux of 22Na+ and 86Rb+ in Arbacia sperm occur predominantly by passive transport. The 22Na+ uptake by Arbacia oocytes showed a steady increase after an initial rapid uptake. A slight but significant inhibition of 22Na+ uptake was observed with ouabain. However, 86Rb+ uptake by the oocytes reached an early equilibrium and was not affected by ouabain. The uptake of Rb+ by Arbacia oocyte is by passive transport while that of Na+ is both by passive and active transport. 相似文献
9.
Agalakova NI Gusev GP 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2003,134(1):11-20
The treatment of frog erythrocytes incubated in standard nitrate medium with 100 nM phorbol ester (PMA) induced a sharp increase in the 22Na uptake by the cells and intracellular Na(+) concentration. The PMA-induced enhancement in 22Na uptake was stimulated by the addition of 0.1 mM ouabain to the incubation medium and completely blocked by 1 mM amiloride. The time course of 22Na uptake by frog red cells in the presence of PMA showed a lag phase ( approximately 5 min), after which was linear within 5-15 min. The calculated Na(+) influx in erythrocytes treated with PMA was 49.4+/-3.7 mmol l(-1) cells h(-1) as compared with 1.2+/-0.25 mmol l(-1) h(-1) for control cells. 5-(N-ethyl-N-isopropyl)-amiloride, selective blocker of NHE1, caused a dose-dependent inhibition of the PMA-induced Na(+) influx with IC(50) of 0.27 microM. The PMA-induced Na(+) influx was almost completely inhibited by 0.1 microM staurosporine, protein kinase C blocker. Pretreatment of frog red blood cells for 5, 10 or 15 min with 10 mM NaF, non-selective inhibitor of protein phosphatase, led to a progressive stimulation of the PMA effect on Na(+) influx. Both amiloride and NaF did not affect the basal Na(+) influx in frog erythrocytes. The data indicate that the Na(+)-H(+) exchanger in the frog erythrocytes is quiescent under basal conditions and can be markedly stimulated by PMA. 相似文献
10.
Heinke Jäger María José Alencastro Martin Kaupenjohann Ingo Kowarik 《Plant and Soil》2013,368(1-2):629-640
Background and aims
Salinity is an increasing problem for agricultural production worldwide. Understanding how Na+ enters plants is important if reducing Na+ influx, a key component of the regulation of Na+ accumulation in plants and improving salt tolerance of crop plants, is to be achieved. Our previous work indicated that two distinct low-affinity Na+ uptake pathways exist in the halophyte Suaeda maritima. Here, we report the external NaCl concentration at which uptake switches from pathway 1 to pathway 2 and the kinetics of the interaction between external K+ concentration and Na+ uptake and accumulation in S. maritima in order to determine the roles of K+ transporters or channels in low-affinity Na+ uptake.Methods
Na+ influx, Na+ and K+ accumulations in S. maritima exposed to various concentrations of NaCl (0–200 mM) were analyzed in the absence and presence of the inhibitors TEA and Ba+ (5 mM TEA or 3 mM Ba2+) or KCl (0, 10 or 50 mM).Results
Our earlier proposal was confirmed and extended that there are two distinct low-affinity Na+ uptake pathways in S. maritima: pathway 1 might be mediated by a HKT-type transporter under low salinity conditions and pathway 2 by an AKT1-type channel or a KUP/HAK/KT type transporter under high salinity conditions. The external NaCl concentration at which two distinct low-affinity Na+ uptake switches from pathway 1 to pathway 2, the ‘turning point’, is between 90 and 95 mM. Over a short period (12 h) of Na+ and K+ treatments, a low concentration of K+ (10 mM) facilitated Na+ uptake by S. maritima under high salinity (100–200 mM NaCl), whether or not the plants had been subjected to a longer (3 d) period of K+ starvation. The kinetics suggests that low concentration of K+ (10 mM) might activate AKT1-type channels or KUP/HAK/KT-type transporters under high salinity (100–200 mM NaCl).Conclusions
The turning-point of external NaCl concentrations for the two low-affinity Na+ uptake pathways in Suaeda maritima is between 90 and 95 mM. A low concentration of K+ (10 mM) might activate AKT1 or KUP/HAK/KT and facilitate Na+ uptake under high salinity (100–200 mM NaCl). The kinetics of K+ on Na+ uptake and accumulation in S maritima are also consistent with there being two low-affinity Na+ uptake pathways. 相似文献11.
《生物化学与生物物理学报:生物膜》1985,814(2):347-355
The kinetics of uptake and loss of Na+ have been studied using radioisotopic tracer techniques in cells of the cyanobacterium Synechocystis PCC6714 exposed to hyperosmotic stress. Cells transferred from a fresh-water-based medium to NaCl at 300–1000 mmol·dm−3 showed net Na+ uptake during the first 2 min following transfer, with the intracellular Na+ level at 2 min increasing as a direct function of the external NaCl concentration. Further incubation of cells in low-level hypersaline media (350–500 mmol · dm−3 NaCl) led to a marked reduction in cell Na+ within 20 min, indicating an efficient active Na+ extrusion system. In contrast, cells maintained in more extreme hypersaline media showed little (750 mmol · dm−3 NaCl) or no (1000 mmol · dm−3 NaCl) net Na+ extrusion following upshock. Cells grown in a saline medium (with NaCl at 500 mmol · dm−3 showed a greatly reduced net Na+ uptake after 2 min in media containing higher levels of NaCl. However, net Na+ uptake was also observed when these cells were downshocked to media containing 50–200 mmol · dm−3 NaCl. This is the first demonstration of downshock-induced Na+ accumulation in a microbial cell. Time-courses for Na+ extrusion in cells downshocked from 500 mmol · dm−3 to 100 mmol · dm−3 NaCl were similar to those for cells upshocked from freshwater to 500 mmol · dm−3 NaCl, requiring approximately 30 min to reach their lowest values. Net Na+ extrusion in upshocked cells was found to be markedly sensitive to the external K+ concentration, with limited net Na+ extrusion in the absence of external K+ and maximal reductions in cell Na+ in media containing K+ at 1–10 mmol · dm−3. Temperature was also shown to affect uptake and loss of cell Na+ during upshock: cells maintained at 5°C showed no capacity for net Na+ extrusion, while higher temperatures (up to 35°C) led to a progressive reduction in the amount of cell Na+ at 2 min following upshock and also in the rate of net Na+ extrusion after this time. 相似文献
12.
A. O. Sherstobitov A. A. Lapin V. V. Glazunov A. A. Nikiforov 《Journal of Evolutionary Biochemistry and Physiology》2010,46(3):235-240
Mechanisms of transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis were studied by using 204Tl. The Tl+ transport in lamprey oocytes has been shown to be realized by at least two pathways: through Na/K-pump and by mechanisms
of Na,K,Cl-cotransport. In the standard Ringer solution (mM): 4 KCl, 140 NaCl, 0.5 CaCl2, 5 glucose, 10 Tris-HCl-in the presence of ouabain, the coefficient of the 204Tl stationary distribution (cell/medium) was within the range of 2.3–2.5, while the time necessary to reach its 50% value
amounted to 40–45 min at 20°C. In potassium-free media, transport of 204Tl via Na/K-pump was described by simple kinetics with saturation and was characterized by the value V
max = 520 pmol/(cell h) and K
M = 0.3 mM. In the presence of 4 mM K+ and 0.1 mM/1 Tl+, the ouabain-sensitive Tl+ flux decreased to 75 pmol/(cell h). At activation of the mechanism of Na,K,Cl-cotransport by the outer Na+ (in Na-NMDG media of different composition) the total influx of Tl+ reached 193 ± 20 pmol/(cell h), while the bumetanide-sensitive component—119 ± 12 pmol/(cell h) with K
M for Na+ about 20 mM. In the incubation media with variable concentration of chloride ions (replacement of Cl− by NO3) the total Tl+ flux reached 220 ± 21, while via the mechanisms of Na,K,Cl-cotransport—87 ± 8 pmol/(cell h). Under our experimental conditions,
mechanisms of active transport and Na,K,Cl-cotransport accounted for 94% of the Tl+ influx. The potassium channels that usually are permeable also to monovalent thallium ions were not revealed. 相似文献
13.
The influence of salt status of root tissue of Zea mays on influxof 84Rb and 22Na and net accumulation of K+ and Na+ was studied.Low-salt roots were grown in 0.5 mM CaCl2, and high-salt rootsin 2.5 mM KC1 + 7.5 mM NaCl + 0.5 mM CaCl2. High-salt statusgreatly reduced (approx. 90 per cent inhibition) both 22Na and86Rb influxes in the low concentration range isotherm (i.e.at external concentrations below 1 mM). A less marked inhibitionwas observed in the higher concentration range isotherm (130mM), indicating that the uptake in this range is less affectedby the salt status of the tissue. During transition from low- to high-salt status there was anet accumulation of K+ but not of Na+ despite the presence ofa measurable 22Na+ influx at all times. The presence of a continuous22Na influx but no net accumulation implies an Na+ efflux frommaize root tissue. The results differ significantly from thosepreviously published for barley and a possible explanation ofthese differences is discussed. 相似文献
14.
The activity of the β-cell Na+/K+ pump was studied
by using ouabain-sensitive (lmM ouabain) 86Rb+
influx in β-cell-rich islets of Umeå-ob/ob mice as an
indicator of the pump function. The present results
show that the stimulatory effect of glucose on
ouabain-sensitive 86Rb+ influx reached its approximate
maximum at 5mM glucose. Pre-treatment of
the islets with 20mM glucose for 60 min strongly
reduced the glucose-induced stimulation of the Na+/K+ pump. Pre-treatment (60 or 180 min) of islets at
0mM glucose, on the other hand, did not affect the
magnitude of the glucose-induced stimulation of 86Rb+ influx dunng the subsequent 5-min incubation.
Glibenclamide stimulated the ouabain-sensitive
86Rb+ uptake in the same manner as glucose.
The stimulatory effect, showed its apparent maximum
at 0.5μM. Pre-treatment (60 min) of islets with
1μM glibenclamide did not reduce the subsequent
stimulation of the ouabain-sensitive 86Rb+ influx.
The stimulatory effect of glibenclamide and D-glucose
were not .additive, suggesting that they
may have the same mechanism of action. No direct
effect of glibenclamide (0.01-1μM) was observed
on the Na+/K+ ATPase activity in homogenates of
islets. Diazoxide (0.4mM) inhibited the Na+/K+
pump. This effect was sustained even after 60 min of
pre-treatment of islets with 0.4mM diazoxide. The
stimulatory effect of glibenclamide and D-glucose
were abolished by diazoxide. It is concluded
that nutrient as well as non-nutrient insulin secretagogues activate the Na+/K+ pump, probably
as part of the membrane repolarisation process. 相似文献
15.
Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with 22Na to determine the unidirectional Na+ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na+ transport into erythrocytes. The stimulation of Na+ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na+ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na+ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na+ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (~12%), but statistically significant decrease of intracellular Na+ content. A small decrease of intracellular K+ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na+ transport across the lamprey erythrocyte membrane in both directions. 相似文献
16.
E.J.J. Van Zoelen L.G.J. Tertoolen J. Boonstra P.T. Van Der Saag S.W. De Laat 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(3):223-234
1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na++K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na++K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2·10?6 M. Neuro-2A cells contain (3.5±0.7)·105 ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10?20 mol K+/min per copy of (Na++K+)-ATPase at room temperature. 相似文献
17.
T. I. Ivanova A. O. Sherstobitov G. P. Gusev 《Journal of Evolutionary Biochemistry and Physiology》2001,37(5):528-534
To study H+ transport, the lamprey red blood cells were acidified to pH 6.0 by a pretreatment with an ionophore, nigericin. Incubation of the acidified cells in NaCl-medium at pH 8.0 was accompanied by a rapid H+ efflux from the erythrocytes. There was a tenfold decrease of the H+ efflux rate on addition to NaCl-medium of dimethylamiloride or on replacing Na+ in the medium (KCl-medium, pH 8.0). A high rate of Na+ influx into the acidified erythrocytes occurred only in the presence of H+ gradient (pH medium 8.0), but not in its absence (pH medium 6.0). The Na+-dependent H+ efflux from the cells and H+-dependent Na+ influx into the cells were quantitatively similar (about 700 mmol/l cells/h). A rapid elevation of the intracellular Na+ concentration as measured by flame photometry was also observed during incubation of the acidified cells in NaCl-medium (pH 8.0). The H+-dependent Na+ influx and an increase of the Na+ content in the acidified cells were significantly inhibited by amiloride. The data obtained for the first time prove with certainty the presence of the Na+/H+ exchanger in erythrocytes of the river lamprey. 相似文献
18.
Mitzy Canessa José R. Romero Nelson Ruiz-Opazo Victoria L. M. Herrera 《The Journal of membrane biology》1993,134(2):107-122
The properties of the α1 Na+-K+ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive luxes (mmol/liter cell x hr = FU, Mean ± se) in red blood cells (RBCs) and varying internal ( i ) and external ( o ) Na+ and K+ concentrations. Kinetic parameters of several modes of operation, i.e., Na+/ K+, K+/K+, Na+/Na+ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n=12 rats of each strain) into fresh cells incubated in 140 mm Na+ + 5 mm K+, ouabain-sensitive K+ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na+ efflux and Na i were similar in both strains. Thus, the coupling ratio between unidirectional Na+∶K+ fluxes was significantly higher in DS than in DR cells at similar RBC Na+ content. In the presence of 140 mm Na o , activation of ouabain-sensitive K+ influx by K o had a lower K m and V max in DS as estimated by the Garay equation (N=2.70 ± 0.33, K m 0.74 ± 0.09 mm; V max 2.87 ± 0.09 FU) than in DR rats (N=1.23 ± 0.36, K m 2.31 ± 0.16 mm; v max 5.70 ± 0.52 FU). However, the two kinetic parameters were similar following Na o removal. The activation of ouabain-sensitive K+ influx by Na i had significantly lower V max in DS (9.3 ± 0.4 FU) than in DR (14.5 ± 0.6 FU) RBCs but similar K m. These data suggest that the low K+ influx in DS cells is caused by a defect in modulation by Na o and Na i . Na+ efflux showed no differences in Na i activation or trans effects by Na o and K o , thus accounting for the different Na+∶K+ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na+ and K+ fluxes, where the net ouabain-sensitive Na+ losses showed similar magnitudes in the two Dahl strains while the net ouabainsensitive K+ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na+ influx and K+ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na+ influx by K o was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, K o could activate higher K+ influx in DR pumps without a complete inhibition of ouabain-sensitive Na+ influx. This behavior is consistent with K o interaction with distinct Na+ and K+ transport sites. In addition, the inhibition of K+ efflux by Na, was different between Dahl strains. Ouabain-sensitive K+ efflux at Na i level of 4.6 mmol/liter cell, was significantly higher in DS (3.86 ± 0.67 FU) than in DR (0.86 ± 0.14 FU) due to a threefold higher K50 for Na i -inhibition 9.66 ± 0.41 vs. 3.09 ± 0.11 mmol/liter cell. This finding indicates that Na+ modulation of K+ transport is altered at both sides of the membrane. The dissociation of Na+ modulatory sites of K+ transport from Na+ transport sites observed in RBCs of Dahl strains suggests that K+ transport by the Na+-K+ pump is controlled by Na+ allosteric sites different from the Na+ transport sites. The alterations in K+ transport may be related to the amino acid substitution (Leu/Gln276) reported for the cDNA of the α1 subunit of the Na+-K+ pump in the DS strain or to post-translational modifications during RBC maturation. These studies were supported by the following grants: NIH (HL-35664, HL-42120, HL-18318, HL-39267, HL-01967). J.R.R. is a Ford Foundation Predoctoral Fellow. A preliminary report of this work was presented at the International Conference on the Na+-K+ pump and 44th Annual Meeting of the Society of General Physiologists held at Woods Hole, MA, September 5–9, 1990, and published as an abstract in the J. Gen. Physiol. 96:70a, 1990. 相似文献
19.
A. Corriat N. Moatti A. Lemonnier 《In vitro cellular & developmental biology. Plant》1983,19(7):522-528
Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions.
The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at
substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system
(K
m
=0.25 mmol/l, V
max
=17 nmol/mg protein per min) and a nonsaturable component of influx (K
d
=1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na+ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The
largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These
results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na+ independent system L. The differences between this cell type and others studied previously are discussed.
This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France). 相似文献
20.
Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na+ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg2+ and can be increased by 40-fold by loading cells with Mg2+ to 10 mm. The purpose of this study was to verify the connection of AS Na+ influx with Na-dependent, amiloride-sensitive Mg2+ efflux and to utilize AS Na+ influx to explore that pathway.Determination of unidirectional influx of Na+ and net loss of Mg2+ in parallel sets of cells showed that activation by extracellular [Na+] follows a simple Michaelis-Menten relationship for both processes with a K
m
of 105–107 mm and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg2+] with a [Mg2+]0.5 of 2.1–2.3 mm and a Hill coefficient of 1.8. Comparison of Vmax for both sets of experiments indicated a stoichiometry of 2 Na: l Mg. Amiloride inhibits Na+ influx and Mg2+ extrusion in parallel (K
i
= 0.3 mm). Like Mg2+ extrusion, amiloride-sensitive Na+ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na+ influx in hamster red cells appears to be through the Na-Mg exchange pathway.There was no amiloride-sensitive Na+ efflux in hamster red cells loaded with Na+ and incubated with high [Mg2+] in the medium with or without external Na+, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier. 相似文献