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1.
Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca2+ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca2+ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca2+ indicator dye. The uptake of Ca2+ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca2+. At 100 nm Ca2+, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H+-ATPase, or by NH4Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca2+ increased as the Ca2+ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca2+ concentrations. At 700 nm Ca2+, the rate and extent of uptake were lower in the absence of K+ than in the presence of K+. The inhibitors of Ca2+-activated K+-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K+-dependent Ca2+ uptake at 700 nm Ca2+. Thus the uptake of Ca2+ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H+-gradient and (ii) the K+ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca2+ and contribute to regulation of cytosolic Ca2+ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999  相似文献   

2.
Summary The whole-cell patch-clamp method has been used to measure Ca2+ influx through otherwise K+-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K+ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K+ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K+ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca2+ is more negative than or equal to the K+ equilibrium potential (–47 mV), indicating that the current is due to Ca2+ influx through the K+ channels prior to their closure. Decreasing internal [Ca2+] (Ca i ) from 200 to 2 nm or increasing the external [Ca2+] (Ca o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca2+ influx also decrease the amplitude of time-activated current, indicating that Ca2+ permeation is slower than K+ permeation, and so causes a partial block. Increasing Ca o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K+ channels in Zea mays guard cells also appear to have a Ca i -, and Ca o -dependent ability to mediate Ca2+ influx. We suggest that the inwardly rectiying K+ channels are part of a regulatory mechanism for Ca i . Changes in Ca o and (associated) changes in Ca i regulate a variety of intracellular processes and ion fluxes, including the K+ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.  相似文献   

3.
The activation of Ca2+-dependent K+ channel by propranolol or by ascorbate-phenazine methosulphate stimulates Na+-dependent transport of α-aminoisobutyric acid. This stimulation arises from a membrane hyperpolarization due to the specific increase of membrane K+ conductance. The same treatment does not modify the Na+-independent uptake of the norbornane amino acid.  相似文献   

4.
Summary Intracellular Ca2+ has been suggested to play an important role in the regulation of epithelial Na+ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca2+-free medium enhanced Na+ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na+ entry across the apical membrane by intracellular Ca2+. In the present study, we have examined the effect of Ca2+-free preincubation on apical membrane Na+ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca2+ medium exhibited higher Na+ uptake than those derived from proximal tubules incubated in Ca2+-free EGTA medium. Such effect of Ca2+ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba2+ (1mm). Ca2+ in the preincubation medium did not affect Na+-dependent BBM glucose uptake, and its effect on BBM Na+ uptake was pH gradient dependent and amiloride (10–3 m) sensitive, suggesting the involvement of Na+/H+ antiport system. Addition of verapamil (10–4 m) to 1mm Ca2+ preincubation medium abolished while ionomycin (10–6 m) potentiated the effect of Ca2+ to increase BBM Na+ uptake, suggesting that the effect of Ca2+ in the preincubation medium is likely to be mediated by Ca2+-dependent cellular pathways and not due to a direct effect of extracellular Ca2+ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na+ uptake was affected by the presence of Ca2+ in the preincubation medium, suggesting that Ca2+ in the preincubation medium did not increase BBM Na+ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10–4 m) to 1mm Ca2+ preincubation medium did not prevent the increase in BBM Na+ uptake. The effect of Ca2+ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10–6 m). In summary, these results show the Ca2+ dependency of Na+ transport by renal BBM, possibly through stimulation of Na+/H+ exchanger by protein kinase C.  相似文献   

5.
Summary The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K+ and Ca2+ transport and respiration (QO2) of proximal tubule suspensions. Hg2+, Cu2+, and Au3+ (10–4 m) each caused a rapid net K+ efflux and a delayed inhibition of QO2. The Hg2+-induced net K+ release represented passive K+ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg2+ and Ag+ promoted a net Ca2+ uptake that was nearly coincident with the onset of the net K+ efflux. A delayed inhibition of ouabainsensitive QO2 and nystatin-stimulated QO2, indicative of Na+, K+-ATPase inhibition, was observed after 30 sec of exposure to Hg2+. More prolonged treatment (2 min) of the tubules with Hg2+ resulted in a 40% reduction in the CCCP-uncoupled QO2, indicating delayed injury to the mitochondria. The net K+ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmaleimide (10–4 m) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10–4 m). In addition, both DTT and GSH immediately reversed the Ag+-induced net Ca2+ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na+, K+-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.  相似文献   

6.
Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(K m (Ca2+)=0.4 m) and ATP(K m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl or NO 3 . Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO 4 3– which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.  相似文献   

7.
The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.  相似文献   

8.
We have previously reported the presence of two Ca2+ influx components with relatively high (KCa= 152 ± 79 μm) and low (KCa= 2.4 ± 0.9 mm) affinities for Ca2+ in internal Ca2+ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432:105–111]. We have also reported the presence of a high affinity Ca2+ influx component with KCa= 279 ± 43 μm in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289–296]. The present studies show that a low affinity Ca2+ influx component is also present in BLMV with KCa= 2.3 ± 0.41 mm (Vmax= 16.36 ± 4.11 nmoles of Ca2+/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca2+ influx component that is activated by internal Ca2+ store depletion in dispersed parotid gland acini by the following criteria: (i) similar KCa for calcium flux, (ii) similar IC50 for inhibition by Ni2+ and Zn2+; (iii) increase in KCa at high external K+, (iv) similar effects of external pH. The high affinity Ca2+ influx in cells is different from the low affinity Ca2+ influx component cells in its sensitivity to pH, KCl, Zn2+ and Ni2+. The low and high affinity Ca2+ influx components in BLMV can also be distinguished from each other based on the effects of Zn2+, Ni2+, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca2+ influx pathway in BLMV which displays characteristics similar to the low affinity Ca2+ influx component detected in parotid acinar cells following internal Ca2+ store depletion. Received: 19 March 1997/Revised: 25 November 1997  相似文献   

9.
It is concluded that Ca2+ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na+/Ca2+-exchange mechanism that is driven by the (transmembrane) Na+ gradient established by Na+/K+-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na+/Ca2+-exchanger next to the Ca2+-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca2+ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na+ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca2+ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca2+ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na+/K+-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996  相似文献   

10.
The effects of cadmium and lead on the internal concentrations of Ca2+ and K+, as well as on the uptake and translocation of K(86Rb+) were studied in winter wheat (Triticum aestivum L. a. MV-8) grown hydroponically at 2 levels of K+ (100 uM and 10 mM). Cd2+ and Pb2+ were applied in the nutrient solution in the range of 0.3 to 1000 u.M. Growth was more severely inhibited by Cd2+ and in the high-K+ plants as compared to Pbz+ and low-K+ plants. Ions of both heavy metals accumulated in the roots and shoots, but the K+ status influenced their levels. Ca2+ accumulation was increased by low concentrations of Cd2+ mainly in low-K+ shoots, whereas it was less influenced by Pb2+. The distribution of Cd2+ and Ca2+ in the plant and in the growth media indicated high selectivity for Cd2+ in the root uptake, while Ca2+ was preferred in the radial and/or xylem transport. Cd2+ strongly inhibited net K+ accumulation in high-K+ plants but caused stimulation at low K+ supply. In contrast, the metabolis-dependent influx of K+(86Rb+) was inhibited in low-K+ plants, while the passive influx in high-K+ plants was stimulated. Translocation of K+ from the roots to the shoots was inhibited by Cd2+ but less influenced in Pb2+-treated plants. It is concluded that the effects of heavy metals depend upon the K+-status of the plants.  相似文献   

11.
Summary 86Rb+ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba2+ required for half-maximal inhibition was 0.6mm. The effects of externally added cations on86Rb+ influx and efflux have established that this pathway is conductive, with a selectivity for K+, Rb+ and Cs+ over Na+ and Li+. the Rb+ uptake is inversely dependent on external pH, but not significantly affected by internal Ca2+ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb+ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K+ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na+ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca2+ activity directly blocks apical Na+ entry.  相似文献   

12.
Summary The activation of rabbit aortic smooth muscle was studied by two most widely used vascular smooth muscle stimulants: -adrenoceptor activation by norepinephrine (NE) and high-K+ depolarization. This was studied by measurements of isometric contractions and net as well as unidirectional Ca2+ fluxes. These parameters showed markedly differential sensitivities towards two smooth muscle inhibitors used in this study: D 600 and amrinone. By choosing an appropriate concentration of D 600 or amrinone, Ca2+ uptake or Ca2+ influx induced by high K+ or NE could be selectively inhibited. Furthermore, by using unidirectional flux measurements it was demonstrated that Ca2+ influx stimulated by NE and high K+ were additive in nature. The data from the addivity experiment exclude the interpretation of a common Ca2+ pathway with two separate mechanisms for opening it. The data on three criteria employed in this study provide evidence for the existence of two independent Ca2+ pathways, one for each mode of activation, for Ca2+ influx known to be associated with these contractions.  相似文献   

13.
Summary Using Ca2+- and K+-selective microelectrodes, the cytosolic free Ca2+ and K+ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca2+ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca2+ concentration were observed in the submicromolar ranges. During the Ca2+ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca2+ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca2+ concentration down to 10–7 m or by applying a Ca2+ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca2+-activated K+ channel, sizable Ca2+ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K+ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K+ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca2+ level, which, in turn, periodically stimulate Ca2+-activated K+ channels.  相似文献   

14.
Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca2+-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca2+-binding and -release, respectively (rate constants >>103 sec−1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 103 sec−1). With 10 mm K+ and 1 mm Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mm Ca2+) and for conductance (1.2 mm Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles. Received: 6 June 1995/Revised: 12 September 1995  相似文献   

15.
Cadmium (Cd2+) interferes with the uptake, transport and utilization of several macro‐ and micronutrients, which accounts, at least in part, for Cd2+ toxicity in plants. However, the mechanisms underlying Cd2+ interference of ionic homeostasis is not understood. Using biophysical techniques including membrane potential measurements, scanning ion‐selective electrode technique for non‐invasive ion flux assays and patch clamp, we monitored the effect of Cd2+ on calcium (Ca2+) and potassium (K+) transport in root hair cells of rice. Our results showed that K+ and Ca2+ contents in both roots and shoots were significantly reduced when treated with exogenous Cd2+. Further studies revealed that three cellular processes may be affected by Cd2+, leading to changes in ionic homeostasis. First, Cd2+‐induced depolarization of the membrane potential was observed in root hair cells, attenuating the driving force for cation uptake. Second, the inward conductance of Ca2+ and K+ was partially blocked by Cd2+, decreasing uptake of K+ and Ca2+. Third, the outward K+ conductance was Cd2+‐inducible, decreasing the net content of K+ in roots. These results provide direct evidence that Cd2+ impairs uptake of Ca2+ and K+, thereby disturbing ion homeostasis in plants.  相似文献   

16.
The charge translocation associated with sarcoplasmic reticulum (SR) Ca2+ efflux is compensated for by a simultaneous SR K+ influx. This influx is essential because, with no countercurrent, the SR membrane potential (Vm) would quickly (<1 ms) reach the Ca2+ equilibrium potential and SR Ca2+ release would cease. The SR K+ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K+ countercurrent during release. To better define the physiological role of the SR K+ channel, we compared SR Ca2+ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K+ channel function. Specifically, we reduced SR K+ channel conduction 35 and 88% by replacing cytosolic K+ for Na+ or Cs+ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca2+ reloading, and caffeine-evoked Ca2+ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K+ (TRIC) channels is not required to support SR Ca2+ release (or uptake). Because K+ enters the SR through RyRs during release, the SR K+ (TRIC) channel most likely is needed to restore trans-SR K+ balance after RyRs close, assuring SR Vm stays near 0 mV.  相似文献   

17.
Summary The Ca2+-activated K+ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na+ outside, K+ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K+-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca2+-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca2+ concentration in contact with the membrane inside ([Ca2+]i) to 1.5×10–7 m had little effect on the relationship between membrane potential andP. When [Ca2+]i was increased to 3×10–7 m and 6×10–7 m smaller potential changes were required to open the channels. Increasing [Ca2+]i further to 8×10–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca2+]i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K+ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca2+]i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca2+ influx through voltage-activated Ca2+ channels.  相似文献   

18.
Summary Kinetic properties of Na+–Ca2+ exchange in a renal epithelial cell line (LLC-MK2) were assessed by measuring cytosolic free Ca2+ with fura-2 and45Ca2+ influx. Replacing external Na+ with K+ produced relatively small increases in free Ca2+ and45Ca2+ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na+ and strongly potentiated the effect of replacing external Na+ with K+ on free Ca2+ and45Ca2+ uptake.45Ca2+ influx in 140mm K+ or N-methyl-d-glucamine minus influx in 140mm Na+ was used to quantify Na+–Ca2+ exchange activity of Na+-loaded cells. The dependence of exchange on cell Na+ was sigmoidal; theK 0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na+ partly account for the small increase in Ca2+ influx when all external Na+ is replaced by K+. Besides raising cell Na+ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg2+ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na+. Potassium also increased theV max of exchange by 86% and had no effect on theK m for Ca2+. The exchanger does not cause detectable22Na+–Mg2+ exchange and does not appear to require K+ or transport86Rb+. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na+–Ca2+ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.  相似文献   

19.
20.
Summary We have examined the effect of second messengers on ATP-driven H+ transport in an H+ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H+ transport. Acridine orange fluorescence in the presence of 0.5mm Ca2+ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H+ transport by Ca2+ was concentration dependent; 0.25 and 0.5mm Ca2+ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca2+ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca2+ did not affect ATP hydrolysis. ATP-dependent Br uptake was virtually unchanged in the presence of 0.5mm Ca2+ (+1mm EGTA). These vesicles were also shown to transport Ca2+ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca2+ uptake. These results are consistent with the co-existence of an H+ ATPase and an H+/Ca2+ exchanger on these endosomes, the latter transport system using the H+ gradient to energize Ca2+ uptake. Attempts to demonstrate an H+/Ca2+ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca2+ uptake could be demonstrated. A Ca2+-dependent increase in H+ permeability and an ATP-dependent Ca2+ uptake might have important implications for the regulation of vacuolar H+ ATPase activity as well as the homeostasis of cytosolic Ca2+ concentration.  相似文献   

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