共查询到20条相似文献,搜索用时 515 毫秒
1.
Paul P. M. Schnetkamp 《The Journal of membrane biology》1985,88(3):263-275
Summary The permeability properties of the plasma membrane of intact rod outer segments purified from bovine retinas (ROS) were studied with the aid of the optical probe neutral red as described in the companion paper. The following observations were made: (1) Electrical shunting of ROS membranes greatly stimulated Na+ and K+ transport, suggesting that this transport reflects Na+ and K+ currents, respectively. The dissipation of a Na+ gradient across the plasma membrane occurred with a half-time of 30 sec at 25°C. (2) The Na+ permeability was progressively inhibited when the external Ca2+ concentration was raised from 1 m to 20mm. A similar Ca2+ dependence was observed for H+ and Li+ transport. The Na+ permeability was not affected when the total internal Ca2+ content of ROS was varied between 0.1 mol Ca2+/mol rhodopsin and 7 mol Ca2+/mol rhodopsin, or when the free internal Ca2+ concentration was varied between 0.1 and 50 m. (3) The K+ permeability was progressively stimulated when the external Ca2+ concentration was raised from 0.001 to 1 m, whereas a further increase to 20mm was without effect. A similar Ca2+ dependence was observed for Rb+ and Cs+ transport. (4) At an external Ca2+ concentration in the micromolar range the rate of transport decreased in the order: Na+>K+=H+>Cs+>Li+. (5) Na+ fluxes depended in a sigmoidal way on the external Na+ concentration, suggesting that sodium ions move in pairs. The concentration dependence of uniport Na+ transport and that of Na+-stimulated Ca2+ efflux (exchange or antiport transport) were very similar. 相似文献
2.
Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca2+ transport have been examined in intact human red blood cells. Active Ca2+ efflux was determined from the initial rate of45Ca2+ loss after CoCl2 was added to block Ca2+ loading via the ionophore A23187. Ca2+-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca2+ in the presence of A23187. The apparent Ca affinity of active Ca2+ efflux (K
0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca2+-ATPase assay (K
0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca2+ efflux and Ca2+-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg2+-depleted cells, and completely restored by reintroduction of intracellular Mg2+. Active Ca2+ efflux was inhibited almost completely by raising external CaCl2 (but not MgCl2) to 20mm, probably by interaction of Ca2+ at the externally oriented E2P conformation of the pump. Cd2+ was more potent than Ca2+ in this inhibition, while Mn2+ was less potent and 10mm Ba2+ was without effect. A Ca2+: proton exchange mechanism for active Ca2+ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca2+ efflux at least twofold above the efflux rate at pH 7.8 Ca2+ transport was not affected by decreasing the membrane potential across the red cell. 相似文献
3.
Summary The effects of cAMP, ATP and GTP on the Ca2+-dependent K+ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca2+-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K+ ghosts were incubated in an isotonic Na+ medium, the rate constant of Ca2+-dependent K+ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg2+ was added together with ATP, and it was abolished by raising free Ca2+ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K+ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K+ loss when the ghost free-Ca2+ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg2+ and it, was not overcome by raising internal Ca2+. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K+ loss. Although this effect was observed in the absence of added Mg2+ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg2+. The K+ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF2 (10mm), or MgF2 (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-32P)ATP under various conditions affecting K+ channel activity, was in direct correspondence to their effect on K+ efflux. The results suggest that the K+ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins. 相似文献
4.
Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule 总被引:1,自引:0,他引:1
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
5.
The ionic requirements for K+-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca2+ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K+-induced efflux. The Ca2+-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K+. In addition, verapamil did not antagonize 50 mM K+-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K+-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K+ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K+-evoked release, replacing Na+ with choline abolished the taurine efflux seen in response to K+ stimulation. Together, these findings suggest that neuronal N- and L-type Ca2+- and voltage-dependent Na+-channels are not involved in the influx of Ca2+ which appears to be necessary for K+-evoked taurine efflux, and that in addition to Ca2+, extracellular Na+ is also required. 相似文献
6.
Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line 总被引:1,自引:0,他引:1
Xinjun He Xiaozai Wu R. James Turner Bruce J. Baum 《The Journal of membrane biology》1990,115(2):159-166
Summary We have investigated muscarinic receptor-operated Ca2+ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca2+ release and extracellular Ca2+ entry. The carbachol (Cch) dose response of intracellular Ca2+ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K
0.510 or 30 m, depending on the method for [Ca2+]
i
determination). However, similar data for Ca2+ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca2+ release and a second higher affinity site withK
0.52.5 m. In addition, the Ca2+ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K+ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca2+ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca2+]
i
of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca2+ channel blocker La3+ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca2+ in these cells by increasing Ca2+ entry. Organic Ca2+ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca2+ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K+ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP3) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP3 formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca2+ entry in HSG-PA cells. One is associated with IP3 formation and intracellular Ca2+ release and is independent of membrane potential; the other is less dependent on IP3 formation and intracellular Ca2+ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating. 相似文献
7.
Shmuel Muallem Timothy Beeker Stephen J. Pandol 《The Journal of membrane biology》1988,102(2):153-162
Summary The relative contributions of the Na+/Ca2+ exchange and the plasma membrane Ca2+ pump to active Ca2+ efflux from stimulated rat pancreatic acini were studied. Na+ gradients across the plasma membrane were manipulated by loading the cells with Na+ or suspending the cells in Na+-free media. The rates of Ca2+ efflux were estimated from measurements of [Ca2+]
i
using the Ca2+-sensitive fluorescent dye Fura 2 and45Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca2+ efflux is described by a single exponential function under control, Na+-loaded, and Na+-depleted conditions. Manipulation of Na+ gradients had no effect on the hormone-induced increase in [Ca2+]
i
. The results indicate that Ca2+ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca2+ pump. The effects of several cations, which were used to substitute for Na+, on cellular activity were also studied. Choline+ and tetramethylammonium+ (TMA+) released Ca2+ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na+, TMA+, choline+, or N-methyl-d-glucamine+ (NMG+) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG+, choline+, and TMA+ inhibited amylase release by 55, 40 and 14%, respectively. NMG+ also increased the Ca2+ permeability of the plasma membrane. Thus, to study Na+ dependency of cellular function, TMA+ is the preferred cation to substitute for Na+. The stimulatory effect of TMA+ can be blocked by atropine. 相似文献
8.
Summary This study concerns the properties of rapid K+ and Cl transport pathways that are present in the (H++K+)-ATPase membrane from stimulated, and secreting, gastric oxyntic cells. Ion permeabilities in the isolated stimulation-associated vesicles were monitored via the rates of H+ efflux under conditions of exclusive H+/K+ counterflux or H+–Cl co-efflux, as well as by comparison of equilibration rates for86Rb and36Cl under conditions of equilibrium exchange and unidirectional salt flux. These latter studies suggest that Rb+ and Cl pathways are conductive and independent. In spite of the functional independence of the ion pathways, several divalent cations inhibit Rb+ and Cl isotopic exchange as well as the H+ efflux that is dependent on either K+ or anion (Cl, SCN, NO2) fluxes. Zn2+ is the more potent inhibitor, reducing by 50% the sensitive component of K+, Cl, and NO2 fluxes at about 20 m; Mn2+ has a similar effect at 200 m. Ni2+ and Co2+ were roughly equipotent to Mn2+ while Mg2+ and Ca2+ had not inhibitory effect. These results suggest that the stimulation-induced permeabilities, while functioning independently, may be physically linked, i.e., residing within a single entity. In similar studies carried out in (H++K+)-ATPase vesicles obtained from nonstimulated cells, no vestiges of sensitivity to the inhibitory divalent cations could be detected. The implications of these findings for the physiology of the oxyntic cell in the context of a model for membrane fusion are discussed. 相似文献
9.
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
10.
Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex 总被引:1,自引:0,他引:1
M. P. E. van Heeswijk J. A. M. Geertsen C. H. van Os 《The Journal of membrane biology》1984,79(1):19-31
Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca2+-ATPase, ATP-dependent Ca2+ transport and Na+/Ca2+ exchange have been studied with special emphasis on the relative transport capacities of the two Ca2+ transport systems. The kinetic parameters of Ca2+-ATPase activity in digitonin-treated membranes are:K
m
=0.11 m Ca2+ andV
max=81±4 nmol Pi/min·mg protein at 37°C. ATP-dependent Ca2+ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca2+/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca2+ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca2+ transport, a stoichiometry of 0.7 mol Ca2+ transported per mol ATP is found for the Ca2+-ATPase. In the presence of 75mm Na+ in the incubation medium ATP-dependent Ca2+ uptake is inhibited 22%. When Na+ is present at 5mm an extra Ca2+ accumulation is observed which amounts to 15% of the ATP-dependent Ca2+ transport rate. This extra Ca2+ accumulation induced by low Na+ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na+–K+)-ATPase generates a Na+ gradient favorable for Ca2+ accumulation via the Na+/Ca2+ exchanger. In the absence of ATP, a Na+ gradient-dependent Ca2+ uptake is measured which rate amounts to 5% of the ATP-dependent Ca2+ transport capacity. The Na+ gradient-dependent Ca2+ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na+/Ca2+ exchange system for Ca2+ is between 0.1 and 0.2 m Ca2+, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca2+ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca2+-ATPase system exceeds the capacity of the Na+/Ca2+ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca2+ homeostasis of rat kidney cortex cells. 相似文献
11.
Paul P. M. Schnetkamp 《The Journal of membrane biology》1985,88(3):249-262
Summary The nature of the Ca2+ buffer sites in intact rod outer segments isolated from bovine retinas (ROS) was investigated. The predominant Ca2+ buffer in intact ROS was found to be negatively charged groups confined to the surface of the disk membranes. Accordingly, Ca2+ buffering in ROS was strongly influenced by the electrostatic surface potential. The concentration of Ca2+ buffer sites was about 30mm, 80% of which were located at the membrane surface in the intradiskal space. A comparison with observations in model systems suggests that phosphatidylserine is the major Ca2+ buffer site in ROS. Protons and alkali cations could replace Ca2+ as mobile counterions for the fixed negatively charged groups. At physiological ionic strength, the total number of these diffusible, but osmotically inactive, counterions was as large as the number of osmotically active cations in ROS. The surface potential is dependent on the concentration of cations in ROS and can be measured with the optical dye neutral red. Addition of cations to the external solution led to the release of the internally bound dye as the cations crossed the outer membrane. The chemical and spectral properties of the dye enable its use as a real-time indicator of cation transport across the outer envelope of small particles in suspension. In this study, the dye method is illustrated by the use of well-defined ionophores in intact ROS and in liposomes. In the companion paper this method is used to describe the cation permeabilities native to ROS. 相似文献
12.
Summary Regulation of passive potassium ion transport by the external calcium concentration and temperature was studied on cell cultures of 3T3 mouse cells and their DNA-virus transformed derivatives. Upon lowering of external calcium concentration, passive potassium efflux generally exhibits a sharp increase at about 0.1mm. The fraction of calcium-regulated potassium efflux is largely independent of temperature in the cases of the transformed cells, but shows a sharp increase for 3T3 cells upon increasing temperature above 32°C. In the same range of temperature, the 3T3 cells exhibit the phenomenon of high-temperature inactivation of the residual potassium efflux at 1mm external calcium. At comparable cellular growth densities, the transformed cell lines do not show high-temperature inactivation of residual potassium efflux. These results are consistent with the notion of a decisive role of the internal K+ concentration in the cell-density dependent regulation of cell proliferation. In particular, the growth-inhibiting effect of lowering the external Ca2+ concentrations is considered as largely due to a rise of passive K+ efflux and a subsequent decrease of internal K+ concentration. The experimental data on the Ca2+ dependence of passive K+ flux are quantitatively described by a theoretical model based on the constant field relations including negative surface charges on the external face of the membrane, which cooperatively bind Ca2+ ions and may concomitantly undergo a lateral redistribution. The present evidence is consistent with acidic phospholipids as representing these negative surface charges.This work is dedicated to the memory of Max Delbrück (deceased March 10, 1981), in whose laboratory in 1966 the earlier version of the present theoretical model was developed by one of the authors. 相似文献
13.
Summary The voltage- and time-dependent K+ current,I
K
+
out
, elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La3+) to the extracellular medium. These results suggested that the influx of external Ca2+ was necessary for K+ current activation. The IC50, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca2+ actuated the K+ current, we altered either the Ca2+ buffering capacity or the free Ca2+ concentration of the intracellular medium (pipette filling solution). By these means,I
K
+
out
could be varied over a 10-fold range. Increasing the free Ca2+ concentration from 40 to 400nm also shifted the activation of the K+ current toward more negative potentials. Maintaining cytoplasmic Ca2+ at 500nm with 40nm EGTA resulted in a more rapid activation of the K+ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca2+ on depolarization. Increasing intracellular Ca2+ to 500nm or 1 m also led to inactivation of the K+ current within a few minutes. It is concluded thatI
K
+
out
is regulated by cytosolic Ca2+, which is in turn controlled by Ca2+ influx through dihydropyridine-, and phenylalkylamine-sensitive channels. 相似文献
14.
Summary The K+-stimulated, ouabain-insensitive ATPase activity present in vesicles of microsomal fractions from hog gastric mucosa can be demonstrated in fresh preparations by adding Ca2+ (M range) to the incubation medium. Ca2+ effect is similar but not additive to the effect of gramicidin or freezing. High Ca2+ concentrations (1 mM) produce an inhibotory effect on the K+-stimulated ATPase activity. This effect, is not seen in the presence of gramicidin. Calcium increases the magnitude of ATP-driven H+ uptake in vesicles exposed to K+ for periods of time up to 60 min. At longer times of exposure (120 min) the response does not differ from controls. It is concluded that Ca2+ at low concentrations (m range) enhances the K+ permeability of the vesicular membrane. At higher concentrations (mm range), Ca2+ becomes inhibitory to the K+ permeability. A role for Ca2+ as a second messenger in stimulus-secretion coupling in the parietal cell is discussed. 相似文献
15.
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
16.
Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium,and by other divalent ions 总被引:4,自引:0,他引:4
Summary Direct inhibitory effects of Ca2+ and other ions on the epithelial Na+ channels were investigated by measuring the amiloride-blockable22Na+ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol.
81:643–666, 1983) we found that the presence of micromolar concentrations of Ca2+ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive22Na+ uptake could not be blocked by Ca2+ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba2+, Sr2+, or VO2+, but not by Mg2+. The Ca2+ inhibition constant was a strong function of pH, and varied from 0.04 m at pH 7.8 to >10 m at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of22Na+ uptake in vesicles that contained 0.5 m Ca2+. This Ca2+ activity produced a maximal inhibition of22Na+ uptake at pH7.4 but had no effect at pH7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data is compatible with the model that Ca2+ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself inhibit transport. The fact that the apical Na+ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca2+, rather than, or in addition to changes in the cytoplasmic level of free Ca2+. 相似文献
17.
The Ca2+-dependent K+ efflux from rat submandibular gland was studied using a K+-sensitive electrode. A K+ efflux was induced by either adrenalin or by using the divalent cation ionophore A23187 plus added Ca2+ to bypass the receptor mechanism. Trifluoperazine, which was used to investigate the role of calmodulin, was found to block the adrenalin-induced K+ efflux but not the A23187/Ca2+-induced K+ efflux. The adrenalin-induced K+ efflux was abolished by quinidine and the A23187/Ca2+-induced K+ efflux was significantly reduced by quinidine. In other experiments, the presence of indomethacin did not inhibit the adrenalin-induced K+ efflux, and exogenously added arachidonic acid did not induce a K+ efflux. It is concluded that neither prostaglandin synthesis, nor a cytosolic Ca2+-calmodulin complex is involved in the agonist-induced K+ efflux from rat submandibular gland. A similarity between the Ca2+-dependent K+ efflux mechanism of erythrocyte ghosts and submandibular tissue is indicated by their common response to quinidine. 相似文献
18.
Franco Turrini Ivan Sabolić Zoran Zimolo Brunhilde Moewes Gerhard Burckhardt 《The Journal of membrane biology》1989,107(1):1-12
Summary In the presence of inhibitors for mitochondrial H+-ATPase, (Na++K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl–) or entry of a cation, e.g., Na+ via electrogenic Na+/d-glucose and Na+/l-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane. 相似文献
19.
Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves 总被引:3,自引:0,他引:3
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
20.
Tomoji Hata Naoki Makino Hironobu Nakanishi Takashi Yanaga 《Molecular and cellular biochemistry》1988,84(1):65-76
Summary The purpose of this study was to examine the effect of three classes of Ca2+ antagonists, diltiazem, verapamil and nifedipine on Na+-Ca2+ exchange mechanism in the sarcolemmal vesicles isolated from canine heart. Na+-Ca2+ exchange and Ca2+ pump (ATP-dependent Ca2+ uptake) activities were assessed using the Millipore filtration technique. sarcolemmal vesicles used in this study are estimated to consist of several subpopulations wherein 23% are inside-out and 55% are right side-out sealed vesicles in orientation. The affect of each Ca2+ antagonist on the Na+-dependent Ca2+ uptake was studied in the total population of sarcolemmal vesicles, in which none of the agents depressed the initial rate of Ca2+ uptake until concentrations of 10 M were incubated in the incubation medium. However, when sarcolemmal vesicles were preloaded with Ca2+ via ATP-dependent Ca2+ uptake, cellular Ca2+ influx was depressed only by verapamil (28%) at 1 M in the efflux medium with 8 mM Na+. Furthermore, inhibition of Ca2+ efflux by verapamil was more pronounced in the presence of 16 mM Na+ in the efflux medium. The order of inhibition was; verapamil > diltiazem > nifedipine. These results indicate that same forms of Ca2+-antagonist drugs may affect the Na+-Ca2+ exchange mechanism in the cardiac sarcolemmal vesicles and therefore we suggest this site of action may contribute to their effects on the myocardium. 相似文献