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1.
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   

2.
Summary Human red cells were prepared with various cellular Na+ and K+ concentrations at a constant sum of 156mm. At maximal activation of the K+ conductance,g K(Ca), the net efflux of K+ was determined as a function of the cellular Na+ and K+ concentrations and the membrane potential,V m , at a fixed [K+]ex of 3.5mm.V m was only varied from (V m E K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g K(Ca) as a function of cellular Na+ and K+ concentrations atV m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na+. Since the present Ca2+-activated K+ channels have been shown to be of the multi-ion type, the experimental data from each set of Na+ and K+ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na+ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na+ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca2+ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg K(Ca) for outward currents at (V m E K)>25 25 mV reflects a voltage-dependent Na+ block of the Ca2+-activated K+ channels.  相似文献   

3.
A partially purified preparation of the lobster muscle Na+/Ca2+ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca2+ efflux from 45Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na+ (Na ev )-dependent Ca2+ efflux depended directly upon the extravesicular Ca2+ concentration ([Ca2+] ev ) with a half-maximal activation at [Ca2+] ev = 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca2+, as in most other species. In contrast, at low [Na+] ev , the Ca ev -binding site (i.e., on the cytoplasmic surface) for Ca2+ transported via Ca2+/Ca2+ exchange was half-maximally activated by about 7.5 μm Ca2+. Mild proteolysis of the Na+/Ca2+ exchanger by α-chymotrypsin also upregulated the Na ev -dependent Ca2+ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca2+. Proteolytic digestion in the presence of 1.9 μm free ev Ca2+, however, induced only a 1.6-fold augmentation of Ca2+ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca2+ also inhibited proteolytic degradation of the Na+/Ca2+ exchanger measured by immunoblotting. These data suggest that Ca2+, bound to a high affinity binding site, protects against the activation of the Na+/Ca2+ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na+/Ca2+ exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids. Received: 20 October 1999/Revised: 13 January 2000  相似文献   

4.
Summary Micromolar concentrations of silver ion activate large Ca2+ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag+-induced Ca2+ efflux from intact ROS depended on the Ag+ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag+ the rate of Ca2+ efflux was 7×106 Ca2+/outer segment/sec; this represents a change in total intracellular Ca2+ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag+-induced Ca2+ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag+-induced Ca2+ efflux when gramicidin was present. In the presence of gramicidin, Ag+-induced Ca2+ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca2+ flux across ROS disk membranes. The ion selectivity of the Ag+-induced pathway proved to be broad with little discrimination between the alkali cations Li+, Na+, K+, and Cs+ or between Ca2+ and Mg2+. The properties of the Ag+-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.  相似文献   

5.
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   

6.
Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na+-Ca2+ exchange. Therefore, we investigated the effect of -alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2,4-dimethylbenzamil), a Na+-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3 Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 M of the blocker of the taurine-Na+ cotransporter, -alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of -alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na+-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of -alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na+-Ca2+ exchanger.  相似文献   

7.
Paramecium Na+ channels, which were Ca2+-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10–5 to 10–3 m Ca2+ in the bath. They became much less active in the presence of 10–6 m Ca2+, and their activity subsided completely at 10–8 m Ca2+. A Hill plot showed a dissociation constant of 6 m for Ca2+ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mm Mg2+. The channels also exhibited a mild voltage dependence. When exposed to 10–8 m Ca2+ for an extended period of 2–4 min, channels were further inactivated even after bath Ca2+ was restored to 10–4 m. Whereas neither high voltage (+100 mV) nor high Ca2+ (10–3 m) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca2+ to the bath restored channel activity without a requirement of additional Mg2+ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca2+ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na+ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na+ channel activation, and is the Ca2+ sensor of the Na+-channel gating machinery.We thank C. Kung for his kind support, and A. Boileau for critical reading. Supported by grants from National Institutes of Health GM 22714-20 and 36386-09.  相似文献   

8.
Summary The inhibition of Ca2–-ATPase, (Na++K+)-ATPase and Na+/Ca2+ exchange by Cd2+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca2+ uptake into inside-out membrane vesicles displayed a K m for Ca2+ of 88±17 nm, and was extremely sensitive to Cd2+ with an IC50 of 8.2±3.0 pM Cd2+, indicating an inhibition via the Ca2+ site. (Na++K+)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd2+, displaying an IC50 of 2.6±0.6 m Cd2+. Cd2+ ions apparently compete for the Mg2+ site of the (Na +K+)-ATPase. The Na+/Ca2+ exchanger was inhibited by Cd2+ with an IC50 of 73±11 nm. Cd2+ is a competitive inhibitor of the exchanger via an interaction with the Ca2+ site (K i = 11 nm). Bepridil, a Na+ site specific inhibitor of Na+/Ca2+ exchange, induced an additional inhibition, but did not change the K i of Cd2+. Also, Cd2+ is exchanged against Ca2+, albeit to a lesser extent than Ca2+. The exchanger is only partly blocked by the binding of Cd2+. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na+/Ca2+ exchanger. We conclude that intracellular Cd2+ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca2+ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO).  相似文献   

9.
Summary This communication reports the kinetics of the Na+/ Ca2+ exchanger and of the plasma membrane (PM) Ca2+ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca2+ extrusion and its Na+ dependence for concentrations of cytoplasmic free Ca2+ ([Ca2+]cyt) in the 1–10-m range. The PM Ca2+ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca2+]cyt] 1.5 m with the fluorescent Ca2+ indicator quin2 (K d= 115 nm). That study determined that the PM Ca2+ pump in the basal state has a V max = 0.098 mm/min, a K m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca2+]cyt with the intracellular Ca2+ probe, rhod2 (K d= 500 nm), which has almost a fivefold lower affinity for Ca2+. An Appendix also describes the Mg2+ and pH dependence of the K dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca2+ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca2+ extrusion into a Ca2+-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca2+ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca2+ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca2+ with a K m= 0.97 ± 0.31 m and Vmax = 1.0 ± 0.6 mm/ min. (ii) At high [Ca2+]cyt, the exchanger works at a rate 10 times as large as the basal V max of the PM Ca2+ extrusion pump. (iii) The exchanger can work in reverse after Na+ loading of the cytoplasm by monensin. (iv) The PM Ca2+ extrusion pump is activated by exposure to [Ca2+]cyt 1.5 m for 20–50 sec. Activation raises the pump V max to 1.6 ± 0.6 mm/min and the K mto 0.55 ± 0.24 m. (v) The Ca2+ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca2+]cyt. In summary, the results show that the human platelet can extrude Ca2+ very rapidly at high [Ca2+]cyt. Both the Na+/Ca2+ exchanger and Ca2+ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca2+ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca2+]cyt cytoplasmic Ca2+ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or Vextrusion true rate of Ca2+ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - I fraction of high-affinity rhod2 complexed with Ca2+ - F the observed fluorescence - Fmin the minimal fluorescence observed in the absence of Ca2+ - Fmax the maximal fluorescence observed when the dye is saturated with Ca2+ - X1 the fraction of high-affinity dye - K d,1 dissociation constant of high-affinity dye - K d,2 dissociation constant of the low-affinity dye - -d1/dt rate of Ca2+ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - Fmax (Fmax — Fmin)cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F50 fluorescence of the high-affinity form ofrhod2for[Ca2+]cyt=50 nM - [Ca2+]0 external Ca2+concentration - K p proportionality constant between the total number of Ca2+ ions moved and the change in high-affinity rhod2 complexation to Ca2 - (d[Ca2+]cyt, T)/dt rate of Ca2+ influx obtained with maximal levels of ionomycin - kleak rate constant for passive inward Ca2+ leakage - kinno rate constant for ionomycin-mediated Ca2+ influx - T total - [rhod2]cyt,T total intracellular rhod2 concentration - [quin2]cyt,T total intracellular quin2 concentration - [B]T total cytoplasmic buffering capacity - A[Ca2+]cyt,T total number of Ca2+ ions moved into the cytoplasm - [rhod2-Ca]cyt, T change in concentration of total intracellular high-affinity rhod2 complexed to Ca2+ - [B-Ca]T change in concentration of total cytoplasmic binding sites complexed to Ca2+ - [quin2]cyt, T change in concentration of total intracellular quinl complexed to Ca2+ - change in the degree of intracellular quin2 saturation - 1 change in degree of saturation of cytoplasmic high-affinity rhod2 - 1-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - Vobs observed rate of Ca2+ removal from the rhod2-Ca complex - V8.3 m the rate of Ca2+ removal from the high affinity rhod2-Ca complex at [Ca2+]cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca2+]cytT/t initial linear rate of ionomycin-mediated Ca2+ influx - EC50 effective concentration giving a half-maximal effect - [Na+]cyt cytoplasmic Na+ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid  相似文献   

10.
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   

11.
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   

12.
Na+/Ca2+ exchange (NCX) is a major Ca2+ extrusion system in cardiac myocytes, but can also mediate Ca2+ influx and trigger sarcoplasmic reticulum Ca2+ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na+]i may lead to a rise in [Ca2+]i through NCX, causing Ca2+ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca2+ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca2+ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca2+ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na+-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca2+]i and abolished spontaneous Ca2+ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca2+ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca2+ influx via NCX may be critical in causing myocardial Ca2+ overload and triggered activities induced by cardiac glycoside or reoxygenation.  相似文献   

13.
Summary Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia,Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca2+ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca2+ pump (V m :0.63 nmol·min–1 mg–1,K m : 27nm Ca2+) is calculated to be 2.17 nmol·min–1·mg–1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na+/Ca2+ exchanger with high affinity for Ca2+ (V m :7.2 nmol·min–1·mg–1,K m : 181nm Ca2+) is calculated to be 13.6 nmol·min–1·mg–1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na+/Ca2+ exchanger. The high affinity for Ca2+ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na+/Ca2+ exchanger will contribute substantially to Ca2+ extrusion in the fish enterocyte. Further evidence for an important contribution of Na+/Ca2+ exchange to Ca2+ extrusion was obtained from studies in which the simultaneous operation of ATP-and Na+-gradient-driven Ca2+ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca2+ transporting cell, in which Na+/Ca2+ exchange activity with high affinity for Ca2+ extrudes Ca2+ from the cell.  相似文献   

14.
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   

15.
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   

16.
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   

17.
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   

18.
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   

19.
Summary The countertransport of Ca2+ and Na+ across the membranes of the unicellular fresh-water algaChlamydomonas reinhardtii CW-15 and twoDunaliella species differing in salt tolerance was studied. All algae used are devoid of cell walls. The calcium uptake by twoDunaliella species depended markedly on the intracellular sodium concentration. This calcium uptake was accompanied by Na+ release. For 15 and 30 s after artificial gradient formation (Naint + greater than Naext +) the ratio of released Na+ to absorbed Ca2+ was 31 and 41, respectively. For the extremely halotolerantD. salina, the apparent Michaelis constant of the Ca2+ uptake was 33 M, and for the marine halotolerant algaD. maritima, it was equal to 400 M, presuming more efficient Na+-for-Ca2+ exchange inD. salina cells. Ouabain, an inhibitor of Na+/K+-ATPase, suppressed Na+ transfer by 25%, whereas the agents blocking Ca2+-channels did not affect the transport of Ca2+ and Na+. The oppositely directed transmembrane Ca2+ and Na+ transfer was shown to depend on the external concentrations of Na+ and H+. In the fresh-water algaC. reinhardtii CW-15 (Naext + greater than Naint +), the direction of Ca2+ and Na+ fluxes across the plasma membrane was opposite to those described for Dunaliella cells. The results obtained point to the ability of the Na+-Ca2+ exchanger function in plasma membranes of algal cells.  相似文献   

20.
Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPS) affects ATP-activated Ca2+-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K+ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na+ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na+ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca2+ Ba2+ Mn2+ Sr2+ Na+ K+ = 68 30 26 10 3.5 1. Slope conductances (in pS) for permeant ions rank as follows: Ca2+ Sr2+ Na+ Mn2+ Ba2+ = 19 18 14 12 10. Unitary Ca2+ currents display a tendency to saturate with the Ca2+ concentration increase with apparent dissociation constant (K d ) of 10 mm. No block of Na+ permeation by extracellular Ca2+ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca2+-permeable channels on the other cell types.DeceasedThe authors are grateful to K. Kiselyov and A. Mamin for technical assistance. The work was supported by the Russian Basic Research Foundation, Grant N 93-04-21722 and was made possible in part by Grant N R4A000 from the International Science Foundation.  相似文献   

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