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1.
Exogenous tritiated -aminobutiric acid ([3H]GABA) is retained in two compartments in sheep cortex synaptosomes, corresponding to cytoplasmic and vesicular spaces, assuming that freeze-thawing the synaptosomes loaded with [3H]GABA releases the cytoplasmic [3H]GABA (81±3.9%), and that subsequent solubilization of the synaptosomes with 1% sodium cholate releases the vesicular [3H]GABA (19±3.9%). Depolarization of synaptosomes with 40 mM K+ in a Na+-medium, in the absence of Ca2+, releases 20.3±2.7% of the [3H]GABA retained in the synaptosomes. The [3H]GABA released under these conditions comes predominantly from the cytoplasm. The presence of 1 mM Ca2+ during depolarization releases and additional 13% (a total of about 33.5±9.9%) of the releasable [3H]GABA, and the [3H]GABA release which is Ca2+-dependent also comes mostly from the cytoplasmic compartment. When choline replaces external Na+, the [3H]GABA release is absolutely Ca2+-dependent, and the [3H]GABA released also comes mostly from the cytoplasmic pool. Therefore, it appears that [3H]GABA taken up by synaptosomes is accumulated mostly in the cytoplasmic compartment from which it is released upon depolarization. The technique described permits distinguishing the effect of different factors on the two pools of accumulated [3H]GABA.  相似文献   

2.
The Ca2+-dependent, presumably exocytotic fraction of the [3H]GABA released by depolarization is dissected from the depolarization-induced Na+-dependent, carrier-mediated fraction of [3H]GABA release in mouse brain synaptosomes. GABA homoexchange is prevented by the [3H]GABA carrier blocker, DABA. The absence of external Na+ completely abolishes the release of the carrier-mediated, presumably cytoplasmic release of [3H]GABA induced by homoexchange and heteroexchange with GABA and DABA, respectively. The carrier-mediated, Na+-dependent fraction of the depolarization-induced release of [3H]GABA is resistant to tetrodotoxin (TTX) but is sensitive to amiloride and verapamil. The Ca2+-dependent fraction of the [3H]GABA released by high K+ depolarization is also completely abolished by amiloride (from 300 M) and sensitive to verapamil (30 M), but in contrast is insensitive to the absence of external Na+ and to DABA. On the basis of these results we conclude that amiloride and verapamil inhibit high K+-induced release of [3H]GABA by antagonizing the entrance of Ca2+ (and possibly Na+ when external Ca2+ is absent) through a population of voltage sensitive presynaptic Ca2+ channels activated by depolarization.Depto. de Biología Molecular Instituto de Investigaciones Biomédicas UNAM.  相似文献   

3.
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   

4.
The involvement of different subtypes of voltage-sensitive Ca2+ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [3H]acetylcholine ([3H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [3H]ACh were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca2+ channel blocker -conotoxin GVIA (-CgTX, 0.01–1 M) reduced the stimulation-evoked release of ATP and [3H]ACh in a dose-dependent manner. Similarly, the P-type Ca2+ channel antagonist -agatoxin IVA (-Aga IVA) (0.05 M) and the inorganic Ca2+ channel blocker Cd2+ (0.2 mM) inhibited the outflow of both transmitters, while Ni2+ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca2+ antagonists. Long-term perfusion (i.e., 90 min) with Ca2+ free solution inhibited the evoked-release of ATP and [3H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [3H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca2+]0 condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca2+ chelators, and dipyridamole (2 M), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca2+-free media. Tetrodotoxin (TTX, 1 M), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca2+]0 conditions. In summary, we showed that both N- and P-type Ca2+ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [3H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [Ca2+]0 conditions an additional release of ATP occurs, which is not associated with action potential propagation.  相似文献   

5.
The effect of a protein kinase inhibitor, staurosporine, on Ca2+-dependent and Ca2+-independent release of [14C]GABA in isolated rat brain synaptosomes was studied. Calcium-dependent [14C]GABA release was stimulated by depolarization with a K+ channel blocker, 4-aminopyridine (4-AP), or high K+ concentration. It has been shown that the effect of 4-AP is Ca2+-dependent, while high K+ is able to evoke [14C]GABA release in both Ca2+-dependent and Ca2+-independent manners. In addition, Ca2+-independent [14C]GABA release was studied using α-latrotoxin (LTX) as a tool. Pretreatment of synaptosomes with staurosporine resulted in pronounced inhibition of 4-AP-stimulated Ca2+-dependent [14C]GABA release. The inhibitory effect of staurosporine on [14C]GABA release was not due to modulation of 4-AP-promoted45Ca2+ influx into synaptosomes. If the process of [14C]GABA release occurred in the Ca2+-independent manner irrespectively of what, LTX or high K+, stimulated this process, it was not inhibited by staurosporine. Considering the above findings, it is reasonable to assume that the absence of Ca2+ in the extracellular medium created conditions for activation of the process of neurotransmitter release without Ca2+-dependent dephosphorylation of neuronal phosphoproteins; as a consequence, regulation of exocytotic process was modulated in such a manner that inhibition of protein kinases did not disturb exocytosis.  相似文献   

6.
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   

7.
Summary Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration ([Ca2+] i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+] i response to these agonists was examined by four methods. Low concentrations of TPA (2×10–10 m) had no effect on Ca2+ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca2+] i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca2+] i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF- resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10–8 m) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca2+] i , showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca2+] i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca2+ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca2+] i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca2+ for EGF-mediated Ca2+ release which also respond differently to TPA.  相似文献   

8.
[3H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca2+-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [3H]purine release. Nimodipine only at the concentration of 10 M modified [3H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [3H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of45Ca2+ by the cultures. The treatment of cells with the Ca2+ agonist Bay K 8644 did not affect [3H]purine release or the45Ca2+ uptake. The drug did not either alter [Ca2+]i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca2+ discharge, significantly reduced the evoked [3H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca2+ ATPase, was able to increase either the culture [3H]purine release or the [Ca2+]i. Together, the findings indicate that voltage-sensitive calcium channels (VSCCs) of the neuronal N and L-types are not involved in the modulation of [3H]purine release from rat cultured astrocytes whereas Ca2+ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCCs seem to be able to affect [3H]purine outflow with mechanisms other than VSCC gating.  相似文献   

9.
In this study we investigated the role of external monovalent cations, and of intracellular Ca2+ concentration ([Ca2+]i) in polarized and depolarized rat cerebral cortex synaptosomes on the release of [3H]--aminobutyric acid (3H-GABA). We found that potassium-depolarization, in the absence of Ca2+, of synaptosomes loaded with3H-GABA releases 7.4±2.1% of the accumulated neurotransmitter, provided that the external medium contains Na+, and an additional 19.0±2.5% is released upon adding 1.0 mM CaCl2 to the exterior. The Ca2+-independent release component does not occur in a choline medium and it is only 3.4±0.8% of the3H-GABA accumulated in a Li+ medium, but both ions support the Ca2+-dependent release of3H-GABA (13.4±0.6% in choline and 15.4±1.5% in Li+), which suggests that the exocytotic release is independent of the external monovalent cation present, whereas the carrier-mediated release specifically requires Na+ outside. Furthermore, previous release of the cytosolic3H-GABA due to predepolarization in the absence of Ca2+ does not influence the amount of3H-GABA subsequently released by exocytosis due to Ca2+ addition (19.1±2.5% or 19.1±1.1%, respectively). In choline or Li+ medium, the value of the [Ca2+]i is raised by Na+/Ca2+ exchange to 663±75 nM or 782±54 nM, respectively, within three minutes after adding 1.0 mM Ca2+, in the absence of depolarization, and parallel release experiments show no release of3H-GABA in the choline medium, but a substantial release (7.1±2.1%) of3H-GABA occurs in the Li+ medium without depolarization. Subsequent K+-depolarization shows normal Ca2+-dependent release of3H-GABA in the choline medium (14.1±2.0%) but only 8.6±1.1% release in the Li+ medium, which suggests that raising the [Ca2+]i by Na+/Ca2+ exchange, without depolarization, supports some exocytotic release in Li+, but not in choline media. The role of [Ca2+]i and of membrane depolarization in the release process is discussed on the basis of the results obtained and other relevant observations which suggest that both Ca2+ and depolarization are essential for optimal exocytotic release of GABA.Special issue dedicated to Dr. Santiago Grisolia.  相似文献   

10.
The thermodynamic efficiency of the calmodulin-activated form of the Ca2+-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca2+ concentration ([Ca2+]i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][Pi]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg2+, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca2+]o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM Pi, variable [ADP]. Under these conditions, with the pump working near itsK m of 64nM, the [Ca2+]i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca2+]i/[Ca2+]o ratio against [ATP]/([ADP][Pi]) gave a straight line with a slope of 1.5×107M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×107M). These results demonstrate (1) tight coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2 Ca2+ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC50 of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.  相似文献   

11.
Summmary The Ca2+ uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca2+ uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0° or 37°, but the Ca2+ uptake activity was relatively stable at 25°. The decay of Ca2+ uptake activity at 0° could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca2+ uptake activity of homogenates kept on ice but not of homogenates kept at 37°. We also found that release of the CSR from the cellular debris required homogenization in high KCI. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 mol/min-mg in the absence of CSR calcium channel blockers and 0.67 mol/min/mg in the presence of 10 M ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca2+-ATPase rates averaged 1.07 mol/min/mg and 0.88 mol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a crude preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca2+-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function.  相似文献   

12.
The effects of spontaneous and evoked [3H]taurine release from a P2 fraction prepared from rat retinas were studied. The P2 fraction was preloaded with [3H]taurine under conditions of high-affinity uptake and then examined for [3H]taurine efflux utilizing superfusion techniques. Exposure of the P2 fraction to high K+ (56 mM) evoked a Ca2+-independent release of [3H]taurine. Li+ (56 mM) and veratridine (100 M) had significantly less effect (8–15% and 15–30%, respectively) on releasing [3H]taurine compared to the K+-evoked release. 4-Aminopyridine (1 mM) had no effect on the release of [3H]taurine. The spontaneous release of [3H]taurine was also Ca2+-independent. When Na+ was omitted from the incubation medium K+-evoked [3H]taurine release was inhibited by approximately 40% at the first 5 minute depolarization period but was not affected at a second subsequent 5 minute depolarization period. The spontaneous release of [3H]taurine was inhibited by 60% in the absence of Na+. Substitution of Br for Cl had no effect on the release of either spontaneous or K+-evoked [3H]taurine release. However, substitution of the Cl with acetate, isethionate, or gluconate decreased K+-evoked [3H]taurine release. Addition of taurine to the superfusion medium (homoexchange) resulted in no significant increase in [3H]taurine efflux. The taurine-transport inhibitor guanidinoethanesulfonic acid increased the spontaneous release of [3H]taurine by approximately 40%. These results suggest that the taurine release of [3H]taurine is not simply a reversal of the carrier-mediated uptake system. It also appears that taurine is not released from vesicles within the synaptosomes but does not rule out the possibility that taurine is a neurotransmitter. The data involving chloride substitution with permeant and impermeant anions support the concept that the major portion of [3H]taurine release is due to an osmoregulatory action of taurine while depolarization accounts for only a small portion of [3H]taurine release.  相似文献   

13.
We studied the release of [3H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca2+-dependent and Ca2+-independent release of [3H]d-aspartate. In Ca2+-free (no added Ca2+) Na+ medium, the agonists of the glutamate receptors induced the release of [3H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl2 the release of [3H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca2+-free medium. NMDA was the most potent agonist in stimulating the Ca2+-dependent release of [3H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca2+-dependent release of [3H]d-aspartate evoked by kainate was dependent on the influx of Ca2+ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca2+ channels (VSCC). The exocytotic release of [3H]d-aspartate evoked by AMPA relied exclusively on Ca2+ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca2+ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca2+ entry modulating vesicular release may be selectively recruited.  相似文献   

14.
Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs+-conducting medium. We found that the pharmacology, Cs+ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10–7 to 10–3 M Ca2+, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca2+, respectively, the cardiac SR channel remained activated in the presence of high [Ca2+]. In correlation to these studies, [3H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca2+] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [3H]ryanodine binding of cardiac SR at millimolar Ca2+ in the absence of adenine nucleotides.When Ca2+-induced Ca2+ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca2+-induced inactivation at high [Ca2+] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca2+ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca2+-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.  相似文献   

15.
A boron-containing antibiotic, boromycin (BM), was found to influence the Ca2+ homeostasis in both excitable and non-excitable cells. In non-excitable cells (human erythrocytes and leucocytes) it inhibited the resting passive45Ca2+ transport in 10–6–10–5 mol/L concentrations. In human erythrocytes, the passive 45Ca2+ transport induced by the presence of 1 mmol/L NaVO3 was inhibited by boromycin (90% inhibition) as well. The inhibitory effect of BM on the NaVO3-induced passive 45Ca2+ transport was diminished in the presence of inhibitory concentrations of nifedipine (10 mol/L – 60% inhibition) or of those of K+ o (75 mmol/L – 20% inhibition). On the other hand, in rat brain synaptosomes, and rat cardiomyocytes, BM stimulated the passive 45Ca2+ transport in resting cells at similar concentrations. In rat cardiomyocytes the stimulation was transient. The stimulatory effect on the passive 45Ca2+ transport in rat brain synaptosomes was accompanied with the increase of cytoplasmic Ca2+ concentration measured by means of the entrapped fluorescent Ca2+ chelator fura-2. The stimulatory effect of BM was diminished when synaptosomes were pre-treated with veratridine (10 mol/L) which itself stimulated the passive 45Ca2+ transport. At saturating concentrations of veratridine, no stimulatory effect of BM was observed. These results could be explained by the indirect interaction of BM with both Ca2+ and Na+ transport systems via transmembrane ionic gradients of monovalent cations and could be useful in determining whether the cells belong to excitable, or non-excitable cells.  相似文献   

16.
The effects of the Na+-Ca2+ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943) on depolarization-induced Ca2+ signal and [3H]noradrenaline release were examined in SH-SY5Y cells. KB-R7943 at 10 M significantly inhibited high K+-induced increase in intracellular Ca2+ concentration. KB-R7943 also inhibited high K+-evoked release of [3H]noradrenaline from the cells. These findings suggest that the Na+-Ca2+ exchanger in the reverse mode is involved at least partly in depolarization-induced transmitter release.  相似文献   

17.
Mitochondria in Ca2+ Signaling and Apoptosis   总被引:8,自引:0,他引:8  
Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa2+ ([Ca2+]c) signals. Mitochondria are endowed with multiple Ca2+ transport mechanismsby which they take up and release Ca2+ across their inner membrane. These transport processesfunction to regulate local and global [Ca2+]c, thereby regulating a number of Ca2+-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca2+ effluxpathway from mitochondria. In addition, Ca2+ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na+/Ca2+ exchanger. Duringcellular Ca2+ overload, mitochondria take up [Ca2+]c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (m) and cell death. In apoptosis signaling,collapse of ;m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed.  相似文献   

18.
Ca2+ transients and the rate of Ca2+ release (dCaREL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca2+ indicators. Single pulse experiments with different returning potentials showed that Ca2+ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca2+ removal (dCaREM/dt) was studied by fitting the decaying phase of the Ca2+ transient (Melzer, Ríos & Schneider, 1986) and dCaREL/dt was calculated as the difference between dCa/dt and dCaREM/dt. The fast Ca2+ release decayed as a consequence of Ca2+ inactivation of Ca2+ release. Double pulse experiments showed inactivation of the fast Ca2+ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca2+ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca2+]i and the inhibited amount of Ca2+ release was 0.98. The [Ca2+]i for 50% inactivation of dCaREL/dt was 0.25 m, and the minimum number of sites occupied by Ca2+ to inactivate the Ca2+ release channel was 3.0. These data support Ca2+ binding and inactivation of SR Ca2+ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.  相似文献   

19.
Summary The relationship between Ca2+ release from sarcoplasmic reticulum, induced by elevated pH, tetraphenylboron (TPB) or chemical modification, and the change in the surface charge of the membranes as measured by the fluorescence intensity of anilinonaphthalene sulfonate (ANS) is examined. The stimulated Ca2+ release is inhibited by dicyclohexylcarbodiimide and external Ca2+. TPB, but not tetraphenylarsonium (TPA+), causes a decrease in ANS fluorescence, with 50% decrease occurring at about 5 m TPB. The decrease in ANS fluorescence as well as the inhibition of Ca2+ accumulation induced by TPB are prevented by TPA+. A linear relationship between the decrease in membrane surface potential and the extent of the Ca2+ released by TPB is obtained. Similar levels of [3H]TPB bound to sarcoplasmic reticulum membranes were obtained regardless of whether or not the vesicles have taken up Ca2+. The inhibition of Ca2+ accumulation and the [3H]TPB incorporation into the membranes were correlated. Ca2+ release from sarcoplasmic reticulum, by pH elevation, chemical modification or by addition of NaSCN (0.2 to 0.5m) or the Ca2+ ionophore ionomycin, is also accompanied by a decrease in ANS fluorescence intensity. However, chemical modification and elevated pH affects the surface potential much less than SCN or TPB do. These results suggest that the enhancement of Ca2+ release by these treatments is not due to a general effect on the membrane surface potential, but rather through the modification of a specific protein. They also suggest that membrane surface charges might play an important role in the control mechanism of Ca2+ release.  相似文献   

20.
The effect of the alcohol-deterrent drug, disulfiram, on mitochondrial Ca2+ content was studied. Addition of this drug (20 µM) to mitochondria induces a complete loss of accumulated Ca2+. The calcium release is accompanied by a collapse of the transmembrane potential, mitochondrial swelling, and a diminution of the NAD(P)H/NAD(P) radio. These effects of disulfiram depend on Ca2+ accumulation; thus, ruthenium red reestablished the membrane and prevents the oxidation of pyridine nucleotides. The binding of disulfiram to the membrane sulfhydryls appeared to depend on the metabolic state of mitochondria, as well as on the mitochondrial configuration. In addition, it is shown that modification of 9 nmol -SH groups per mg protein suffices to induce the release of accumulated Ca2+.  相似文献   

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