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1.
The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca 2+ levels ([Ca 2+] i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca 2+ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca 2+] i in a concentration-dependent manner. The [Ca 2+] i signal was biphasic with an initial rise and a slow decay. Ca 2+ removal inhibited the Ca 2+ signal by 41%. Adding 3 mM Ca 2+ increased [Ca 2+] i in cells pretreated with clomiphene in Ca 2+-free medium, confirming that clomiphene induced Ca 2+ entry. In Ca 2+-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca 2+ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca 2+ release. Conversely, pretreatment with 50 μM clomiphene in Ca 2+-free medium abolished the [Ca 2+] i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca 2+release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca 2+] i increases in PC3 cells by releasing store Ca 2+ from multiple stores in an phospholipase C-independent manner, and by activating Ca 2+ influx; and clomiphene was of mild cytotoxicity. 相似文献
2.
To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs ( Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO 4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10 −8 M). Such an inhibitory effect of DFB on Ca 2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na +, and Li + did not affect the calcium uptake. Among ionophores, agents disrupting H + gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca 2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and γ-S-GTP stimulated Ca 2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca 2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca 2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells. 相似文献
3.
Light-dependent Ca 2+ efflux via the Ca 2+/H + antiport in the photosynthetic purple sulfur bacterium Chromatium vinosum was inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca 2+ uptake by C. vinosum in the dark nor any effect on the light-dependent efflux of either Na + or Tl + catalyzed, respectively, by the C. vinosum Na +/H + or K +/H + antiports. Ruthenium red and LaCl 3, neither of which inhibited light-dependent Ca 2+ efflux in C. vinosum, markedly inhibited Ca 2+ uptake in the dark by C. vinosum cells. Ruthenium red had no effect on the uptake of either Na +or the K + analog T1 + by C. vinosum cells in the dark. These results have been interpreted in terms of two separate Ca 2+ transport systems in C. vinosum: (i) a phenothiazine-sensitive and ruthenium red, La 3+-insensitive Ca 2+/H + antiport responsible for Ca 2+ efflux in the light; and (ii) a ruthenium red and La 3+-sensitive but phenothiazine-insensitive Ca 2+ uptake system. 相似文献
4.
Utilizing the whole-cell configuration of the patch-clamp technique the effect of calmodulin (CaM) on thapsigargin-induced Ca 2+ current has been studied. Addition of several concentrations of CaM to the patch pipette induced concentration-dependent inhibition of thapsigargin-induced Ca 2+ current in bovine aortic endothelial cells. The effect of CaM was Ca 2+ dependent and was not observed when the intracellular Ca 2+ was buffered to 1 nM with EGTA. CaM produced two major effects on the thapsigargin-induced Ca 2+ current. First CaM slow down activation of the current by thapsigargin from a control value of 16 ± 5 to 31 ± 6 s with 1 μM CaM in the pipette solution. The second effect of CaM was to reduce the current amplitude in a concentration-dependent manner. The inhibition of Ca 2+ current was observed at the peak of the current and at the sustained current level. The reduction of current at the sustained level was observed 15–20 s after onset of the thapsigargin response. The half inhibitory concentration determined from these experiments was 0.1 μM. These results indicate that CaM can modulate thapsigargin-induced Ca 2+ current in this endothelium, suggesting a possible role for CaM in the regulation of store-operated Ca 2+ influx. 相似文献
5.
In this study we seek to elucidate the interaction of capsaicin with the calmodulin mediated signal pathways in macrophages, by comparing its action on macrophage functions with a known calmodulin antagonist, fluphenazine. Kinetics of capsaicin uptake by macrophages (10 3 cells) revealed that a maximum of 200 μM capsaicin was taken up within 10 min. Ca 2+ ionophore triggered generation of superoxide anion and hydrogen peroxide by macrophages was inhibited in a dose-dependent manner by fluphenazine (IC 50, 20 μM and 12 μM, respectively) and also by capsaicin (IC 50, 30 μM and 9 μM, respectively), suggesting an involvement of calmodulin in the regulation of NADPH oxidase. In vitro both fluphenazine and capsaicin inhibited Ca 2+-Mg 2+ ATPase and cAMP-phosphodiesterase from macrophages and this inhibition was reversed by exogenous addition of calmodulin. Fluorescence studies revealed a direct Ca 2+ dependent interaction of capsaicin with calmodulin. From these results we suggest that capsaicin acts via calmodulin to inhibit stimulus-induced macrophage oxidative burst and also that calmodulin regulates the oxidative burst in macrophages. 相似文献
6.
The intracellular free Ca 2+ ion concentration ([Ca 2+]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca 2+]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca 2+ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca 2+]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in [Ca 2+]i were rapid, transient and were blocked by atropine at 10 -6M. A brief exposure to 50 mM K + resulted in a transient increase in [Ca 2+]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10 -4M or 10 -3M) induced [Ca 2+]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca 2+ free medium, the source of the increase in [Ca 2+]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca 2+]i. These data strongly suggest that ACh exerted a direct action on [Ca 2+]i in normal rat pancreatic β-cells and support a role for Ca 2+ as a second messenger in the action of ACh. 相似文献
7.
Ca 2+ uptake by rat brain mitochondria was studied under different experimental conditions. The most rapid uptake of Ca 2+ occurred in the presence of ATP, succinate and P i. ATP alone also supported Ca 2+ uptake. In contrast, no Ca 2+ uptake occurred with succinate and P i when no ATP was added. Oligomycin and atractylate completely inhibited ATP-supported Ca 2+ uptake but produced only a partial inhibition of Ca 2+ transport in the presence of ATP, succinate and P i. ATP plays a dual role in its action on brain mitochondria; it can support Ca 2+ uptake by itself and it serves a function in allowing respiration-dependent Ca 2+ uptake to proceed. The latter role of ATP does not involve transfer of energy from the nucleotide. 相似文献
8.
Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H 2O 2 and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca 2+. The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane potential and influx of Ca 2+. Treatment of the cells with H 2O 2, and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca 2+. These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca 2+ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS. 相似文献
9.
Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca 2+ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca 2+ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2',7'-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N'-diphenyl- p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca 2+ concentration, which was completely prevented by the antioxidants. An intracellular Ca 2+ chelator, bis-( o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca 2+ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca 2+ increase and induction of apoptosis were significantly inhibited by an extracellular Ca 2+ chelator or Na +/Ca 2+ exchanger blockers (bepridil and benzamil), whereas neither Ca 2+ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca 2+ through the activation of reverse mode of Na +/Ca 2+ exchanger. However, tBOOH decreased intracellular Na + concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca 2+ concentration by direct activation of the reverse mode of Na +/Ca> 2+ exchanger, rather than indirect elevation of intracellular Na + levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca 2+ may mediate this action of tBOOH. These results further suggest that Na +/Ca 2+ exchanger may be a target for the management of oxidative hepatotoxicity. 相似文献
10.
1. 1. (Mg2+ + Ca2+) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg2+ + Ca2+) ATPase seen during development was greatest in the synaptic membrane preparations. 2. 2. At 37°C both Na+ or K+ at concentrations higher than 30 mM inhibited the microsomal Mg2+ ATPase, but the (Mg2+ + Ca2+) ATPase was stimulated by both Na+ and K+. Synaptic membrane Mg2+ ATPase was inhibited by concentrations higher than 100 mM K+; Na+ however stimulated this enzyme at all concentrations. Much of this Na+ stimulated activity was ouabain sensitive. Synaptic membrane (Mg2+ + Ca2+) ATPase was stimulated by Na+ or K+, this stimulation follows approximate saturation kinetics with an apparent Km of 18.8 mM Na+ or K+. 3. 3. Arrhenius plots of microsomal (Mg2+ + Ca2+) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole−1 above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg2+ + Ca2+) ATPase from both immature and adult brain.
Author Keywords: Brain; ATPase; temperature; development; synaptic membranes 相似文献
11.
The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca 2+ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca 2+–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca 2+ levels ([Ca 2+] i) without changing 25 μM clomiphene-induced [Ca 2+] i increase. 17β-estradiol and tamoxifen increased [Ca 2+] i by causing Ca 2+ influx and Ca 2+ release because their responses were partly reduced by removing extracellular Ca 2+. In contrast, clomiphene solely induced Ca 2+ release. The effect of the lignans on these two Ca 2+ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca 2+] i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca 2+ release, and 17β-estradiol-induced Ca 2+ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca 2+ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca 2+ signaling in human neutrophils in a multiple manner. 相似文献
12.
The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na + channel blockade by 1 μM tetrototoxin, removal of Ca 2+ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice. 相似文献
13.
Regulation of the increase in inositol phosphate (IP) production and intracellular Ca 2+ concentration ([Ca 2+] i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca 2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC 50) and maximal inhibition of BK induced an increase in [Ca 2+] i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca 2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the KD and Bmax of the BK receptor for binding (control: KD = 1.7 ± 0.2 nM; Bmax = 47.3 ± 4.4 fmol/ mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca 2+ mobilisation in VSMCs. 相似文献
14.
The Ca 2+-mobilizing metabolite cyclic ADP-ribose (cADPR) has been shown to release Ca 2+ from ryanodine-sensitive stores in many cells. We show that this metabolite at a concentration of 17μM, but not its precursor β-NAD + nor non-cyclic ADPR at the same concentration, is active in releasing Ca 2+ from rabbit skeletal muscle sarcoplasmic reticulum. The release was not sensitive to Ruthenium red (1μM) nor to the ryanodine receptor-specific scorpion toxin Buthotus1-1 (10 μM). In planar bilayer single channel recordings, concentrations up to 50μM cADPR did not increase the open probability of Ruthenium red and toxin-sensitive Ca 2+ release channels. Thus Ca 2+ release induced by cADPR in skeletal muscle sarcoplasmic reticulum may not involve opening of ryanodine receptors. 相似文献
15.
We previously demonstrated that oxysterols added to the culture medium of NRK 49F cells labelled with [ 14C] arachidonic acid potentiated arachidonic acid (AA) release and prostaglandin (PG) E 2 biosynthesis induced by the activation of these cells with fetal calf serum (FCS). In the absence of FCS, oxysterols had no effect on AA release. As phospholipase (Plase) A 2 activity is Ca 2+-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca 2+ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca 2+ concentration; when external Ca 2+ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca 2+ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca 2+ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 μm/ml), oxysterols are not able to increase, mimmediately or after a short time of contact (90 min) the concentration of intracellular free Ca 2+ ([Ca 2+]) i measured by fluorescence of Quinn-2; at very high concentration of oxysterol (25 μm/ml), [Ca 2+] i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca 2+ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect o oxysterols on cell Ca 2+ level. 相似文献
16.
Ca 2+ mobilization elicited by simulation with brief pulses of high K + were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca 2+ changes across the cell, suggesting the presence of significant spatial Ca 2+ gradients. Treatment with 20 μM ryanodine, an inhibitor of Ca 2+ release from the SR, and 10 μM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca 2+] i elicited by membrane depolarization. The overall spatial distribution of [Ca 2+] i changes appeared unchanged. Ca 2+ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 μM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the
exchange mechanism. Conversely, when the reversal potential of the
exchange was shifted to negative potentials by lowering [Na +] 0 or by increasing [Na +] i by treatment with 20 μM monensin, the amplitude of these Ca 2+ transients increased. Ca 2+ transients elicited by membrane depolarization and largely mediated by reverse operation of Na +-Ca 2+ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These findings suggest that in intact guinea pig cardiac cells, Ca 2+ influx through the
exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling. 相似文献
17.
Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K +-induced [ 3H] 5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3H] 5-HT release induced by the Ca 2+-ionophore A 23187 or Ca 2+-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K +-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [ 3H] nitrendipine to the dihydropyridine-sensitive L-type Ca 2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K +-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K +-induced increase in intracellular free Ca 2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K +-induced [ 3H] 5-HT release by antagonizing voltage-dependent Ca 2+ entry into nerve terminals. 相似文献
18.
The relationships between pyruvate and derived citrate metabolism and acetylcholine (ACh) synthesis in synaptosomes were examined. In the presence of 30 mM KCl, 0.1 mM Ca 2+ caused 31 and 63% inhibition of pyruvate utilization and citrate accumulation, respectively. Verapamil and EGTA (0.5 mM) brought about no change in pyruvate consumption but increased rate of citrate accumulation, and overcame inhibitory effect of Ca 2+. The rates of citrate accumulation in the presence of verapamil or EGTA were three to six times, respectively, higher than those in the presence of Ca 2+. (−) Hydroxycitrate increased rate of citrate accumulation under all experimental conditions. The value of this activation appeared to be stable (0.20–0.28 nmol/min/mg of protein) and independent of changes in the basic rate of citrate accumulation. Ca 2+ caused no significant changes in [ 14C]ACh synthesis, but it inhibited 14CO 2 production by synaptosomes. These activities were inhibited by verapamil by 33 and 60%, respectively. Ca 2+ did not modify these effects of the drug. On the other hand, (−)hydroxycitrate resulted in 22 and 29% inhibition of [ 14C]ACh synthesis in Ca 2+ free and Ca 2+ supplemented medium, respectively. These data indicated that rates of acetyl-CoA synthesis in synaptoplasm, via ATP-citrate lyase and probably by another pathways are independent of Ca-evoked changes in pyruvate oxidation and citrate supply from intraterminal mitochondria. This property might play a significant role in maintenance of stable level of ACh in active cholinergic nerve endings. 相似文献
19.
Increase in cytoplasmic cyclic AMP concentration stimulates Ca 2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca 2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca 2+ influx. Cyclic AMP evoked discharge of Ca 2+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 μM of free Ca 2+, while Ca 2+ lower than 0.1 μM did not affect the Ca 2+-release. The Ca 2+ flux across plasma membrane was restored from this Ca 2+-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca 2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca 2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca 2+ channel. 相似文献
20.
In this study we investigated the release of Ca 2+ in brain microsomes after Ca 2+ loading by the Ca 2+-ATPase or by the Na +/Ca 2+ exchanger. The results show that in microsomes loaded with Ca 2+ by the Ca 2+-ATPase, Ins(1,4,5)P 3 (5 μM) release 21±2% of the total Ca 2+ accumulated, and that in the microsomes loaded with Ca 2+ by the Na 2+/Ca 2+ exchanger, Ins(1,4,5)P 3 released 28±3% of the total Ca 2+ accumulated. These results suggest that receptors of Ins(1,4,5)P 3 may be co-localized with the Na 2+/Ca 2+ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P 3 receptors in the plasma membrane where the Na 2+/Ca 2+ exchanger is normally present, or both. We also found that Ins(1,4,5)P 3 inhibited the Ca 2+-ATPase by 33.7%, but that it had no significant effect on the Na 2+/Ca 2+ exchanger. 相似文献
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