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1.
M Inagaki T Tanaka Y Sasaki H Hidaka 《Biochemical and biophysical research communications》1985,130(1):200-206
We found that ionophore A23187 interacted reversibly with calmodulin (CaM), in a calcium-dependent fashion. It was found that A23187 interacts selectively with CaM, among calcium binding proteins (such as troponin C and S-100 protein) and other proteins. However, apparently differing from W-7, A23187 did not suppress CaM-dependent enzyme activity such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase. Our observations suggest that there are novel calcium-dependent regions of CaM which can be monitored using ionophore A23187 and may not be related to enzyme activation. 相似文献
2.
Rat eggs treated with the calcium ionophore A23187 and subjected to long-term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time-lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore-treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD-phallacidin, the distribution of F-actin in ionophore-activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5-h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD-phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore-induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg. 相似文献
3.
The role of Ca2+ on 32Pi incorporation into polyphosphoinositides (PPI) of rat cortical synaptosomes was studied. Stimulation of muscarinic receptor by carbachol (1 mM) resulted in a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphophaphate (TPI) and phosphatidylinositol-4-phosphate (DPI), and an increase in 32Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA), whereas no significant effect on other membrane phospholipids was found. This response could be blocked by atropine (1 microM). The stimulatory effect of carbachol required Ca2+ in the medium; the presence of 0.5 mM EGTA blocked the effect of carbachol on PPI turnover completely. Calcium ionophore A23187, at 1 microM, had a similar effect on PPI turnover by carbachol (1 mM). At higher concentrations (10-100 microM) of A23187, the PPI turnover rate was much enhanced. Depolarization of the membrane by high potassium (60 mM) in the presence of calcium resulted in an enhanced PPI turnover, which was similar to the results of the carbachol (1 mM) effect but to a lesser extent. Calcium antagonists, diltiazem and trifluoperazine, at 10 microM could block the carbachol effect on 32Pi incorporation into PPI in this preparation. Our results suggest that the enhancement of PPI turnover in rat cortical synaptosomes by carbachol, calcium ionophore or high potassium requires Ca2+, and it can be blocked by compounds which interfere with the availability of this ion, such as EGTA or calcium antagonists. 相似文献
4.
The effect of calcium ionophore A23187 on the release of nonmetabolizable glutamate analogues [3H]D-aspartate and the exocytosis registered by fluorescent dyes in synaptosomes was investigated. It was shown that A23187 is able to induce neurotransmitter release both in calcium-containing and calcium-free medium, the effect in the latter case being more pronounced. Calcium ionophore is able to induce exocytosis registered by acridine orange and FM 2-10. The influence of A23187 on the fluorescence of acridine orange was mainly calcium-independent, whereas the change in the fluorescence of FM 2-10 was calcium-dependent. It was suggested that the calcium-independent increase in acridine orange fluorescence is related to the dissipation of pH gradient in synaptic vesicles. Probably, the calcium-independent release of D-aspartate is also associated with the dissipation of pH gradient and subsequent leakage of neurotransmitters. 相似文献
5.
The effect of the calcium ionophore A23128 on calcium fluxes from Y-1 adrenal cortical cells was investigated. Conditions were chosen which are known to result in an inhibition of steroidogenesis (6 . 10(-6) M ionophore and 3 . 10(-4) M extracellular calcium). Calcium efflux from Y-1 cells exhibited two distinct phases. A fast phase which was insensitive to the mitochondrial poison sodium azide and a slow, azide-sensitive phase. The ionophore brought about a rapid increase in the rate of calcium efflux and an 84% reduction in the size of the calcium pool which was associated with the slow efflux phase as well as a reduction in its rate constant. A decrease in the size of the rapidly exchanging calcium pool was also detected. Ethanol, the solvent which was used for the ionophore, slightly increased the rate constant of the rapidly exchanging pool. Conditions which resulted in diminished steroidogenic capacity also brought about a reduction in the size of an energy dependent, intracellular pool. The data is interpreted as being consistent with a hypothesis that the ionophore-induced inhibition of steroidogenesis may be causatively related to the loss of intracellular calcium or to the mechanism which brings about the loss. 相似文献
6.
Effects of respiratory inhibitors and ionophore A23187 on frog gustatory nerve responses 总被引:1,自引:0,他引:1
S Nagahama K Kurihara 《Comparative biochemistry and physiology. A, Comparative physiology》1984,79(3):431-435
An increase in Ca concentration in a frog taste cell by application of respiratory inhibitors and ionophore A23187 to Ringer solution perfusing the lingual artery led to a large suppression of the taste nerve responses to quinine, ethanol and acids. The responses to CaCl2, L-threonine, D-galactose and distilled water were unchanged or increased. 相似文献
7.
The effect of an increase in intracellular Ca2+ concentration on tight-junctional permeability in rat liver was studied by using the calcium ionophore A23187. Infusion of 100 microliters of dimethyl sulphoxide containing various amounts of A23187 over 30 min into isolated perfused livers was followed by a pulse of horseradish peroxidase (HRP) under single-pass conditions. The first biliary HRP peak, a measure of junctional permeability, was increased 4-fold with 100 micrograms of A23187. There were, however, no significant effects on bile flow or on aspartate aminotransferase leakage as compared with the control at this dosage, and thus the increase in junctional permeability was occurring without evidence of appreciable cholestatic or hepatocellular damage. Higher dosages of A23187, however, caused not only an increase in HRP peak height but also changes in bile flow and increases in aminotransferase leakage, indicating more extensive effects at these higher dosages. A second peak of HRP secretion, occurring 20-25 min after the HRP pulse, was also elevated approx. 3.5-fold; this may indicate that pinocytosis and transcellular movement of HRP are also increased under these conditions. 相似文献
8.
9.
Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria 总被引:5,自引:0,他引:5
D T Wong J R Wilkinson R L Hamill J S Horng 《Archives of biochemistry and biophysics》1973,156(2):578-585
A23187, a new antibiotic with ionophore properties, uncoupled oxidative phosphorylation in mitochondria which oxidized either malate plus glutamate or succinate. Ca2+, but not Mg2+, enhanced the uncoupling effect. Fluorescence of ANS1 was increased by A23187 suggesting the mitochondrial membranes were de-energized. This de-energization was presumably by activation of the energy-dependent uptake of Ca2+. The steady-state measurements of murexide-divalent cation complexes showed that A23187 caused mitochondria to release the accumulated Ca2+ to the medium. This reduced the transmembrane Ca2+ gradient even though normal active Ca2+ uptake could take place. A23187 inhibited activity of ATPase induced by 2,4-dinitrophenol, valinomycin, and Ca2+. The addition of Mg2+ could prevent this inhibition presumably by maintaining the endogenous Mg2+ concentration. The above metabolic events could be explained by the fact that molecules of A23187 function in the mitochondrial inner membrane as mobile carriers for divalent cations. 相似文献
10.
Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells
B K Tsang D F Mattice M Li E K Asem 《Canadian journal of physiology and pharmacology》1988,66(7):960-963
The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production. 相似文献
11.
The administration of the ionophore A23187 to superfused rat adrenal cortical tissue slices resulted in a significant elevation in corticosterone production. Removal of calcium from the superfusion medium prevented this ionophore induced corticosteroidogenesis. Threshold amounts of ionophore potentiated the steroidogenic action of 1 mU but not 10 mU ACTH under conditions. This potentiation by ionophore on ACTH stimulated steroid production was not observed when calcium was omitted from the superfusing medium. Potentiation by the ionophore on dbCAMP or CAMP stimulated steroid formation was not observed for any dose of cyclic nucleotide employed. 相似文献
12.
The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca2+ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca2+ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both 45Ca2+ fluxes, Ca2+ content and amylase release. A23187 increased the initial rates of both 45Ca2+ uptake and washout. In addition, the content of both exchangeable 45Ca2+ and total Ca2+ were reduced. These results indicated, therefore, that A23187 increases Ca2+ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca2+. In the absence of extracellular Ca2+, however, A23187 caused a rapid fall in acinar Ca2+ and subsequent amylase release was abolished. Depletion of intracellular Ca2+ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca2+ which in turn mediates their actions. 相似文献
13.
The tissue/medium distribution of the nonmetabolized glucose analog [14C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in 45Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca2+. However, both sugar and 45Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca2+, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca2+ from internal storage sites into the cytoplasm. Increasing internal Na+ through Na+ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or 45Ca, indicating Na+-Ca2+ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca2+ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca2+ stimulating hexose transport. Experiments with low Mg++ and high K+ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca2+-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca2+ fluxes in these cells. 相似文献
14.
The distribution of (14C)-3-0-methyl-D-glucose and of (45Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca2+ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K+-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca2+ in the regulation of sugar transport in muscle. However, increased influx of Ca2+ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca2+ from intracellular storage and binding sites. 相似文献
15.
The present study was performed to evaluate the effects of calcium ionophore A23187 on adenosine 3',5'-monophosphate (cyclic AMP) and testosterone production in rat interstitial cells. Interstitial cells were incubated in Krebs-Ringer solution with varying amounts of luteinizing hormone, pregnenolone, or A23187. Cyclic AMP and testosterone were measured in the incubation medium after 4 h incubation. A23187 (0.01--10 microgram/ml) caused progressive increases of cyclic AMP formation (from 0.18 +/- 0.02 (S.E.) pmol/10(6) cells for the control of 0.42 +/- 0.02 pmol/10(6) cells, P less than 0.025), while testosterone production remained unaltered. When varying amounts of A23187 were added concomitantly with luteinizing hormone (5 IU/l), A23187 inhibited luteinizing hormone-induced steroidogenesis in a dose-dependent manner, but it had no effect on luteinizing hormone-induced cyclic AMP formation. When pregnenolone (10(-6) M) was added to the cells, testosterone formation increased from 1.50 +/- 0.22 to 8.46 +/- 1.65 ng/10(6) cells. A23187 (1 microgram/ml) had no discernable effect on the conversion of pregnenolone to testosterone. The main effect of increased cytosol calcium on steroidogenesis seems to be at the steps beyond adenylate cyclase-cyclic AMP. These results suggest that calcium is important for the conversion of cholesterol to pregnenolone, while the steps beyond pregnenolone are relatively independent of Ca2+. 相似文献
16.
Pretreatment of cultured human fibroblasts with the calcium ionophore A 23187 resulted in a decrease in low-density lipoprotein internalization. This effect was dose-dependent and did not occur in a medium devoid of calcium. About 2-fold reduction was observed with 10(-5)M A 23187. In contrast, the low-density lipoprotein binding was only slightly affected. The incorporation of [14C]acetate and [14C]oleate into all classes of lipids (sterol, triacylglycerols and phospholipids) was strikingly reduced by ionophore pretreatment. 相似文献
17.
J C Garrison 《The Journal of biological chemistry》1978,253(19):7091-7100
Recent experiments have demonstrated that stimulation of rat hepatocyte alpha-adrenergic receptors alters the activity of enzymes known to be regulated by cycles of phosphorylation and dephosphorylation. These events apparently occur without an increase in the activity of adenosine 3':5'-monophosphate-dependent protein kinase. The present study compared the effects of glucagon and catecholamines on the incorporation of radioactive phosphate into cytosolic proteins obtained from intact rat hepatocytes. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis resolved 27 phosphorylated bands in the molecular weight range 220,000 to 29,000. Treatment of the intact hepatocytes with glucagon or cyclic nucleotides increased the phosphorylation of 12 of these bands. Incubation of unlabeled cytoplasmic proteins with the catalytic subunit of protein kinase and [gamma-32P]ATP leads to the phosphorylation of 11 proteins. The molecular weights of these proteins were very similar to those altered by glucagon treatment of intact cells. Stimulation of the alpha-receptor with norepinephrine, epinephrine, or phenylephrine in the presence of 20 micrometer propranolol caused an increase in the phosphorylation of at least 10 of the same 12 phosphorylated bands stimulated by glucagon. The increase in phosphorylation mediated by alpha-receptors was only 50 to 60% of that observed with glucagon and occurred in the absence of any change in the level of adenosone 3':5'-monophosphate. The effects of alpha-receptor stimulation could be completely antagonized by 20 micrometer ergotamine or 20 micrometer phentolamine. Treatment of the cells with the Ca2+ ionophore A23187 in an attempt to mimic alpha-receptor function increased the phosphorylation of 4 of the phosphoproteins altered by glucagon or catecholamines. The effects of the ionophore depended on the presence of extracellular Ca2+ ion and were similar in magnitude to those of catecholamines. It is concluded that alpha-receptor occupation alters the activity of an adenosin 3':5'-monophosphate-independent protein kinase or phosphatase with a specificity similar to those affected by cyclic nucleotides. 相似文献
18.
A postulated mechanism for the coordinate effects of ionophore A23187 on calcium uptake and cell viability in rat thymocytes 总被引:1,自引:0,他引:1
Human beta-thromboglobulin, low affinity platelet factor 4 and platelet basic protein have been purified to homogeneity from the material released by thrombin-stimulated platelets. Purification steps included isoelectric focusing and heparin-agarose chromatography. Antibodies against each of these proteins have been raised in rabbits. Antigenic identity of the proteins has been demonstrated in radioimmunoassay using 125I-labelled platelet basic protein or 125I-labelled low affinity platelet factor 4 and a variety of antibodies. The molecular weight of platelet basic protein estimated by gel filtration in 6 M guanidine hydrochloride using Sepharose 6B corresponded to approx. 10 000 daltons, slightly higher than that of beta-thromboglobulin (8851 daltons) and low affinity platelet factor 4 (9278 daltons). These findings raise the possibility that the formation of low affinity platelet factor 4 beta-thromboglobulin may be a consequence of the action of proteolytic enzymes on platelet basic protein. 相似文献
19.
P W Reed 《The Journal of biological chemistry》1976,251(11):3489-3494
A23187 transports calcium rapidly into rat erythrocytes, apparently by an electroneutral exchange for intracellular magnesium and protons. When red cells are incubated in the absence of any added divalent cations, A23187 transports internal magnesium out of the cells, in exchange for extracellular protons. Magnesium uptake into erythrocytes is produced by A23187, providing the extracellular concentration of this cation exceeds intracellular levels, and the ionophore also transports strontium, but not barium, into red cells. A23187 produces a rapid and extensive loss of intracellular potassium from erythrocytes during uptake of calcium or strontium, but not magnesium. When red cells are incubated in the absence of any exogenous divalent cations, A23187 still produces a potassium efflux and this is inhibited completely by small amounts of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and restored by the addition of calcium in excess of the chelator. Although EDTA enhances the extent of magnesium release from erythrocytes incubated with A23187, it prevents the potassium efflux. Dipyridamole and 4-acetamid-4'-isothiocyano-stilbene-2,5'-disulfonic acid, which decrease chloride premeability of erythrocytes, inhibit the A23187-induced potassium loss from red cells. Rutamycin, peliomycin, venturicidin, and A23668B also inhibit potassium efflux from intact cells incubated with A23187, but this effect is not correlated with their abilities to inhibit various ATPases in red cell membrane preparations. It is concluded that A23187 does not transport potassium directly across the erythrocyte plasma membrane, but permits small amounts of endogenous calcium to interact with some membrane component to enhance potassium permeability of the cell. 相似文献
20.
The effect of ionophore A23187 on calcium ion fluxes and alpha-adrenergic-agonist action in perfused rat liver. 总被引:4,自引:4,他引:0
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The effect of ionophore A23187 on cellular Ca2+ fluxes, glycogenolysis and respiration was examined in perfused liver. At low extracellular Ca2+ concentrations (less than 4 microM), A23187 induced the mobilization of intracellular Ca2+ and stimulated the rate of glycogenolysis and respiration. As the extracellular Ca2+ concentration was elevated, biphasic cellular Ca2+ fluxes were observed, with Ca2+ uptake preceding Ca2+ efflux. Under these conditions, both the glycogenolytic response and the respiratory response also became biphasic, allowing the differentiation between the effects of extracellular and intracellular Ca2+. Under all conditions examined the rate of Ca2+ efflux induced by A23187 was much slower than the rate of phenylephrine-induced Ca2+ efflux, although the net amounts of Ca2+ effluxed were similar for both agents. The effect of A23187 on phenylephrine-induced Ca2+ fluxes, glycogenolysis and respiration is dependent on the extracellular Ca2+ concentration. At concentrations of less than 50 microM-Ca2+, A23187 only partially inhibited alpha-agonist action, whereas at 1.3 mM-Ca2+ almost total inhibition was observed. The action of A23187 at the cellular level is complex, dependent on the experimental conditions used, and shows both differences from and similarities to the hepatic action of alpha-adrenergic agonists. 相似文献