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1.
The pathways for cytosolic Ca++ increase under A23187 stimulation of H+ secretion were studied in the isolated gastric mucosa of the toad . A23187 produced a more potent stimulation of secretion when added to the mucosal side which did not contain calcium. Measurements of ionophore incorporation by fluorometric methods indicated that A23187 incorporates into oxyntic cells intracellularly. The presence of divalent cations inhibited incorporation. This may be the reason for a more potent action when A23187 was added from the mucosal side. With-drawal of calcium from serosal solution largely inhibited the secretory response to A23187 added to the mucosal side. Reintroduction of calcium into the serosal side in the presence of ionophore elicited H+ secretion. The results are consistent with an uptake of A23187 from the mucosal side into cellular organelles and basolateral membranes. Calcium entry through the serosal side may be responsible for triggering secretion. Although A23187 likely releases calcium from intracellular stores, its rate of release may not be sufficient to bring about a full stimulation of secretion in serosal-Ca++-free conditions. 相似文献
2.
Pancreatic acinar cells: use of Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling 总被引:5,自引:0,他引:5
The Ca++ ionophore A23187 had no effect on the release of amylase by mouse pancreas fragments in the absence of Ca++ but when Ca++ was re-added to the medium amylase release was observed in a pattern which mimicked that produced by normal stimulants. Uptake of 45Ca++ by pancreatic fragments was increased by A23187. Tetracaine and dinitrophenol at concentrations which block cholinergic stimulated enzyme release blocked ionophore induced release whereas atropine did not. None of the inhibitors studied affected the ionophore induced Ca++ uptake. 相似文献
3.
Andrew Ricci Karen M. Sanders Julia Portmore William G. Van der Kloot 《Life sciences》1975,16(1):177-184
1. The ionophore X-537A increases the rate of catecholamine release from the frog adrenal.2. The ratio of epinephrine/norepinephrine measured during X-537A stimulation was the same as that during spontaneous release.3. Even when Ca++ was removed from the Ringer, X-537A stimulated catecholamine release, but depolarization by elevated extra-cellular K+ was no longer effective.4. X-537A also increases the release of dopamine β-hydroxylase, suggesting that the ionophore acts, at least in part, by stimulating the exocytosis of the chrommaffin granule contents.5. Therefore, it is questionable whether the release of catecholamines by X-537A is owing to its action as a Ca++- ionophore.6. The divalent cation ionophore, A-23187 (50μM), did not affect the rate of catecholamine release. 相似文献
4.
Viablse, purely cholinergic synaptosomes were prepared from the electric organ of Torpedo ocellata and partially purified by differential and sucrose density centrifugation. The synaptosomes contain acetylcholine (ACh), synaptic vesicles, cytoplasmic markers and mitochondria. No adherent postsynaptic membranes were detected. K+ depolarization as well as the ionophore A23187 mediate Ca2+ permeation into the synaptosomes and the consequent release of ACh. Mg2+ does not evoke ACh release whereas Sr2+ and Ba2+ can replace Ca2+ in evoking K+ depolarization induced ACh secretion. In accordance with the calcium hypothesis of stimulus–secretion coupling, both K+ depolarization and the ionophore A23187 seem to mediate the release of the same population of ACh molecules. The mode of action of the ionophore X537A differs from that of A23187. X537A acts independently of Ca2+ and induces the release of a larger fraction of the ACh contained in the fractionated nerve terminals. These results demonstrate that the Torpedo synaptosomes contain the neurosecretion apparatus in a functional active state. This preparation extends the utility of synaptosomes for structural and functional biochemical studies of neurotransmission, as it uniquely contains only one neurosecretion system (cholinergic). 相似文献
5.
Ca++ fluxes in resealed synaptic plasma membrane vesicles 总被引:5,自引:0,他引:5
The effect of the monovalent cations Na+, Li+, and K+ on Ca++ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K2/succinate and 45CaCl2, rapidly released approximately 60% of the intravesicular Ca++ when exposed to NaCl or to the Ca++ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca++. The EC50 for Na+ activation of Ca++ efflux from the vesicles was approximately 6.6mM. exposure of the Ca++-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca++ from the vesicles, but the Na+-induced efflux could still be detected above this K+ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid 45Ca uptake with an estimated EC50 for Ca++ of 7–10 μM. This Ca++ uptake was blocked by dissipation of the Na+ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na+Ca++ exchange process and of a K+ - sensitive Ca++ flux across the membranes. 相似文献
6.
Summary We have measured the effects of the carboxylic Ca++ ionophore A23187 on muscle tension, resting potential and 3-O-methylglucose efflux. The ionophore produces an increase in tension that is dependent on external Ca++ concentration since (a) the contracture was blocked by removing external Ca++ and (b) its size was increased by raising outside Ca++. Neither resting potential nor resting and insulin-stimulated sugar efflux were modified by the ionophore. These data imply that the action of insulin is not mediated by increasing cytoplasmic [Ca++]. Additional support for this conclusion was obtained by testing the effects of caffeine on sugar efflux. This agent, which releases Ca++ from the reticulum, did not increase resting sugar efflux and inhibited the insulin-stimulated efflux. Incubation in solutions containing butyrated derivatives of cyclic AMP or cyclic GMP plus theophylline did not modify the effects of insulin on sugar efflux. Evidence suggesting that our experimental conditions increased the cytoplasmic cyclic AMP activity was obtained. 相似文献
7.
Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced 45Ca++ efflux and 2) a dependence on the extracellular Na+ concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on 45Ca++ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release 45Ca++. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na+ was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the 45Ca++ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na+. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na+ followed by release of intracellularly stored calcium. 相似文献
8.
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
9.
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
10.
John A. Williams Jørgen Hedemark Poulsen Mark Lee 《The Journal of membrane biology》1977,33(1):185-194
Summary Compounds with membrane stabilizing activity were studied as to their ability to affect pancreatic amylase release and the steps in the stimulus-secretion coupling process. Chlorpromazine, propranolol, and thymol were all found to inhibit bethanechol-stimulated amylase release and at slightly higher concentrations to induce release regardless of the presence of the secretagogue. This biphasic effect was similar to that found previously for the local anesthetic tetracaine. Release by high concentrations of propranolol and tetracaine was accompanied by ultrastructural evidence of cell damage. Membrane stabilizers at concentrations which inhibited amylase release were shown to block bethanechol-induced depolarization and stimulation of45Ca++ efflux although the drugs alone partially depolarized pancreatic cells. Release of amylase induced by Ca++ introduced by the ionophore A23187 was also abolished. These findings indicate that membrane stabilizers independently inhibit the steps leading to a rise in intracellular Ca++ and the subsequent Ca++-activated amylase release. 相似文献
11.
Summary Cultured epithelial cells (Intestine 407) derived from fetal human small intestine exhibited spontaneous oscillations of membrane potential between the resting level of about –20 mV and the activated level of about –75mV. The cells were hyperpolarized to the latter level in response to mechanical or electrical stimuli. The hyperpolarizing responses were also elicited by the application of intestinal secretagogues: acetylcholine, histamine, serotonin and vasoactive intestinal polypeptide (VIP). The spontaneous oscillation of membrane potential became prominent and long-lasting in the presence of acetylcholine, histamine, serotonin or VIP. These secretagogue-induced responses were mediated by individual independent receptors on the cell membrane. Muscarinic receptors were responsible for the acetylcholine response, and H1-receptors for the histamine response. The cells also responded with a slow hyperpolarization to calcium ionophore A23187, which is known to induce intestinal secretion. The spontaneously occurring hyperpolarizing responses and those induced by stimuli were both due to an increase in the K+ conductance of the cell membrane. Since acetylcholine, histamine, serotonin and A23187 are known to promote mobilization of cellular Ca2+ ions in intestinal secretory cells, it is hypothesized that these electrical activities of the cell are closely related to the receptor stimulation which leads to the Ca2+-mediated intestinal secretion. 相似文献
12.
The possibility of interactions between calcium and cyclic AMP (cAMP) in the mechanism of stimulation of H+ transport by A23187 was studied in the isolated gastric mucosa of the toad Bufo marinus. A23187 stimulated H+ secretion and histamine release. The amount of histamine released by A23187 did not explain the degree of stimulation. Metiamide partially inhibited the response to A23187. Ca++ ionophore produced an overstimulation of secretion after H+ transport had been induced by supramaximal effective concentrations of histamine (10-4 M). In the presence of metiamide, IMX potentiated the response to A23187. Also, in the same condition (metiamide treated) the effects of db-cAMP and A23187 were additive. The results are consistent with an interaction between Ca++ and ionophore-released histamine at the oxyntic cell in the stimulation by A23187. The stimulatory response may be the result of a potentiation between calcium and cAMP at the intracellular level. 相似文献
13.
Yvette Morot-Gaudry 《Neurochemistry international》1984,6(4):553-561
The effects of trimethyl-tin (anion-hydroxyde ionophore, inhibiting oxydative phosphorylation and H+-ATPase) probenecid (inhibitor of anion transport in neural cells) and phenylglyoxal (arginine-specific reagent, inhibiting chloride exchanges in erythrocytes) were examined in Torpedo synaptosomes prepared from electric organ. All drugs significantly reduced the stimulated release of acetylcholine triggered by depolarization of nerve endings with high-K+ and/or gramicidin D. In contrast, trimethyl-tin, probenecid and phenylglyoxal did not affect the ionophore A23187-induced release of acetylcholine from the synaptosomes. The inhibitory potency of the compound trimethyl-tin was found to be similar to that of probenecid and phenylglyoxal on depolarization-induced acetylcholine release. This leads us to suggest that a relationship exists between modification of anion distribution during depolarization and acetylcholine release process. Moreover, since the release of ACh by calcium-ionophore A23187 was unaffected by trimethyl-tin, probenecid or phenylglyoxal, such compounds may also have an action on voltage-dependent Ca2+ flux across presynaptic membrane. 相似文献
14.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1997,1356(2):198-206
Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca2+]i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca2+]i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [3H]glucosamine. Caffeine by itself failed to increase [Ca2+]i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca2+]i rise, resulting in inhibiting activation of Ca2+-dependent K+ current (IK.Ca) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca2+]i rise and activation of IK.Ca induced by A23187 or inositol trisphosphate (IP3). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca2+]i rise in human gastric epithelial cells, probably through the blockade of receptor-IP3 signaling pathway, which may affect the mucin secretion. © 1997 Elsevier Science B.V. All rights reserved. 相似文献
15.
The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells 总被引:5,自引:0,他引:5
Robert D. Burgoyne 《Bioscience reports》1984,4(7):605-611
The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca2+]i, in bovine adrenal chromaffin cells was determined. At 10–4M carbamylcholine maximal release occurred with an accompanying increase i n [Ca2+]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca2+]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca2+]i. 相似文献
16.
David E. McClain Mildred A. Donlon Stephen Chock George N. Catravas 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,763(4):419-425
To investigate the role of the Ca2+-binding protein calmodulin on histamine release in the rat peritoneal mast cell, we exposed cells to exogenous calmodulin in the presence of a variety of histamine secretagogues. Histamine release stimulated by compound
, polymyxin B and ionophore A23187 was inhibited while concanavalin A-stimulated release was not affected. Calmodulin in the presence of the secretagogues did not affect cell viability and calmodulin alone had no effect on histamine release. No direct interaction between calmodulin and the secretagogues was observed. Exogenous calmodulin does not appear to be incorporated into the cell. The inhibition of histamine release by calmodulin can be explained as a labile interaction between the protein and the cell that requires externally-bound Ca2+. These experiments demonstrate the use of exogenous calmodulin as a probe in the study of the mechanism of histamine release. 相似文献
17.
Summary We have shown that a Ca++-ionophore activity is present in the (Ca+++Mg++)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA
71:3522). Methylmercuric chloride inhibited the (Ca+++Mg++)-ATPase and Ca++ transport, but had no effect on the activity of the Ca++ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca++ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca++ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca++ ionophore and that mercuric chloride inhibited the Ca++ ionophore activity by competition with Ca++ for the ionophoric site. Blockage of Ca++ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca++ ionophoric activity. 相似文献
18.
Dr. John A. Williams 《Cell and tissue research》1978,186(2):287-295
Summary The effects of the Ca2+ ionophore A 2317 on pancreatic amylase and lactate dehydrogenase (LDH) release, cellular electrolyte balance and ultra-structure were studied with the use of incubated pancreatic fragments. A 23187 (0.3 M) in the presence of Ca2+, increased amylase release but at higher concentrations (1–10 M) also increased LDH release and increased uptake of 14C-sucrose with concomitant loss of tissue K+ and gain in Na +. The ultrastructure of the majority of acini appeared normal and showed depletion of zymogen granules. Microtubules and microfilaments which have been implicated in the release process were normal or increased in number. In the absence of Ca+ the ionophore had no effect on secretion, cellular integrity or ultrastructure. It is concluded that A 23187 in the presence of Ca2+ increases amylase release by a mechanism comparable to the terminal steps in stimulussecretion coupling induced by physiological secretagogues. This provides further evidence that amylase release is mediated by a rise in cell Ca2+ although the mechanisms of the ionophore- and physiological secretagogue-induced rise in Ca+ are probably different. High concentrations of ionophore (> 1 M) also induce Ca2+ dependent damage in a fraction of the cells.Supported by grants from the NIH (GM 19998) and the Cystic Fibrosis FoundationI am indebted to Drs. Douglas Chandler and John Heuser for discussion and advice and to M. Lee and E. Roach for technical assistance 相似文献
19.
Summary Light-induced degradation of photoreceptor membrane in the crayfish was studied by quantitative light and electron microscopy. The production of lysosomal organelles within the photoreceptor cells was enhanced by presenting the light stimulus intermittently (i.e., flicker) or by doubling its intensity. The enhancement was seen primarily as an increase in the number and size of multivesicular bodies. As these stimulus conditions are likely to facilitate intracellular Ca++ fluxes, the results are compatibl with recent speculations that Ca++ ions may regulate membrane degradation. To test the possibility that Ca++ acts as a signal coupling receptor stimulation with membrane loss, retinas were incubated in the dark with the ionophore A23187 in the presence or absence of external Ca++. The results demonstrate that A23187 produces a Ca++-dependent increase in lysosomal organelles, predominantly multivesicular bodies. These data are consistent with a role for intracellular Ca++ in the degradative process; however, the exact locus of the effect is unclear.Supported by a grant (BNS 8004587) from the National Science Foundation to G.S.H. The authors gratefully acknowledge the helpful discussions and expert technical assistance of Thomas R. Tokarski 相似文献
20.
Summary Human erythrocyte Ca2+-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca2+ inside the vesicles, both in the absence and in the presence of the Ca2+-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca2+ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca2+/2H+ ionophore A23187 (9 to 10-fold). A Ca2+ ionophore unable to translocate H+, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca2+ ionophore, the electroneutral K+(Na+)/H+ ionophoretic exchanger, nigericin, or the electroneutral Na+(K+)/H+ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca2+-ATPase not only translocates Ca2+ but also H+ in the opposite direction. 相似文献