Generation of hydrogen peroxide by cerebral-cortex synaptosomes. Stimulation by ionomycin and plasma-membrane depolarization

Guinea-pig cerebral cortex synaptosomes steadily release H2O2 into the suspending medium, at the rate of 20-30 pmol min-1 mg protein-1. A transient increase of the H2O2 release is induced by the addition of 1 mM Ca2+, which declines within 60-90 s to a rate identical or slightly higher than that before Ca2+. The extra H2O2 following Ca2+ addition varies between 40-100 pmol/mg protein and is insensitive to verapamil. The H2O2 release increases strongly (up to 250 pmol min-1 mg-1) upon depletion of the synaptosomal glutathione by treatment with 1-chloro-2,4-dinitrobenzene, a substrate for glutathione transferase. This treatment however has no effect on the Ca2+-induced H2O2 transient. In these treated synaptosomes a further increase of the output of H2O2 is rapidly induced upon addition of the Ca2+ ionophore ionomycin. This increase (about 100 pmol min-1 mg-1) lasts several minutes and requires the presence of Ca2+. A similar, though less pronounced increased H2O2 release is obtained (also in the absence of Ca2+) upon depolarization of the synaptosomal plasma membrane with KCl or with veratridine.     

Degranulation in human neutrophils primes the cells for subsequent responsiveness to the chemoattractant N-formylmethionylleucylphenylalanine but does not increase the sensitivity of the NADPH-oxidase to an intracellular calcium rise

Both the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) and the calcium-specific ionophore ionomycin can activate the NADPH-oxidase in human neutrophils. However, since ionomycin and FMLP activity differ in their requirement for azide, a potent inhibitor of the hydrogen peroxide consuming enzymes catalase and myeloperoxidase, we propose that the two stimuli can activate different pools of the oxidase. Degranulation, induced in vitro by sn-1,2-dedecaoylglycerol or in vivo by an exudation process, resulted in a priming of the cells using FMLP as stimulating agent as well as in a reduced capacity to generate H2O2 in response to ionomycin. The sensitivity of the plasma membrane-bound NADPH-oxidase to an intracellular [Ca2+] rise, induced by the ionophore was, however, not changed by the degranulation. From these results we propose that FMLP activates the plasma membrane-bound oxidase, whereas the ionophore is capable of activating a granule-bound pool of the oxidase.     

Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes

The exocytotic release of L-glutamate from guinea-pig cerebral cortical synaptosomes can be extensively inhibited by preincubation with botulinum neurotoxin type A at 37 degrees C for 1-2 h. The toxin has no effect on synaptosomal respiratory control, respiratory capacity, ATP synthesis, plasma-membrane 86Rb+ permeability or plasma-membrane potential, does not inhibit the entry of 45Ca2+ into the synaptosome upon depolarization and does not alter the ability of intrasynaptosomal mitochondria to sequester Ca2+. The blockade of Ca2+-dependent glutamate release may be totally reversed by the Ca2+/2 H+-exchange ionophore ionomycin, but not by increasing extracellular Ca2+ concentration. It is suggested (a) that exocytosis is triggered by the penetration of Ca2+ into an intracellular hydrophobic milieu; (b) that this stage is blocked by the toxin and (c) that ionomycin is able to bypass this block and deliver Ca2+ to the exocytotic apparatus.     

Regulation of calcium-dependent [3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton.

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.     

Inhibition by iodide of iodide binding to proteins: the "Wolff-Chaikoff" effect is caused by inhibition of H2O2 generation

H2O2 generation is limiting the oxidation and binding to proteins of iodide. In dog thyroid slices thyrotropin and carbamylcholine greatly enhance protein iodination and H2O2 generation. The action of thyrotropin is mimicked by dibutyryl cyclic AMP and forskolin which suggests that it is mediated by cyclic AMP. The action of carbamylcholine was mimicked by ionomycin and by phorbol myristate ester. This suggests that the effect of carbamylcholine is mediated by the two intracellular signals generated by the Ca++ phosphatidylinositol cascade: Ca++ and diacylglycerol. The Wolff-Chaikoff effect is the inhibition by iodide of its own organification. In dog thyroid slices, iodide greatly inhibited H2O2 generation stimulated by thyrotropin and by carbamylcholine. Iodide decreased the production of intracellular signals induced by TSH and carbamylcholine but it also inhibited the action of probes of these intracellular signals (dibutyryl cAMP, forskolin, ionomycin, phorbol-myristate ester) on the H2O2 generating system itself. These effects were suppressed by methimazole an inhibitor of iodide oxidation.     

Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells.

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca2+]i) is influenced by hydrogen peroxide (H2O2) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H2O2 resulted in an increase in [Ca2+]i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H2O2 induced an oscillatory pattern, whereas 1 mmol H2O2/L caused a slow and sustained increase in [Ca2+]i. H2O2 abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H2O2/L, but depletion of both stores abolished it. Additionally, lower H2O2 concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 micromol H2O2/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H2O2 reduced amylase release stimulated by CCK-8. Finally, either the H2O2-induced calcium mobilization or the inhibitory effect of H2O2 on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H2O2 at micromolar concentrations induces calcium release from agonist-sensitive stores, and at millimolar concentrations H2O2 can also evoke calcium release from the mitochondria. The action of H2O2 is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.     

Further observations on the effect of calcium ionophores on ascites tumor cells

The effect of the Ca2+ ionophore ionomycin on neoplastic thymocytes in comparison to its effect on normal thymus cells was studied. Ionomycin increases intracellular Ca2+ in normal lymphocytes but fails to increase Ca2+ in neoplastic thymocytes. In these cells the ionophore causes a transient increase in cytosolic free Ca2+. The lack of effect of ionomycin reproduces that of A23187, but it does not depend on reduced availability of intracellular Mg2+ to exchange with Ca2+; it appears to depend on the strong activity of the plasma membrane Ca2+-extruding pump that counteracts ionomycin permeabilization and that can be partly inhibited by the calmodulin inhibitor R24571 (calmidazolium). Neoplastic thymocytes show a high content of magnesium, the intracellular binding of which is efficiently regulated by endogenous ATP. The data show also an interesting correlation between the regulation of energy metabolism (aerobic glycolysis) and cation homeostasis in the neoplastic cells studied.     

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Priming of neutrophils and macrophages for enhanced release of superoxide anion by the calcium ionophore ionomycin. Implications for regulation of the respiratory burst

Phagocytic cells can be primed for enhanced stimulated release of superoxide anion (O2-) by exposure to a variety of biologic agents, including gamma-interferon and lipopolysaccharide. We examined the role of calcium ion in this priming, using the calcium ionophore ionomycin. Preincubation with ionomycin, 1 to 10 nM, primed human neutrophils to release up to 7-fold more O2- during stimulation with 1 microM formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). With 160 nM phorbol myristate acetate as stimulus, ionomycin caused a doubling of O2- production in mouse peritoneal macrophages. Incubation of phagocytes with ionomycin at priming concentrations did not directly stimulate O2- release. Priming of neutrophils occurred in 1-2 min and was associated with a marked reduction in the lag time for O2- release after f-Met-Leu-Phe stimulation and with an increase in the rate of O2- production. Kinetic analysis of NADPH-dependent O2(-)-producing activity in sonicates of resting human neutrophils incubated with sodium dodecyl sulfate suggested that modification of the enzyme responsible for the respiratory burst was not responsible for priming. Priming of neutrophils with ionomycin had no apparent effect on either the activity or subcellular distribution of protein kinase C. The effect of ionomycin on the cytosolic free calcium concentration ([Ca2+]c) was assessed in neutrophils using the calcium-sensitive fluorescent dye fura-2. Ionomycin at priming concentrations caused an approximate doubling of the base-line [Ca2+]c. When neutrophils were exposed to various concentrations of ionomycin, a parallel rise in [Ca2+]c and priming was observed. A rise in [Ca2+]c of approximately 0.8 microM caused half-maximal priming. These results suggest that an increase in [Ca2+]c is not sufficient to initiate release of O2-, but they support the concept that Ca2+ can serve as a second messenger in this event.     

Calcium-dependent intracellular acidification dominates the pH response to mitogen in human T cells

The effects of a phorol ester and a mitogenic lectin on the intracellular pH (pHi) of human T lymphocytes was investigated. In contrast to the cytoplasmic alkalinization induced by 12-0-tetradecanoylphorbol-13-acetate, an acidification was recorded in cells treated with phytohemagglutinin. This decrease in pHi was magnified in Na+-free medium or in the presence of amiloride analogues, suggesting that activation of Na+/H+ exchange partially counteracts the phytohemagglutinin-induced acidification. The decrease in pHi was dependent on a sustained increase in cytosolic free Ca2+ and could be mimicked by addition of the divalent cation ionophore, ionomycin. The elevation of cytosolic free Ca2+ leads to metabolic H+ (equivalent) generation with consequent cytoplasmic acidification, which in human T cells predominates over the concurrent activation of the Na+/H+ antiport. These findings argue against the notion that activation of Na+/H+ exchange is a signal for the initiation of proliferation.     

Antioxidant-induced release of calcium in biological membranes

The effect of synthetic (BHT, 2,2,5,7,8-pentamethyl-6-hydroxychroman) and natural (alpha-tocopherol) antioxidants on Ca++-transporting systems was compared in platelets, brain synaptosomes, and skeletal muscle sarcoplasmic reticulum. It was shown that synthetic antioxidants, in contrast to alpha-tocopherol, induced Ca++-release manifested in platelet aggregation, stimulation of 5-hydroxytryptamine release by synaptosomes, synaptosome depolarization and inhibition of Ca++-transport and Ca++-ATPase activity in the sarcoplasmic reticulum. The disturbances of Ca++-homeostasis induced by synthetic antioxidants are considered as molecular mechanisms of complications encountered upon their application.     

Glutamate release by an Na+ load and oxidative stress in nerve terminals: relevance to ischemia/reperfusion

Previously we have reported that oxidative stress induced by hydrogen peroxide exacerbates the effect of an Na+ load in isolated nerve terminals, with a consequence of an ATP depletion, [Ca2+]i and [Na+]i deregulation, and collapse of mitochondrial membrane potential. In the present study, the release of glutamate in response to a combined effect of an [Na+] load and oxidative stress was measured in isolated nerve terminals over an incubation for 15 min. Exposure to hydrogen peroxide (100 micro m) had no effect on the release of glutamate, but significantly enhanced the Ca2+-independent glutamate release induced by a small [Na+] load achieved with 10 micro m veratridine. The effect of a larger Na+ load induced by 40 micro m veratridine was not further increased by hydrogen peroxide; in contrast the external Ca2+-dependent glutamate release was completely eliminated by the oxidant under this condition. The effects of oxidative stress superimposed on a Na+ load are consistent with at least two factors: (i) a relatively modest Na+ load induced by veratridine is augmented by H2O2 giving rise to an increased Ca2+-independent release of glutamate (ii) oxidative stress in combination with a larger Na+ load causes severe ATP depletion limiting the Ca2+-dependent vesicular glutamate release. Given the concurrent presence of an Na+ load and oxidative stress in ischemia/reperfusion these results indicate that the extent of the Na+ load developing during the ischemic period could determine the release of glutamate induced by an oxidative stress during reperfusion.     

Intracellular production of reactive oxygen species in human neutrophils following activation by the soluble stimuli FMLP, dioctanoylglycerol and ionomycin.

The stimuli, sn-1, 2-dioctanoylglycerol; (DG8) the calcium specific ionophore, ionomycin, and the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) can interact with normal human neutrophils and activate their superoxide/hydrogen peroxide generating NADPH-oxidase. In response to the peptide as well as DG8, the neutrophils produced both superoxide (O2-) and hydrogen peroxide (H2O2). Since interaction between the cells and ionomycin was not associated with any notable superoxide production and hydrogen peroxide was induced only in the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, we conclude that this stimulus can generate oxygen metabolites intracellularly. Since the DG8-induced production of hydrogen peroxide was increased in the presence of azide, whereas the FMLP-induced response was largely unaffected, we concluded that the three stimuli differ in their capacity to generate oxygen metabolites intracellularly. The use of sn-1,2-didecanoylglycerol (DG10) as stimulating agent did not result in any detectable activation of the NADPH-oxidase. However, preincubation caused an increased (primed) response during stimulation with the chemotactic peptide FMLP. The response of primed neutrophils to FMLP proceeds with a time-course different from that seen in normal cells. From the results presented on FMLP-induced activity in the presence of azide, we conclude that FMLP causes normal cells to produce oxygen radicals which are released from the cells. However, the primed cells are also capable of generating oxygen metabolites that are retained inside the cells. In fact, measurement of the intracellularly generated metabolites discloses this to be the predominant part of the response.     

The control of mediator release from RBL-2H3 cells: roles for Ca2+, Na+, and protein kinase C1

Antigen-stimulated rat basophilic leukemia (RBL-2H3) cells release serotonin and other inflammatory mediators by a process that requires Ca2+ influx and increased cytoplasmic Ca2+ levels, and is mimicked by Ca2+ ionophores. We report here that the Ca2+ response to antigen and to ionomycin has two components, a Ca2+ spike and a Ca2+ plateau. In nominally Ca2+-free medium, both components of the Ca2+ response are inhibited and secretion does not occur. In Na+-free medium, the initial Ca2+ spike induced by antigen or ionomycin occurs, but the plateau is again absent and secretion is inhibited by 30 to 50%. Secretion is also reduced by 10(-4) M amiloride, an inhibitor of Na+ transport pathways, and by 10(-5) M concentrations of two amiloride analogs with greater activity than amiloride, respectively, against Na+ channels and Na+/Ca2+ exchange. Phorbol esters, which stimulate protein kinase C, enhance the Ca2+ plateau and secretion caused by suboptimal amounts of both antigen and ionomycin; this enhancement depends on extracellular Na+. The Na+ ionophore, monensin, mimics the Ca2+ plateau. From these data, we infer that the Ca2+ spike and plateau reflect separate responses of RBL-2H3 cells to antigen or ionomycin. We propose that the Ca2+ plateau results at least in part from the activation of a Na+-dependent Ca2+ influx pathway. One possible mechanism is that antigen binding stimulates a protein kinase C-regulated Na+ transport system. The resulting influx of Na+ may activate a Na+/Ca2+ antiporter that supports the Ca2+ plateau and mediator release.     

Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of Ca2+ in regulating the catabolism of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rabbit platelets.

In the present study we have investigated the effect of changes in the concentration of cytosolic free Ca2+ ([Ca2+]i) on the deacetylation-reacylation of PAF-acether (alkylacetylglycerophosphocholine, alkylacetyl-GPC) by rabbit platelets. Washed platelets were incubated with alkyl[3H]acetyl-GPC ([3H]acetyl-PAF) or [3H]alkylacetyl-GPC ([3H]alkyl-PAF) and [Ca2+]i was subsequently elevated by the addition of the ionophore A23187 or thrombin. The catabolism of PAF-acether was studied by measuring the release of [3H]acetate or the formation of [3H]alkylacyl-GPC. The ionophore inhibited the release of [3H]acetate and the formation of [3H]alkylacyl-GPC with no accumulation of lyso-[3H]PAF, indicating that the deacetylation of PAF-acether was blocked. The effect of ionophore on the deacetylation of PAF-acether was parallel with the increase of [Ca2+]i and could be reversed by the addition of EGTA. In contrast with the prolonged inhibition evoked by ionophore, thrombin, which induced a transient elevation of [Ca2+]i, merely delayed the deacetylation of PAF-acether. Since intact platelets failed to convert exogenous lyso-PAF, the effect of Ca2+ on its acylation was investigated by using platelet homogenates. These experiments showed that the acylation of lyso-PAF was inhibited by the exogenously added Ca2+, with a maximum effect at 1 mM. When the formation of endogenous lyso-PAF from the labelled pool of alkylacyl-GPC was examined, a prolonged increase in the concentration of lyso-PAF with a parallel and equally prolonged decrease in the cellular level of alkylacyl-GPC were observed after the addition of ionophore to intact platelets. The addition of EGTA reversed the effect of ionophore, thus permitting reacylation of lyso-PAF. In contrast, only a transient change in the level of lyso-PAF and alkylacyl-GPC was evoked by the addition of thrombin. Therefore we conclude that the inhibitory effect of Ca2+ on the deacetylation-reacylation of PAF-acether may have an important role in the regulation of its biosynthesis.     

Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils

The intracellularly trapped fluorescent calcium indicator, quin 2, was used not only to monitor changes in cytosolic-free calcium, [Ca2+]i, but also to assess the role of [Ca2+]i in neutrophil function. To increase cytosolic calcium buffering, human neutrophils were loaded with various quin 2 concentrations, and [Ca2+]i transients, granule content release as well as superoxide [O2-] production were measured in response to the chemotactic peptide formyl-methionyl-leucyl- phenylalanine (fMLP) and the calcium ionophore ionomycin. Receptor- mediated cell activation induced by fMLP caused a rapid rise in [Ca2+]i. The extent of [Ca2+]i rise and granule release were inversely correlated with the intracellular concentration of quin 2, [quin 2]i. These effects of [quin 2]i were more pronounced in the absence of extracellular Ca2+. The initial rate and extent of fMLP-induced O2- production were also inhibited by [quin 2]i. The rates of increase of [Ca2+]i and granule release elicited by ionomycin were also inversely correlated with [quin 2]i in Ca2+-containing medium. As the effects of ionomycin, in contrast to those of fMLP, are sustained, the final increase in [Ca2+]i and granule release were not affected by [quin 2]i. A further reduction of fMLP effects was seen when intracellular calcium stores were depleted by incubating the cells in Ca2+-free medium with ionomycin. The specificity of quin 2 effects on cellular calcium were confirmed by loading the cells with Anis/AM, a structural analog of quin 2 with low affinity for calcium which did not inhibit granule release. In addition, functional responses to phorbol myristate acetate (PMA), which stimulates neutrophils without raising [Ca2+]i, were not affected by [quin 2]i. The findings indicate that rises in [Ca2+]i control the rate and extent of granule exocytosis and O2-generation in human neutrophils exposed to the chemotactic peptide fMLP.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.