Calcium-dependent mitochondrial formation of species promoting strand scission of genomic DNA in U937 cells exposed to tert-butylhydroperoxide: the role of arachidonic acid

Treatment of U937 cells with a sublethal concentration of tert-butylhydroperoxide generates DNA single strand breakage in U937 cells and this response is increased by caffeine, ATP, pyruvate or antimycin A. As we previously reported (Guidarelli, Clementi, Brambilla and Cantoni, (1997) Biochem. J. 328, 801-806), the enhancing effects of antimycin A are mediated by inhibition of complex III and the ensuing formation of superoxides and hydrogen peroxide in a reaction in which ubisemiquinone serves as an electron donor. Active electron transport was required in pyruvate-supplemented cells since the increased genotoxic response occurred as a consequence of enforced mitochondrial Ca²⁺ accumulation, a process driven by the increased electrochemical gradient. The enhancing effects of caffeine or ATP were also the consequence of mitochondrial Ca²⁺ accumulation but these responses were independent on electron transport. The increased formation of DNA lesions resulting from exposure to tert-butylhydroperoxide associated with the Ca²⁺-mobilizing agents or the respiratory substrate was mediated by arachidonic acid generated by Ca²⁺-dependent activation of phospholipase A₂. Melittin, a potent phospholipase A₂ activator, and reagent arachidonic acid mimicked the effects of caffeine, ATP or pyruvate on the tert-butylhydroperoxide-induced DNA single strand breakage.     

Chemical Hypoxia-Induced Cell Death in Human Glioma Cells: Role of Reactive Oxygen Species,ATP Depletion,Mitochondrial Damage and Ca2+

This study was undertaken to evaluate whether chemical hypoxia-induced cell injury is a result of reactive oxygen species (ROS) generation, ATP depletion, mitochondrial permeability transition, and an increase in intracellular Ca²⁺, in A172 cells, a human glioma cell line. Chemical hypoxia was induced by incubating cells with antimycin A, an inhibitor of mitochondrial electron transport, in a glucose-free medium. Exposure of cells to chemical hypoxia resulted in cell death, ROS generation, ATP depletion, and mitochondrial permeability transition. The H₂O₂ scavenger pyruvate prevented cell death, ROS generation, and mitochondrial permeability transition induced by chemical hypoxia. In contrast, changes mediated by chemical hypoxia were not affected by hydroxyl radical scavengers. Antioxidants did not affect cell death and ATP depletion induced by chemical hypoxia, although they prevented ROS production and mitochondrial permeability transition induced by chemical hypoxia. Chemical hypoxia did not increase lipid peroxidation even when antimycin A was increased to 50 M, whereas the oxidant t-butylhydroperoxide caused a significant increase in lipid peroxidation, at a concentration that is less effective than chemical hypoxia in inducing cell death. Fructose protected against cell death and mitochondrial permeability transition induced by chemical hypoxia. However, ROS generation and ATP depletion were not prevented by fructose. Chemical hypoxia caused the early increase in intracellular Ca²⁺. The cell death and ROS generation induced by chemical hypoxia were altered by modulation of intracellular Ca²⁺ concentration with ruthenium red, TMB-8, and BAPTA/AM. However, mitochondrial permeability transition was not affected by these compounds. These results indicate that chemical hypoxia causes cell death, which may be, in part, mediated by H₂O₂ generation via a lipid peroxidation-independent mechanism and elevated intracellular Ca²⁺. In addition, these data suggest that chemical hypoxia-induced cell death is not associated directly with ATP depletion and mitochondrial permeability transition.     

Release of arachidonic acid via Ca2+ increase stimulated by pyrophosphonucleotides and bradykinin in mammary tumour cells

The relationship between the increase of intracellular Ca²⁺ and the release of arachidonic acid by bradykinin and pyrophosphonucleotides was studied in cultured mammary tumour cells, MMT060562. Bradykinin, ATP, UTP and UDP induced an increase of intracellular Ca²⁺ and the release of arachidonic acid from phospholipids into the extracellular fluid. Release of arachidonic acid was also induced by the application of the Ca²⁺ ionophore, A23187. Liberation of arachidonic acid by bradykinin and ATP was reduced by mepacrine, a blocker of phospholipase A₂ and W-7, a calmodulin antagonist. It is suggested that the increase in cytosolic Ca²⁺-induced release of arachidonic acid occurs through activation of calmodulin-dependent phospholipase A₂.     

Membrane Phospholipid Alterations In Alzheimer's Disease: Deficiency of Ethanolamine Plasmalogens

The ethanolamine plasmalogens are decreased whereas serine glycerophospholipids are significantly increased in plasma membrane phospholipid in affected regions of brain in Alzheimer's disease. This may be due to stimulation of Ca²⁺-independent plasmalogen-selective phospholipase A₂, which was recently discovered in brain. This phospholipase A₂ differs from other Ca²⁺-independent phospholipases A₂ in response to ATP and various inhibitors. It may be responsible for excess release of arachidonic acid and accumulation of prostaglandins and lipid peroxides in AD. Accumulation of the above lipid metabolites due to abnormal receptor function and signal transduction may contribute to neurodegeneration in AD.     

Involvement of phospholipase A2 and arachidonic acid in the depolarization-evoked accumulation of Ca2+ in hippocampal mossy fiber nerve endings

Depolarization-evoked increases in intraterminal free Ca²⁺ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca²⁺ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca²⁺ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca²⁺ channel blockers. In addition, the K⁺-dependent effects on Ca²⁺ concentrations were attenuated in the presence of phospholipase A₂ inhibitors, but were mimicked by the phospholipase A₂ activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca²⁺ were reduced by voltage-sensitive Ca²⁺ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca²⁺. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A₂ and the subsequent accumulation of unesterified arachidonic acid.     

Mitochondrial Generation of Oxygen Radicals During Reoxygenation of Ischemic Tissues

Ischemia and reperfusion causes severe mitochondrial damage, including swelling and deposits of hyd-roxyapatite crystals in the mitochondrial matrix. These crystals are indicative of a massive influx of Ca²⁺ into the mitochondrial matrix occurring during reoxygenation. We have observed that mitochondria isolated from rat hearts after 90 minutes of anoxia followed by reoxygenation, show a specific inhibition in the electron transport chain between NADH dehydrogenase and ubiquinone in addition to becoming uncoupled (unable to generate ATP). This inhibition is associated with an increased H₂O₂ formation at the NADH dehydrogenase level in the presence of NADH dependent substrates. Control rat mitochondria exposed for 15 minutes to high Ca²⁺ (200 nmol/mg protein) also become uncoupled and electron transport inhibited between NADH dehydrogenase and ubiquinone. a lesion similar to that observed in post-ischem-ic mitochondria. This Ca²⁺ -dependent effect is time dependent and may be partially prevented by albumin, suggesting that it may be due to phospholipase A₂ activation. releasing fatty acids, leading to both inhibition of electron transport and uncoupling. Addition of arachidonic or linoleic acids to control rat heart mitochondria, inhibits electron transport between Complex I and III. These results are consistent with the following hypothesis: during ischemia, the intracellular energy content drops severely, affecting the cytoplasic concentration of ions such as Na⁺ and Ca²⁺. Upon reoxygenation, the mitochondrion is the only organelle capable of eliminating the excess cytoplasmic Ca²⁺ through an electrogenic process requiring oxygen (the low ATP concentration makes other ATP-dependent Ca^?' lransport systems non-operational). Ca²⁺-overload of mitochondria activates phospholipase A₂ releasing free fatty acids, leading to uncoupling and inhibition of the interactions between Complex I and III of the respiratory chain. As a consequence, the NADH-dehydrogenase becomes highly reduced, and transfers electrons directly to oxygen generating O₂.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Lipid-Dependent Modulation of Ca2+ Availability in Isolated Mossy Fiber Nerve Endings

An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca²⁺, since exocytosis requires Ca²⁺ and a potentiation of Ca²⁺ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca²⁺ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca²⁺ accumulation was reduced by inhibitors of phospholipase A₂ and voltage-sensitive Ca²⁺ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A₂ was also indicated by the ability of staurosporine to reduce the Ca²⁺ accumulation induced by arachidonic acid or the phospholipase A₂ activator melittin. Similarly, the synergistic facilitation of evoked Ca²⁺ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca²⁺ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.     

Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes

Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca²⁺ oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca²⁺] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca²⁺ oscillations in the cytosol that are transduced into mitochondrial Ca²⁺ oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP]_mito and [ATP]_cyto. We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca²⁺]_cyto and [ATP]_cyto during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

Characterisation of a plasma membrane-associated phospholipase A2 activity increased in response to cold acclimation

We here demonstrate the presence of a plasma membrane-associated phospholipase A₂ (EC 3.1.1.4; PLA₂) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA₂ activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca²⁺, with full enzyme activity at 10 μmol/L Ca²⁺. The Ca²⁺-dependent PLA₂ activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca²⁺-dependent PLA₂ activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA₂ activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA₂ in cold acclimation in plants.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen speciesgeneration

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca²⁺ uptake. Here we sought to elucidate the effects of extramitochondrial Ca²⁺ (e[Ca²⁺]) on ROS production (measured as H₂O₂ release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl₂ and then energized with the complex I substrate Na⁺ pyruvate or the complex II substrate Na⁺ succinate. Mitochondrial H₂O₂ release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca²⁺] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H₂O₂ (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca²⁺], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H₂O₂ release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca²⁺] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H₂O₂ emission from complex III during ischemia.     

Arachidonic acid release in renal proximal tubule cell injuries and death

Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 μM), the protonophore carbonyl cyanide ρ-trifluoromethoxypheitylhydrazone (1 μM FCCP), the oxidant tertbutyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 μM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-¹⁴C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A₂ activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Imaging of caffeine-inducible release of intracellular calcium in cultured embryonic mouse telencephalic neurons

To gain a better understanding of Ca²⁺-induced Ca²⁺ release in central neurons, we have studied the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by application of caffeine to cells cultured from embryonic mouse telencephalon (hippocampus or cortex). The magnitudes and distributions of changes in [Ca²⁺]_i in neuron somata were measured by quantitative video microscopy. We observed that application of caffeine to pyramidally shaped neurons typically initiated an increase in [Ca²⁺]_i in the cytoplasmic region between the nucleus and the base of a major dendrite. [Ca²⁺] in this region increased over a period of 3 to 6 s and was followed by, with a slight delay, a surge of Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ responses to caffeine were observed in Ca²⁺-containing and nominally Ca²⁺-free external solutions, suggesting that caffeine was inducing Ca²⁺ release from intracellular stores. Ca²⁺ responses to caffeine were potentiated by inducing a tonic Ca²⁺ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors activated by 0.3 μM glutamate and multiple responses to caffeine could be elicited by using this Ca²⁺ influx to refill the intracellular stores. Ryanodine inhibition of caffeine-induced Ca²⁺ release was use- and concentration-dependent; the median effective concentration EC₅₀ for ryanodine declined from 22 μM for the first application of caffeine to 20 nM for the fourth. We conclude, based on these responses to caffeine, that ryanodine-sensitive mechanisms of intracellular Ca²⁺ release are active in hippocampal and cortical neurons and may be involved in generation of directed Ca²⁺ waves that engulf the nucleus. © 1995 John Wiley & Sons, Inc.     

The activation by Ca2+ of platelet phospholipase A2: Effects of dibutyryl cyclic adenosine monophosphate and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate

Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt₂ cyclic AMP) or with 0.6 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt₂ cyclic AMP. The addition of external Ca²⁺ to thrombin-treated platelets incubated with Bt₂ cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt₂ cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca²⁺. Inhibition of arachidonic acid liberation by Bt₂ cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg²⁺ is substituted for Ca²⁺, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt₂ cyclic AMP is slightly enhanced. The phospholipase A₂ activity of platelet lysates is increased by the presence of added Ca²⁺, however, the addition of either A23187 or Bt₂ cyclic AMP is without effect on this activity. We suggest that Bt₂ cyclic AMP may promote a compartmentalization of Ca²⁺, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca²⁺ stores in situ or restrict access of Ca²⁺ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore.     

Osmotic Swelling-Induced Changes in Cytosolic Calcium Do Not Affect Regulatory Volume Decrease in Rat Cultured Suspended Cerebellar Astrocytes

Abstract: Hyposmotic swelling-induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca²⁺]_i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca²⁺ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca²⁺]_i elevation. Omission of external Ca²⁺ or addition of Cd²⁺, Mn²⁺, or Gd³⁺ did not reduce RVD, although it was decreased by La³⁺ (0.1–1 mM). Verapamil did not affect either the swelling-evoked [Ca²⁺]_i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca²⁺ stores, such as treatment (in Ca²⁺-free medium) with 0.2 µM thapsigargin (Tg), 10 µM 2,5-di-tert-butylhydroquinone, 1 µM ionomycin, or 100 µM ATP abolished the increase in [Ca²⁺]_i but did not affect RVD. However, prolonged exposure to 1 µM Tg blocked RVD regardless of ER Ca²⁺ content or cytosolic Ca²⁺ levels. Ryanodine (up to 100 µM) and caffeine (10 mM) did not modify [Ca²⁺]_i or RVD. BAPTA-acetoxymethyl ester (20 µM) abolished [Ca²⁺]_i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca²⁺]_i rise occurs as a consequence of increased Ca²⁺ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.