Nerve growth factor-induced glutamate release is via p75 receptor,ceramide, and Ca(2+) from ryanodine receptor in developing cerebellar neurons

Very little is known about the contribution of a low affinity neurotrophin receptor, p75, to neurotransmitter release. Here we show that nerve growth factor (NGF) induced a rapid release of glutamate and an increase of Ca2+ in cerebellar neurons through a p75-dependent pathway. The NGF-induced release occurred even in the presence of the Trk inhibitor K252a. The release caused by NGF but not brain-derived neurotrophic factor was enhanced in neurons overexpressing p75. Further, after transfection of p75-small interfering RNA, which down-regulated the endogenous p75 expression, the NGF-induced release was inhibited, suggesting that the NGF-induced glutamate release was through p75. We found that the NGF-increased Ca2+ was derived from the ryanodine-sensitive Ca2+ receptor and that the NGF-increased Ca2+ was essential for the NGF-induced glutamate release. Furthermore, scyphostatin, a sphingomyelinase inhibitor, blocked the NGF-dependent Ca2+ increase and glutamate release, suggesting that a ceramide produced by sphingomyelinase was required for the NGF-stimulated Ca2+ increase and glutamate release. This action of NGF only occurred in developing neurons whereas the brain-derived neurotrophic factor-mediated Ca2+ increase and glutamate release was observed at the mature neuronal stage. Thus, we demonstrate that NGF-mediated neurotransmitter release via the p75-dependent pathway has an important role in developing neurons.     

Chronic antidepressants potentiate via sigma-1 receptors the brain-derived neurotrophic factor-induced signaling for glutamate release

Up-regulation of BDNF (brain-derived neurotrophic factor) has been suggested to contribute to the action of antidepressants. However, it is unclear whether chronic treatment with antidepressants may influence acute BDNF signaling in central nervous system neurons. Because BDNF has been shown by us to reinforce excitatory glutamatergic transmission in cultured cortical neurons via the phospholipase-gamma (PLC-gamma)/inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway (Numakawa, T., Yamagishi, S., Adachi, N., Matsumoto, T., Yokomaku, D., Yamada, M., and Hatanaka, H. (2002) J. Biol. Chem. 277, 6520-6529), we examined in this study the possible effects of pretreatment with antidepressants on the BDNF signaling through the PLC-gamma)/IP3/Ca2+ pathway. Furthermore, because the PLC-gamma/IP3/Ca2+ pathway is regulated by sigma-1 receptors (Hayashi, T., and Su, T. P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 491-496), we examined whether the BDNF signaling is modulated by sigma-1 receptors (Sig-1R). We found that the BDNF-stimulated PLC-gamma activation and the ensued increase in intracellular Ca2+ ([Ca2+]i) were potentiated by pretreatment with imipramine or fluvoxamine, so was the BDNF-induced glutamate release. Furthermore, enhancement of the interaction between PLC-gamma and TrkB (receptor for BDNF) after imipramine pretreatment was observed. Interestingly, BD1047, a potent Sig-1R antagonist, blocked the imipramine-dependent potentiation on the BDNF-induced PLC-gamma activation and glutamate release. In contrast, overexpression of Sig-1R per se, without antidepressant pretreatment, enhances BDNF-induced PLC-gamma activation and glutamate release. These results suggest that antidepressant pretreatment selectively enhance the BDNF signaling on the PLC-gamma/IP3/Ca2+ pathway via Sig-1R, and that Sig-1R plays an important role in BDNF signaling leading to glutamate release.     

Neuronal roles of the integrin-associated protein (IAP/CD47) in developing cortical neurons

Little is known about the role of the integrin-associated protein (IAP, or CD47) in neuronal development and its function in the central nervous system. We investigated neuronal responses in IAP-overexpressing cortical neurons using a virus-gene transfer system. We found that dendritic outgrowth was significantly enhanced in IAP (form 4)-transfected neurons. Furthermore, synaptic proteins including synaptotagmin, syntaxin, synapsin I, and SNAP25 (25-kDa synaptosomal associated protein) were up-regulated. In accordance with this finding, the release of the excitatory transmitter glutamate and the frequencies of Ca2+ oscillations (glutamate-mediated synaptic transmission) were increased. Interestingly, the overexpression of IAP activated mitogen-activated protein kinase (MAPK), and this activation was required for the IAP-dependent biological effects. After down-regulation of the endogenous IAP by small interfering RNA, MAPK activity, synaptic protein levels, and glutamate release decreased. These observations suggest that the IAP plays important roles in dendritic outgrowth and synaptic transmission in developing cortical neurons through the activation of MAPK.     

Epidermal growth factor potentiates cholecystokinin/gastrin receptor-mediated Ca2+ release by activation of mitogen-activated protein kinases

Small differences in amplitude, duration, and temporal patterns of change in the concentration of free intracellular Ca2+ ([Ca2+](i)) can profoundly affect cell physiology, altering programs of gene expression, cell proliferation, secretory activity, and cell survival. We report a novel mechanism for amplitude modulation of [Ca2+](i) that involves mitogen-activated protein kinase (MAPK). We show that epidermal growth factor (EGF) potentiates gastrin-(1-17) (G17)-stimulated Ca2+ release from intracellular Ca2+ stores through a MAPK-dependent pathway. G17 activation of the cholecystokinin/gastrin receptor (CCK(2)R), a G protein-coupled receptor, stimulates release of Ca2+ from inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Pretreating rat intestinal epithelial cells expressing CCK(2)R with EGF increased the level of G17-stimulated Ca2+ release from intracellular stores. The stimulatory effect of EGF on CCK(2)R-mediated Ca2+ release requires activation of the MAPK kinase (MEK)1,2/extracellular signal-regulated kinase (ERK)1,2 pathway. Inhibition of the MEK1,2/ERK1,2 pathway by either serum starvation or treatment with selective MEK1,2 inhibitors PD98059 and U0126 or expression of a dominant-negative mutant form of MEK1 decreased the amplitude of the G17-stimulated Ca2+ release response. Activation of the MEK1,2/ERK1,2 pathway either by pretreating cells with EGF or by expression of constitutively active K-ras (K-rasV12G) or MEK1 (MEK1*) increased the amplitude of G17-stimulated Ca2+ release. Although EGF, MEK1*, and K-rasV12G activated the MEK1,2/ERK1,2 pathway, they did not increase [Ca2+](i) in the absence of G17. These data demonstrate that the activation state of the MEK1,2/ERK1,2 pathway can modulate the amplitude of the CCK(2)R-mediated Ca2+ release response and identify a novel mechanism for cross-talk between EGF receptor- and CCK(2)R-regulated signaling pathways.     

Glutamate release from adult primary sensory neurons in culture is modulated by growth factors.

The aim of this study was to examine possible modulatory effects of some trophic molecules, i.e. nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF), on potassium (K(+))-, bradykinin (BK)- or capsaicin (CAPS)-evoked release of glutamate (GLU) from dorsal root ganglion (DRG) neurons in vitro. BK (0.5 and 1 microM) induced a dramatic and significant increase in glutamate release. Neither CAPS nor K(+) (60 mM) produced any significant increase of GLU release vs. basal levels during a 5-min stimulation. The BK-evoked release of GLU was almost completely blocked by HOE 140, a selective BK2-receptor antagonist at high doses. Basal release of GLU was significantly reduced in cultures grown in the presence of bFGF, whereas BDNF and NGF had no significant effect. Incubation with growth factors generally decreased the BK-stimulated GLU release, an effect most pronounced for bFGF, which completely blocked BK-stimulated release. The rise in intracellular [Ca(2+)] following stimulation with BK (100 nM-1 microM), potassium (60 mM) or ATP (10 microM) was also studied using a Ca(2+)-sensitive indicator, Fura-2, in cultures grown in basal medium with or without bFGF. None of the bFGF-treated cells exhibited strong Ca(2+) responses to BK or ATP stimulation, while 10-20% of the responding cells grown in basal medium exhibited strong responses. The K(+)-induced increase of [Ca(2+)] did not vary between the different groups.The present findings suggest that sensory neurotransmission involving glutamate may be modulated by growth factors and that regulation of intracellular Ca(2+) homeostasis may be a contributing factor.     

Calmidazolium selectively inhibits exocytotic glutamate release evoked by P2X7 receptor activation

We previously observed that activation of presynaptic P2X7 receptors located on rat cerebrocortical nerve terminals induced the release of glutamate through different modes: the channel conformation allowing Ca(2+) entry triggered exocytotic release, while the receptor itself functioned as a permeation pathway for the non-exocytotic glutamate release. Considering that exocytotic and non-exocytotic glutamate release evoked by the activation of P2X7 receptors might play a role in the control of glutamatergic synapses, we investigated whether calmidazolium (which has been found to inhibit small cation currents through recombinant P2X7 receptors, but not organic molecule permeation) could distinguish between P2X7-related exocytotic and non-exocytotic modes of glutamate release. We found that calmidazolium inhibited the intrasynaptosomal Ca(2+) response to P2X7 receptor activation and the Ca(2+)-dependent exocytotic glutamate release from rat cerebrocortical nerve terminals, but was ineffective against the Ca(2+)-independent glutamate release. The P2X7 competitive antagonist A-438079 eliminated both exocytotic and non-exocytotic P2X7 receptor-evoked glutamate release. Selective inhibition of exocytotic glutamate release indicates that calmidazolium inhibits events dependent on the function of native rat P2X7 receptors as Ca(2+) channels, and suggests that it can be used as a tool to dissociate P2X7-evoked exocytotic from non-exocytotic glutamate release.     

Occurrence of a Large Ca2+-Independent Release of Glutamate During Anoxia in Isolated Nerve Terminals (Synaptosomes)

Isolated rat cerebral cortical synaptosomes made anoxic by addition of cyanide developed an inhibition of the Ca2+-dependent release of glutamate 2 min after the addition of the metabolic inhibitor when the intrasynaptosomal ATP/ADP ratio decreased below 1.7. In contrast, cyanide induced a continuous efflux of glutamate through a Ca2+-independent pathway that accounted for the release of 25% of total intrasynaptosomal glutamate in 5 min. The results suggest that a Ca2+-independent release of glutamate could be implicated in the neurotoxic action of this amino acid during anoxia.     

Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway.

A sperm-induced intracellular Ca2+ signal ([Ca2+]i) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP3R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca2+ release. IP3R1-mediated Ca2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca2+]i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP3R1 function in eggs. Using mouse and Xenopus eggs, we show that IP3R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP3R1 at at least one highly conserved site, and that its mutation abrogates IP3R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP3R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca2+]i oscillations in response to agonists and show compromised IP3R1 function. These findings identify IP3R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP3R1 function in eggs that serves to optimize [Ca2+]i release at fertilization.     

Genistein inhibits Ca2+ influx and glutamate release from hippocampal synaptosomes: putative non-specific effects

The role of protein tyrosine kinases on glutamate release was investigated by determining the effect of broad range inhibitors of tyrosine kinases on the release of glutamate from rat hippocampal synaptosomes. We found that lavendustin A and herbimycin A did not inhibit glutamate release stimulated by 15 mM KCl, but genistein, also a broad range inhibitor of tyrosine kinases did inhibit the intracellular Ca(2+) concentration response to KCl and, concomitantly, decreased glutamate release evoked by the same stimulus, in a dose-dependent manner. These effects were not observed with the inactive analogue genistin. Therefore, we investigated the mechanism whereby genistein modulates Ca(2+) influx and glutamate release. Studies with voltage-gated Ca(2+) channel inhibitors showed that omega-conotoxin GVIA did not further inhibit glutamate release or the Ca(2+) influx stimulated by KCl in the presence of genistein. This tyrosine kinase inhibitor and omega-agatoxin IVA had a partially additive effect on those events. Nitrendipine did not reduce significantly the KCl-induced responses. Genistein further reduced Ca(2+) influx in response to KCl in the presence of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA, simultaneously. The effect of tyrosine phosphatase inhibitors was also tested on the influx of Ca(2+) and on glutamate release stimulated by KCl-depolarization. We found that the broad range inhibitors sodium orthovanadate and dephostatin did not significantly affect these KCl-evoked events.Our results suggest that genistein inhibits glutamate release and Ca(2+) influx in response to KCl independently of tyrosine kinase inhibition, and that tyrosine kinases and phosphatases are not key regulators of glutamate release in hippocampal nerve terminals.     

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.     

Short communication: unsaturated fatty acids inhibit MP2C, a protein phosphatase 2C involved in the wound-induced MAP kinase pathway regulation

When mechanically injured, plants develop multiple defense systems including the activation of specific genes. These responses are triggered by a complex network of signalling events that include Ca2+ fluxes, the production of free fatty acids from membrane lipids, as well as the activation of mitogen-activated protein kinases (MAPK). In the present paper, we address the question of the regulation of the MAPK pathway by wound-induced Ca2+ and fatty acid signals. We report that MP2C, a serine/threonine protein phosphatase 2C from alfalfa involved in MAPK pathway inactivation, is inhibited specifically in vitro by long-carbon-chain polyunsaturated fatty acids, and alpha-linolenic acid, the primary product of the octadecanoid pathway, was found to be the most potent inhibitor. Ca2+ also inhibits MP2C, but only at high concentrations, and other divalent cations show similar inhibitory effect, making it unlikely that Ca2+ is involved in the regulation of MP2C in vivo. Overall, our data suggest that cross-talk between wound-induced MAPK and octadecanoid pathways may occur at the level of protein phosphatase 2C and linolenic acid.     

Basic fibroblast growth factor inhibits radiation-induced apoptosis of HUVECs. II. The RAS/MAPK pathway and phosphorylation of BAD at serine 112

Radiation-induced endothelial cell apoptosis is involved in the development of many radiation injuries, including radiation-induced skin ulcers. The proangiogenic growth factor basic fibroblast growth factor (bFGF, NUDT6) enhances endothelial cell survival. In the present study, we set up a model of apoptosis in which primary cultured human umbilical vein endothelial cells (HUVECs) were irradiated with (60)Co gamma rays to explore the effects of bFGF on radiation-induced apoptosis of HUVECs and the signaling pathways involved. We found that bFGF inhibited radiation-induced apoptosis of HUVECs, and that the effect was mediated in part by the RAS/MEK/ MAPK/RSK (p90 ribosomal S6 kinase)/BAD pathway. This pathway was activated by exposure of irradiated HUVECs to bFGF, involving phosphorylation of FGFR, MEK and p44/42 MAPK. The survival-enhancing effect of bFGF was partly inhibited by U0126 and PD98059. The fact that the anti-apoptosis effect of bFGF on irradiated HUVECs was not completely abrogated by U0126 and PD98059 suggests that other survival signaling pathways may exist. Transfection of a dominant-negative form of RSK2 (DN RSK2) partly blocked the anti-apoptosis effect of bFGF in irradiated HUVECs. Moreover, we provide evidence for the first time that bFGF induced BAD phosphorylation (at serine 112) and CREB (cAMP response element-binding protein) activation (phosphorylation at serine 133) in gamma-irradiated HUVECs. In our model, inhibition of MAPK signaling-dependent phosphorylation of BAD at serine 112 promoted increased association with BCL-X(L), suggesting that MAPK pathway-dependent serine 112 phosphorylation of BAD is critical for the effect of bFGF on cell survival. These results showed that RAS/MAPK/BAD pathway participated in the bFGF-induced effect on survival of HUVECs exposed to radiation. It is suggested that RAS/ MAPK pathway in tumor vascular endothelium could be a potential therapeutic target to enhance the efficacy of ionizing radiation.     

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.     

Ca2+ entry via AMPA-type glutamate receptors triggers Ca2+-induced Ca2+ release from ryanodine receptors in rat spiral ganglion neurons

Ryanodine receptor (RyR)-gated Ca2+ stores have recently been identified in cochlear spiral ganglion neurons (SGN) and likely contribute to Ca2+ signalling associated with auditory neurotransmission. Here, we identify an ionotropic glutamate receptor signal transduction pathway which invokes RyR-gated Ca2+ stores in SGN via Ca2+-induced Ca2+ release (CICR). Ca2+ levels were recorded in SGN in situ within rat cochlear slices (postnatal day 0-17) using the Ca2+ indicator fluo-4. RyR-gated Ca2+ stores were confirmed by caffeine-induced increases in intracellular Ca2+ which were blocked by ryanodine (100 microM) and were independent of external Ca2+. Glutamate evoked comparable increases in intracellular Ca2+, but required the presence of external Ca2+. Ca2+ influx via the glutamate receptor was found to elicit CICR via RyR-gated Ca2+ stores, as shown by the inhibition of the response by prior depletion of the Ca2+ stores with caffeine, the SERCA inhibitor thapsigargin, or ryanodine. The glutamate analogue AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) elicited Ca2+ responses that could be inhibited by caffeine. Glutamate- and AMPA-mediated Ca2+ responses were eliminated with the AMPA/Kainate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). These data demonstrate functional coupling between somatic AMPA-type glutamate receptors and intracellular Ca(2+) stores via RyR-dependent CICR in primary auditory neurons.     

The MEK inhibitor, U0126, alters fertilization-induced [Ca2+]i oscillation parameters and secretion: differential effects associated with in vivo and in vitro meiotic maturation

Although mitogen-activated protein kinase (MAPK) is a well-known cell cycle regulator, emerging studies have also implicated its activity in the regulation of intracellular calcium concentration ([Ca2+](i)) and secretion. Those studies raise the hypothesis that MAPK activity during oocyte maturation and early fertilization is required for normal egg Ca2+ oscillations and cortical granule (CG) secretion. We extend the findings of [Lee, B., Vermassen, E., Yoon, S.-Y., Vanderheyden, V., Ito, J., Alfandari, D., De Smedt, H., Parys, J.B., Fissore, R.A., 2006. Phosphorylation of IP(3)R1 and the regulation of [Ca2+](i) responses at fertilization: a role for the MAP kinase pathway. Development 133, 4355-4365] by demonstrating acute effects on Ca2+ oscillation frequency, amplitude, and duration in fertilized mouse eggs matured in vitro with the MAPK inhibitor, U0126. Frequency was increased, whereas amplitude and duration were greatly decreased. These effects were significantly reduced in eggs matured in vivo and fertilized in the presence of the inhibitor. Ionomycin studies indicated that intracellular Ca2+ stores were differentially affected in eggs matured in vitro with U0126. Consistent with these effects on [Ca2+](i) elevation, fertilization-induced CG exocytosis and metaphase II exit were also reduced in in vitro-matured eggs with U0126, but not in those similarly treated after in vivo maturation. These results indicate that MAPK targets Ca2+ regulatory proteins during both maturation and fertilization, as well as provide a new hypothesis for MAPK function, which is to indirectly regulate events of early development by controlling Ca2+ oscillation parameters.     

Caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release from the endoplasmic reticulum in honeybee photoreceptors

Light stimulation of invertebrate microvillar photoreceptors causes a large rapid elevation in Cai, shown previously to modulate the adaptational state of the cells. Cai rises, at least in part, as a result of Ins(1,4,5)P3-induced Ca2+ release from the submicrovillar endoplasmic reticulum (ER). Here, we provide evidence for Ca(2+)- induced Ca2+ release (CICR) in an insect photoreceptor. In situ microphotometric measurements of Ca2+ fluxes across the ER membrane in permeabilized slices of drone bee retina show that (a) caffeine induces Ca2+ release from the ER; (b) caffeine and Ins(1,4,5)P3 open distinct Ca2+ release pathways because only caffeine-induced Ca2+ release is ryanodine sensitive and heparin insensitive, and because caffeine and Ins(1,4,5)P3 have additive effects on the rate of Ca2+ release; (c) Ca2+ itself stimulates release of Ca2+ via a ryanodine-sensitive pathway; and (d) cADPR is ineffective in releasing Ca2+. Microfluorometric intracellular Ca2+ measurements with fluo-3 indicate that caffeine induces a persistent elevation in Cai. Electrophysiological recordings demonstrate that caffeine mimics all aspects of Ca(2+)-mediated facilitation and adaptation in drone photoreceptors. We conclude that the ER in drone photoreceptors contains, in addition to the Ins(1,4,5)P3-sensitive release pathway, a CICR pathway that meets key pharmacological criteria for a ryanodine receptor. Coexpression of both release mechanisms could be required for the production of rapid light-induced Ca2+ elevations, because Ca2+ amplifies its own release through both pathways by a positive feedback. CICR may also mediate the spatial spread of Ca2+ release from the submicrovillar ER toward more remote ER subregions, thereby activating Ca(2+)-sensitive cell processes that are not directly involved in phototransduction.     

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.     

Agrin signaling in cortical neurons is mediated by a tyrosine kinase-dependent increase in intracellular Ca2+ that engages both CaMKII and MAPK signal pathways

Agrin has been implicated in multiple aspects of central nervous system (CNS) neuron differentiation and function including neurite formation, synaptogenesis, and synaptic transmission. However, little is known about the signaling mechanisms whereby agrin exerts its effects. We have recently identified a neuronal receptor for agrin, whose activation induces expression of c-fos, and provided evidence that agrin binding to this receptor is associated with a rise in intracellular Ca2+, a ubiquitous second messenger capable of mediating a wide range of effects. To gain further insight into agrin's role in brain, we used Ca2+ imaging to explore agrin signal transduction in cultured cortical neurons. Bath application of either z+ or z-agrin isoforms resulted in marked changes in intracellular Ca2+ concentration specifically in neurons. Propagation of the Ca2+ response was a two-step process characterized by an initial increase in intracellular Ca2+ mediated by ryanodine receptor (RyR) release from intracellular stores, supplemented by influx through voltage-gated calcium channels (VGCCs). Agrin-induced increases in intracellular Ca2+ were blocked by genistein and herbimycin, suggesting that the agrin receptor is a tyrosine kinase. Ca2+ release from intracellular stores activates both calcium/calmodulin-dependent kinase II (CaMKII) and mitogen activated protein kinase (MAPK). Activation of CaMKII is required for propagation of the Ca2+ wave itself, whereas both MAPK and CaMKII play a role in mediating long latency responses such as induction of c-fos. These results suggest that an agrin-dependent tyrosine kinase could play a critical role in modulating levels of intracellular Ca2+ and activity of MAPK and CaMKII in CNS neurons.