Nanodomain coupling between Ca2+ channels and Ca2+ sensors promotes fast and efficient transmitter release at a cortical GABAergic synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.     

Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells.

Intercellular communication of epithelial cells was examined by measuring changes in intracellular calcium concentration ([Ca2+]i). Mechanical stimulation of respiratory tract ciliated cells in culture induced a wave of increasing Ca2+ that spread, cell by cell, from the stimulated cell to neighboring cells. The communication of these Ca2+ waves between cells was restricted or blocked by halothane, an anesthetic known to uncouple cells. In the absence of extracellular Ca2+, the mechanically stimulated cell showed no change or a decrease in [Ca2+]i, whereas [Ca2+]i increased in neighboring cells. Iontophoretic injection of inositol 1,4,5-trisphosphate (IP3) evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. These results support the hypothesis that IP3 acts as a cellular messenger that mediates communication through gap junctions between ciliated epithelial cells.     

PnTx3-6 a spider neurotoxin inhibits K+-evoked increase in [Ca2+](i) and Ca2+-dependent glutamate release in synaptosomes

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.     

Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse

Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca²⁺-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or α-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca²⁺-triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca²⁺-independent release. We propose that synaptotagmin I is the major low affinity Ca²⁺ sensor mediating Ca²⁺ regulation of synchronous neurotransmitter release in hippocampal neurons.     

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O₂^•-, H₂O₂, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death^1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response⁷. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O₂^•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells⁸. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others^9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O₂^•- that are important in the host defense mechanism^11,12. Although paracrine-derived O₂^•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca²⁺ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O₂^•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O₂^•- lead to calcium signaling in adjacent endothelial cells.     

Omega-agatoxin-TK is a useful tool to study P-type Ca2+ channel-mediated changes in internal Ca2+ and glutamate release in depolarised brain nerve terminals

The present study shows that omega-agatoxin-TK, a toxin of the venom of Agelenopsis aperta, which is 10 times more concentrated than the P/Q type Ca(2+) channel blocker, omega-agatoxin-IVA in the venom, inhibits the high K(+) depolarisation-induced rise in internal Ca(2+) (Ca(i), as determined with fura-2) dose dependently in cerebral (striatal and hippocampal) isolated nerve endings, with calculated IC(50)'s of about 60nM. The maximal inhibition exerted by omega-agatoxin-TK in striatal synaptosomes (61 +/- 11%) is 10% larger than in hippocampal synaptosomes, suggesting a larger population of omega-agatoxin-TK-sensitive Ca(2+) channels in striatal than in hippocampal nerve endings. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1muM), inhibits part of the omega-agatoxin-TK-insensitive rise in Ca(i) induced by high K(+). In contrast to the inhibition exerted by omega-agatoxin-TK on the Ca(i) response to high K(+), omega-agatoxin-TK failed to inhibit the tetrodotoxin-sensitive elevations in Ca(i) and in internal Na(+) (Na(i), as determined with SBFI) induced by veratridine, indicating that the Ca(2+) influx activated by veratridine does not involve omega-agatoxin-TK-sensitive channels. High K(+) does not increase Na(i). In [(3)H]Glu preloaded hippocampal synaptosomes super-fused with low Na(+) Krebs Ringer HEPES (a condition that guarantees the elimination of neurotransmitter transporters-mediated release), the release of [(3)H]Glu induced by high K(+) is absolutely dependent on the entrance of external Ca(2+). This exocytotic release of [(3)H]Glu attained in the absence of a chemical Na(+) gradient is inhibited with the same potency and efficacy by omega-agatoxin-TK and by omega-agatoxin-IVA, which is known to differ from omega-agatoxin-TK in its amino terminal moiety. These results indicate that omega-agatoxin-TK represents a good pharmacological tool to study P/Q type Ca(2+) channel-mediated responses in cerebral nerve endings.     

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.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Synchronized Ca2+ signaling by intercellular propagation of Ca2+ action potentials in NRK fibroblasts

Theintercellular propagation of Ca²⁺waves by diffusion of inositol trisphosphate has been shown to be ageneral mechanism by which nonexcitable cells communicate. Here, weshow that monolayers of normal rat kidney (NRK) fibroblasts behave likea typical excitable tissue. In confluent monolayers of these cells,Ca²⁺ action potentials can begenerated by local depolarization of the monolayer on treatment witheither bradykinin or an elevation of the extracellularK⁺ concentration. Theseelectrotonically propagating action potentials travel intercellularlyover long distances in an all-or-none fashion at a speed of ~6.1 mm/sand can be blocked by L-typeCa²⁺ channel blockers. The actionpotentials are generated by depolarizations beyond the threshold valuefor L-type Ca²⁺ channels of about15 mV. The result of these locally induced, propagatingCa²⁺ action potentials is analmost synchronous, transient increase in the intracellularCa²⁺ concentration in largenumbers of cells. These data show that electrically coupled fibroblastscan form an excitable syncytium, and they elucidate a novel mechanismof intercellular Ca²⁺ signaling inthese cells that may coordinate synchronized multicellular responses tolocal stimuli.

     

Effects of staurosporine on Ca2+-dependent and Ca2+-independent [14C]GABA release from rat brain synaptosomes

The effect of a protein kinase inhibitor, staurosporine, on Ca²⁺-dependent and Ca²⁺-independent release of [¹⁴C]GABA in isolated rat brain synaptosomes was studied. Calcium-dependent [¹⁴C]GABA release was stimulated by depolarization with a K⁺ channel blocker, 4-aminopyridine (4-AP), or high K⁺ concentration. It has been shown that the effect of 4-AP is Ca²⁺-dependent, while high K⁺ is able to evoke [¹⁴C]GABA release in both Ca²⁺-dependent and Ca²⁺-independent manners. In addition, Ca²⁺-independent [¹⁴C]GABA release was studied using α-latrotoxin (LTX) as a tool. Pretreatment of synaptosomes with staurosporine resulted in pronounced inhibition of 4-AP-stimulated Ca²⁺-dependent [¹⁴C]GABA release. The inhibitory effect of staurosporine on [¹⁴C]GABA release was not due to modulation of 4-AP-promoted⁴⁵Ca²⁺ influx into synaptosomes. If the process of [¹⁴C]GABA release occurred in the Ca²⁺-independent manner irrespectively of what, LTX or high K⁺, stimulated this process, it was not inhibited by staurosporine. Considering the above findings, it is reasonable to assume that the absence of Ca²⁺ in the extracellular medium created conditions for activation of the process of neurotransmitter release without Ca²⁺-dependent dephosphorylation of neuronal phosphoproteins; as a consequence, regulation of exocytotic process was modulated in such a manner that inhibition of protein kinases did not disturb exocytosis.     

Immunophilin deficiency augments Ca2+-dependent glutamate release from mouse cortical astrocytes

Immunophilins are receptors for immunosuppressive drugs such as the macrolides cyclosporin A (CsA) and FK506; correspondingly these immunophilins are referred to as cyclophilins and FK506-binding proteins (FKBPs). In particular, CsA targets cyclophilin D (CypD), which can modulate mitochondrial Ca(2+) dynamics. Since mitochondria have been implicated in the regulation of astrocytic cytosolic Ca(2+) (Ca(cyt)(2+)) dynamics and consequential Ca(2+)-dependent exocytotic release of glutamate, we investigated the role of CypD in this process. Cortical astrocytes isolated from CypD deficient mice Ppif(-/-) displayed reduced mechanically induced Ca(cyt)(2+) increases, even though these cells showed augmented exocytotic release of glutamate, when compared to responses obtained from astrocytes isolated from wild-type mice. Furthermore, acute treatment with CsA to inhibit CypD modulation of mitochondrial Ca(2+) buffering, or with FK506 to inhibit FKBP12 interaction with inositol-trisphosphate receptor of the endoplasmic reticulum, led to similar reductive effects on astrocytic Ca(cyt)(2+) dynamics, but also to an enhanced Ca(2+)-dependent exocytotic release of glutamate in wild-type astrocytes. These findings point to a possible role of immunophilin signal transduction pathways in astrocytic modulation of neuronal activity at the tripartite synapse.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. III. Block of Ca2+-induced Ca2+ release by organic polyamines

Calcium ions that have been preloaded into isolated SR subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances in a manner that is effectively blocked by ruthenium red and other organic polyamines. Effective blocking substances include certain antibiotics (neomycin, gentamicin, streptomycin, clindamycin, kanamycin, and tobramycin), naturally occurring polyamines (spermine and spermidine), and a number of basic polypeptides and proteins (polylysine, polyarginine, certain histones, and protamine). These agents have only one feature in common: the presence of several amino groups. Ruthenium red, neomycin, spermine, and protamine all appear to act by blocking SR Ca2+ channels since unidirectional 45Ca2+ efflux from the vesicles is strongly inhibited by these agents. Functions ascribable to the SR Ca2+ pump are largely unaffected by these agents. Since inositol 1,4,5-trisphosphate is ineffective at inducing Ca2+ release under these conditions, we conclude that these polyamines may directly block SR Ca2+ channels at very low concentrations by a mechanism unrelated to effects on inositol 1,4,5-trisphosphate production.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. II. Releases involving a Ca2+-induced Ca2+ release channel

Calcium ions that have been preloaded into isolated sarcoplasmic reticulum subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances. We report here that not only caffeine, but also Ca2+ ions, thymol, quercetin, menthol, halothane, chloroform, 1-ethyl-2-methylbenzimidazole, ryanodine, tetraphenylboron, ketoconazole, miconazole, clotrimazole, W-7, doxorubicin, 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and low concentrations of Ag+ induce Ca2+ release from such triadic sarcoplasmic reticulum. All these drugs induce increased undirectional Ca2+ efflux. We believe all these drug-induced Ca2+ releases are mediated by Ca2+ efflux through the same ion channel since these releases are all greatly attenuated when light sarcoplasmic reticulum is substituted for triads and are even more pronounced when transverse tubule-free terminal cisternae are substituted for triads, and all these forms of drug-induced Ca2+ release are inhibited by submicromolar concentrations of ruthenium red, and by submillimolar concentrations of tetracaine, 9-aminoacridine, and Ba2+, yet they are not affected by nifedipine even at a concentration of 50 microM.     

Dihydroorotate induces Ca2+ release from rat liver mitochondria: a contribution to the mechanism of alloxan-induced Ca2+ release

The present work deals with the effects of alloxan on rat liver mitochondria, involving formation of toxic oxygen derivatives and Ca2+ release, and its relations to a physiological pathway, pyrimidine biosynthesis, particularly dihydroorotate dehydrogenation. Ca2+ release by intact isolated mitochondria was studied and redox transfer from solubilized mitochondria to 2,6-dichloroindophenol in the presence of cyanide. In intact mitochondria 5mM dihydroorotate caused a Ca2+ efflux comparable to 2mM alloxan. Both effects were suppressed by orotate, a potent inhibitor of dihydroorotate dehydrogenase, and by ADP, an inhibitor of the alloxan effects. In lysed mitochondria orotate but not ADP inhibited ubiquinone-linked reduction of 2,6-dichloroindophenol with dihydroorotate and with alloxan in a concentration-dependent manner. It is concluded that in vitro part of the redox cycling of alloxan is catalysed by dihydroorotate dehydrogenase whereas the nonsuppressible part reacts nonenzymatically. Without ADP the respiratory control blocks the reoxidation of coenzyme Q via the respiratory chain, thus giving preference to the regeneration by artificial electron acceptors, e.g. oxygen, yielding superoxide radicals and hydrogen peroxide, a notorious inducer of Ca2+ release. In vivo the enzymatic reoxidation of reduced alloxan by dihydroorotate dehydrogenase may be superior to the non-enzymatic pathway since the nonenzymatic fraction of reoxidation decreases with decreasing alloxan concentration.     

Ca2+ release induced by cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is the most potent Ca²⁺-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD⁺ by ADP-ribosyl cyclases. cADPR binds to Ca²⁺ channels in the endoplasmic reticulum membrane to activate a Ca²⁺-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca²⁺ concentrations. Thus, cADPR may function as an endogenous regulator of Ca²⁺-induced Ca²⁺ release, and there is excitement that it may also function as a Ca²⁺-mobilizing second messenger.     

Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2(+)-induced Ca2+ release.

Receptor-mediated inositol 1,4,5-trisphosphate (Ins-(1,4,5)P3) generation evokes fluctuations in the cytoplasmic Ca2+ concentration ([Ca2+]i). Intracellular Ca2+ infusion into single mouse pancreatic acinar cells mimicks the effect of external acetylcholine (ACh) or internal Ins(1,4,5)P3 application by evoking repetitive Ca2+ release monitored by Ca2(+)-activated Cl- current. Intracellular infusion of the Ins(1,4,5)P3 receptor antagonist heparin fails to inhibit Ca2+ spiking caused by Ca2+ infusion, but blocks ACh- and Ins(1,4,5)P3-evoked Ca2+ oscillations. Caffeine (1 mM), a potentiator of Ca2(+)-induced Ca2+ release, evokes Ca2+ spiking during subthreshold intracellular Ca2+ infusion. These results indicate that ACh-evoked Ca2+ oscillations are due to pulses of Ca2+ release through a caffeine-sensitive channel triggered by a small steady Ins(1,4,5)P3-evoked Ca2+ flow.