Inhibition of glutamate release by fluspirilene in cerebrocortical nerve terminals (synaptosomes)

Fluspirilene, a neuroleptic drug which is used clinically to treat schizophrenic patients, is a dopamine D2 receptor antagonist. Besides its well-known actions on the dopamine receptors, fluspirilene also displays calcium channel-blocking activity. The aim of this study was to investigate the effect of fluspirilene on the 4-aminopyridine (4AP)-evoked glutamate release in the cerebrocortical nerve terminals (synaptosomes). Fluspirilene reduced 4AP-evoked glutamate release in a concentration-dependent manner. This inhibitory effect was associated with a decrease in the depolarization-evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]C), which could be completely abolished by the Ca2+ channel blocker omega-CgTX GVIA. Furthermore, fluspirilene did not produce any effect on ionomycin-evoked glutamate release. These results suggest that fluspirilene inhibits glutamate release primarily by reducing presynaptic Ca2+ influx via N-type Ca2+ channels in rat cerebrocortical nerve terminals. This finding implies that presynaptic Ca2+ channel blockade concomitant with inhibition of glutamate release and possibly other neurotransmitters release may contribute to the antischizophrenic action of fluspirilene.     

Intracellular Ca2+ release‐dependent inactivation of Ca2+ currents in thalamocortical relay neurons

Neuronal Ca²⁺ channels are rapidly inactivated by a mechanism that is termed Ca²⁺‐dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca²⁺ release on CDI of high‐voltage‐activated Ca²⁺ channels in rat thalamocortical relay neurons by combining voltage‐clamp, Ca²⁺ imaging and immunological techniques. Double‐pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration‐dependent increase in CDI that was accompanied by an increase in the duration of Ca²⁺ transients. Inhibition of ryanodine receptors and endoplasmic Ca²⁺ pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5‐tris‐phosphate receptors by intracellular application of 2‐aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2‐aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L‐type Ca²⁺ channels was emphasized by the near‐complete block of CDI by nifedipine, an effect only surpassed when Ca²⁺ was replaced by Ba²⁺ and chelated by 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA). Trains of action potential‐like stimuli induced a strong reduction in high‐voltage‐activated Ca²⁺ current amplitude, which was significantly reduced when intracellular Ca²⁺ stores were made inoperative by thapsigargin or Ba²⁺/BAPTA. Western blotting revealed expression of L‐type Ca²⁺ channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross‐signalling between L‐type Ca²⁺ channels and ryanodine receptors that controls the amount of Ca²⁺ influx during neuronal activity.     

Adenosine depresses a Ca(2+)-independent step in transmitter exocytosis at frog motor nerve terminals

The depressant action of adenosine on acetylcholine release at frog motor nerve terminals was studied by intracellular recording and Ca(2+)-imaging techniques. Adenosine (200 microm) quickly and reversibly decreased the amplitude and quantal content of end-plate potentials (EPPs) with no change in quantal size in a low-Ca(2+), high-Mg(2+) solution, and EPP amplitude in normal Ringer containing d-tubocurarine. Likewise, adenosine (200 microm) reduced miniature EPP (MEPP) frequency, but not amplitude, in a high-K(+) (6 mm) solution. Adenosine (40-200 microm), however, did not affect single or repetitive impulse(s)-induced rises in Ca(2+) in the nerve terminals or its basal level. Adenosine (100-200 microm) reduced the Ca(2+)-independent enhancement of MEPP frequency caused by hypertonicity. EPPs induced by tetanic stimulation (33 Hz) in Ringer with d-tubocurarine initially increased in amplitude within 10 stimuli and then declined to the minimum. Adenosine (200 microm) decreased EPP amplitude in the initial phase of the tetanus, but enhanced it in the middle phase, thus prolonging the decay of EPP amplitude. The total sum of these EPPs, reflecting the readily releasable pool of vesicles and its refilling, however, was not changed. The results suggest that adenosine inhibits a Ca(2+)-independent step of transmitter exocytosis at frog motor nerve terminals.     

Astrocytes refill intracellular Ca2+ stores in the absence of cytoplasmic [Ca2+] elevation: a functional rather than a structural ability

Capacitative Ca(2+) entry (CCE) is a phenomenon triggered by depletion of Ca(2+) content in intracellular stores (ICS). Data about this phenomenon in astrocytes are limited. We analyzed CCE in astrocytes by means of fura-2 based digital imaging. We found that in astrocytes CCE is not associated with an increase of cytosolic Ca(2+) concentration ([Ca(2+)](i)), although ICS are efficiently refilled. We used Mn(2+), thapsigargin and prolonged ATP exposure to show that CCE is not associated with cytosolic diffusion of Ca(2+) entering astrocytes. Our data suggest that the ion is being quickly sequestered in the ICS by the smooth endoplasmic reticulum Ca(2+)-ATP-ase (SERCA). Several experiments were carried out with the goal of failing the efficient uptake in the endoplasmic reticulum (ER). In fact, inhibition of SERCA activity, increased extracellular [Ca(2+)](i) or pharmacologic potentiation of CCE all caused [Ca(2+)](i) elevation during CCE, suggesting that the control of this phenomenon could have physiologic and pathological relevance. The molecular components involved in CCE have been proposed to be organized in a multi-molecular complex tethered by cytoskeleton components and arranged via a secretion coupling model. We show here that the efficient routing of Ca(2+) into the ICS in astrocytes is not affected by disruption of cytoskeleton organization or Golgi's function, but it is instead linked to the high efficiency of SERCA. We conclude that depleted ICS in astrocytes are efficiently refilled by CCE activation, although Ca(2+) influx is not accompanied by elevation of [Ca(2+)](i). This ability seems to be functional rather than structural in nature.     

4-Chloro-m-cresol, an activator of ryanodine receptors, inhibits voltage-gated K(+) channels at the rat calyx of Held

4-Chloro-m-cresol (4-CmC) is thought to be a specific activator of ryanodine receptors (RyRs). Using this compound, we examined whether the RyR-mediated Ca(2+) release is involved in transmitter release at the rat calyx of Held synapse in brainstem slices. Bath application of 4-CmC caused a dramatic increase in the amplitude of excitatory postsynaptic currents (TIFCs) with the half-maximal effective concentration of 0.12 mm. By making direct patch-clamp whole-cell recordings from presynaptic terminals, we investigated the mechanism by which 4-CmC facilitates transmitter release. 4-CmC markedly prolonged the duration of action potentials, with little effect on their rise time kinetics. In voltage-clamp recordings, 4-CmC inhibited voltage-gated presynaptic K(+) currents (I(pK)) by 53% (at +20 mV) but had no effect on voltage-gated presynaptic Ca(2+) currents (I(pCa)). In simultaneous pre- and postsynaptic recordings, 4-CmC had no effect on the TIFC evoked by I(pCa). Although immunocytochemical study of the calyceal terminals showed immunoreactivity to type 3 RyRs, ryanodine (0.02 mm) had no effect on the 4-CmC-induced TIFC potentiation. We conclude that the facilitatory effect of 4-CmC on nerve-evoked transmitter release is mediated by its inhibitory effect on I(pK).     

Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5-triphosphate receptors in the neurotoxic effects induced by the amyloid-beta peptide

Studies with in-vitro-cultured neurons treated with amyloid-beta (A beta) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca(2+) homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)-specific apoptotic pathway mediated by caspase-12, which is activated upon the perturbation of ER Ca(2+) homeostasis, may contribute to A beta toxicity. To elucidate the involvement of deregulation of ER Ca(2+) homeostasis in neuronal death induced by A beta peptides, we have performed a comparative study using the synthetic peptides A beta(25-35) or A beta(1-40) and thapsigargin, a selective inhibitor of Ca(2+) uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 microM) increased the intracellular Ca(2+) levels and activated caspase-3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the A beta peptides (A beta(25-35), 25 microM; A beta(1-40), 0.5 microM), we observed a significant increase in [Ca(2+)](i), in caspase-3-like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca(2+) release through ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (IP(3)R) in A beta neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca(2+) release through RyR or IP(3)R, were able to prevent the increase in [Ca(2+)](i) and the activation of caspase-3 and to protect partially against apoptosis induced by treatment with A beta(25-35) or A beta(1-40). In conclusion, our results demonstrate that the release of Ca(2+) from the ER, mediated by both RyR and IP(3)R, is involved in A beta toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to A beta-induced neuronal death. This study suggests that A beta accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca(2+) homeostasis.     

Cell type–specific spatial and functional coupling between mammalian brain Kv2.1 K+ channels and ryanodine receptors

The Kv2.1 voltage‐gated K⁺ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity‐dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca²⁺‐release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy–immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR‐mediated Ca²⁺ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca²⁺/calcineurin‐dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell‐ and circuit‐specific mechanism for coupling intracellular Ca²⁺ release to phosphorylation‐dependent regulation of Kv2.1 to dynamically impact intrinsic excitability. J. Comp. Neurol. 522:3555–3574, 2014. © 2014 Wiley Periodicals, Inc.     

Fluoxetine depresses glutamate exocytosis in the rat cerebrocortical nerve terminals (synaptosomes) via inhibition of P/Q-type Ca2+ channels

Fluoxetine, an antidepressant that is used clinically in the treatment of mood disorders, is a selective serotonin reuptake inhibitor. In the present study we investigated the effects of fluoxetine on 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). Fluoxetine suppressed the release of glutamate evoked by 4AP in a concentration-dependent manner. This effect was associated with a reduction in the depolarization-evoked increase in cytosolic free calcium levels in the absence of significant effect on the synaptosomal membrane potential. In addition, both ionomycin- and sucrose-evoked glutamate releases were not affected by fluoxetine, indicating that fluoxetine-mediated inhibition of glutamate release is not a direct effect on the exocytotic machinery. Furthermore, the inhibitory action of fluoxetine was completely abolished in synaptosomes pretreated with P/Q type Ca(2+) channel blocker omega-agatoxin IVA (omega-AgTX IVA) or protein kinase C (PKC) stimulator 4beta-phorbol 12, 13-dibutyrate (PDBu). These results suggest that, in cerebrocortical nerve terminals, fluoxetine inhibits glutamate release through the suppression of P/Q type Ca(2+) channel activity. The presynaptic action of fluoxetine is mediated by a PKC-sensitive signaling pathway. Synapse 48:170-177, 2003.     

Characterization of ryanodine receptors in oligodendrocytes,type 2 astrocytes,and O-2A progenitors

In this study we have investigated the expression of ryanodine receptors (RyRs), and the ability of caffeine to evoke RyR-mediated elevation of intracellular Ca²⁺ levels ([Ca²⁺]_i) in glial cells of the oligodendrocyte/type 2 astrocyte lineage. Immunocytochemistry with specific antibodies identified ryanodine receptors in cultured oligodendrocytes, type 2 astrocytes, and O-2A progenitor cells, at high levels in the perinuclear region and in a variegated pattern along processes. Glia acutely isolated from rat brain and in aldehyde-fixed sections of cortex were similarly found to express RyRs. Caffeine (5–50 mM) caused an increase in [Ca²⁺]_i in most cultured type 2 astrocytes and in 50% of oligodendrocytes. Responses elicited by caffeine were inhibited by pretreatment with ryanodine (10 μM) or thapsigargin (1 μM), and the peak response was unaffected by removal of [Ca²⁺]_o. O-2A progenitor cells, in contrast, were largely unresponsive to caffeine treatment. Pretreatment with kainate (200 μM) to activate Ca²⁺ entry increased the magnitude of caffeine-evoked [Ca²⁺]_i elevations in type 2 astrocytes and oligodendrocytes, and caused caffeine to activate responses in a significant proportion of previously non-responding O-2A progenitors. In both type 2 astrocytes and oligodendrocytes, caffeine evoked Ca²⁺ changes which propagated as wavefronts from several initiation sites. These wave amplification sites were characterized by significantly higher local Ca²⁺ release kinetics. Our results indicate that several glial cell types express RyRs, and that their functionality differs within different cell types of the oligodendrocyte lineage. In addition, ionotropic glutamate receptor activation fills the caffeine-sensitive Ca²⁺ stores in these cells. J. Neurosci. Res. 52:468–482, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural localization of sorcin,a 22 kDa calcium binding protein,in the rat caudate-putamen nucleus: Association with ryanodine receptors and intracellular calcium release

Sorcin is a 22 kDa calcium binding protein that is widely distributed in mammalian tissues, including brain, and is associated with the ryanodine receptor (RyR) family of intracellular calcium-release channels in the heart. To determine the cellular sites for potential central functions of sorcin, we examined the electron microscopic immunocytochemical localization of antipeptide antisera against sorcin and against cardiac and brain RyR in the rat caudate-putamen nucleus (CPN), one of the few regions expressing high levels of brain RyR. Sorcin-like immunoreactivity (S-LI) was detected in both neurons and glia by using immunoperoxidase and immunogold methods. Of 1,735 profiles containing immunogold-silver labeling for sorcin, almost 50% were dendrites and many of these dendrites were spiny. The remainder were mainly small axons, axon terminals, and, more rarely, glia. Furthermore, analysis of dually labeled tissue sections showed the presence of sorcin in many of the dendrites and some of the axonal and glial processes containing RyR. In dendrites, gold-silver deposits showing S-LI were prominently localized to saccules of smooth endoplasmic reticulum and mitochondria, both of which are known to store calcium. These labeled structures were located near the plasma membrane at sites postsynaptic to excitatory-type asymmetric junctions, as well as non-synaptic portions of the plasma membrane. In axons, S-LI was also often seen at extrasynaptic sites on, or near, the plasma membrane. We conclude that in the rat CPN, sorcin may act independently or, in conjunction with RyR, to modulate cytoplasmic release of calcium, mainly from smooth endoplasmic reticulum and/or mitochondria in neurons. J. Comp. Neurol. 386:625–634, 1997. © 1997 Wiley-Liss, Inc.     

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals

Ca²⁺‐induced Ca²⁺ release (CICR) via type‐3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca²⁺ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular‐free Ca²⁺ ([Ca²⁺]_i: Ca²⁺ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation‐induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.     

Neurotransmitter release from tottering mice nerve terminals with reduced expression of mutated P- and Q-type Ca2+-channels

Neurotransmitter release is triggered by Ca2+-influx through multiple sub-types of high voltage-activated Ca2+-channels. Tottering mice have a mutation in the alpha1A pore-forming subunit of P- and Q-type Ca2+-channels, two prominent sub-types that regulate transmitter release from central nerve terminals. Immunoblotting analysis of purified forebrain terminals from tottering mice revealed an 85% reduction in the protein expression level of the mutated alpha1A subunit compared to expression of the alpha1A subunit in wild-type terminals. In contrast, the expression of the alpha1B subunit of the N-type Ca2+-channels was unchanged. Release of the amino acids glutamate and GABA and of the neuropeptide cholecystokinin (CCK) induced by a short (100 ms) depolarization pulse was unchanged in the terminals of tottering mice. Studies using specific blockers of Ca2+-channels however, revealed a reduced contribution of P- and Q-type Ca2+-channels to glutamate and cholecystokinin release, whereas a greater reliance on N-type Ca2+-channels for release of these transmitters was observed. In contrast, the contribution of the P-, Q- and N-type Ca2+-channels to the release of GABA was not altered in tottering mice. These results indicate that the expression of the alpha1A subunit was decreased in terminals from tottering mice, and that a decreased contribution of P- and Q-type Ca2+-channels to the release of glutamate and cholecystokinin was functionally compensated by an increased contribution of N-type Ca2+-channels.     

Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals

Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4‐aminopyridine (4‐AP)‐evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca²⁺ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4‐AP‐evoked intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. In the presence of N‐, P/Q‐, and R‐type channel blockers, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was markedly reduced; however, the intracellular Ca²⁺‐release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca²⁺/calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABA_A receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca²⁺ influx through N‐, P/Q‐, and R‐type Ca²⁺ channels, through the activation of GABA_A receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca²⁺‐calmodulin/PKA cascade and decreasing 4‐AP‐evoked glutamate release.     

Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia

We have studied modulation of purinergic receptors (P(2Y) and P(2X) subtypes) on changes in intracellular Ca(2+) [Ca(2+)](i) and expression and production of COX-2 in human microglia. Measurements using Ca(2+)-sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca(2+)](i). Application of ATP plus the P(2X) antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), or treatment with adenosine diphosphate-beta-S (ADP-beta-S), a selective P(2Y) agonist, led to a considerable prolongation in [Ca(2+)](i) responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 microM for 8 h). However, treatment using ATP plus PPADS or with ADP-beta-S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca(2+)-free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P(2X) activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOC-mediated influx of Ca(2+).     

Mechanisms underlying the honokiol inhibition of evoked glutamate release from glutamatergic nerve terminals of the rat cerebral cortex

The effect of honokiol, an active component of Magnolia officinalis, on glutamate release from isolated nerve terminals (synaptosomes) was examined. Honokiol potently inhibited 4-aminopyridine (4-AP)-evoked glutamate release in a concentration-dependent manner, and this effect resulted from a reduction of vesicular exocytosis and not from an inhibition of Ca(2+)-independent efflux via glutamate transporter. The inhibitory action of honokiol was not due to decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca(2+) influx, because honokiol did not alter the 4-AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release. Rather, examination of the effect of honokiol on cytosolic [Ca(2+)] revealed that the diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca(2+) influx. Consistent with this, the honokiol-mediated inhibition of 4-AP-evoked glutamate release was completely prevented in synaptosomes pretreated with a wide-spectrum blocker of N-, P-, and Q-type Ca(2+) channels, omega-conotoxin MVIIC. In addition, honokiol modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, in so far as pretreatment of synaptosomes with the PKC inhibitors Ro318220 or GF109203X all effectively occluded the inhibitory effect of honokiol. Furthermore, honokiol attenuated 4-AP-induced phosphorylation of PKC. Together, these results suggest that honokiol effects a decrease in PKC activation, which subsequently attenuates the Ca(2+) entry through voltage-dependent N- and P/Q-type Ca(2+) channels to cause a decrease in evoked glutamate exocytosis. These actions of honokiol may contribute to its neuroprotective effect in excitotoxic injury.     

Unexpected inhibitory regulation of glutamate release from rat cerebrocortical nerve terminals by presynaptic 5-hydroxytryptamine-2A receptors

Presynaptic 5-HT(2A) receptor modulation of glutamate release from rat cerebrocortical nerve terminals (synaptosomes) was investigated by using the 5-HT(2A/2C) receptor agonist (+/-)-1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI). DOI potently inhibited 4-aminopyridine (4AP)-evoked glutamate release. Involvement of presynaptic 5-HT(2A) receptors in this modulation of 4AP-evoked release was confirmed by blockade of the DOI-mediated inhibition by the 5-HT(2A) receptor antagonist ketanserin but not by the 5-HT(2C) receptor antagonist RS102221. Inhibition of glutamate release by DOI was associated with a reduction of 4AP-evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca(2+)](C)) mediated via P/Q- and N-type voltage-dependent Ca(2+) channels (VDCCs). In contrast to the DOI effect on 4AP-evoked release, the agonist had no effect on high external [K(+)] (30 mM)-induced (KCl) stimulation of VDCCs or glutamate release. Likewise, release mediated by direct Ca(2+) entry with Ca(2+) ionophore (ionomycin) or by hypertonic sucrose was unaffected by DOI. Mechanistically, DOI modulation of 4AP-evoked glutamate release appeared to involve a phospholipase C/protein kinase C signaling cascade, insofar as pretreatment of synaptosomes with the phospholipase C inhibitor U73122 or protein kinase C inhibitors Ro320432 or GF109203X all effectively occluded the inhibitory effect of the agonist. Together, these results suggest that presynaptic 5-HT(2A) receptors present on glutamatergic terminals effect an unexpected depression of glutamate release by negatively modulating nerve terminal excitability and downstream VDCC activation through a signaling cascade involving phospholipase C/protein kinase C. These observations invoke presynaptic inhibitory 5-HT(2A) receptor function as a potential target for drugs to mitigate the effects of excessive glutamatergic transmission.     

Calcium dependence of rapid astrocyte death induced by transient hypoxia, acidosis, and extracellular ion shifts

Exposure to hypoxic, acidic, ion-shifted Ringer (HAIR) for 15-40 min has been shown to cause rapid astrocyte death upon reperfusion with normal media. The ion shifts of the HAIR solution included a rise in extracellular K(+) (e.g., [K(+)](o)) and a fall in [Na(+)](o), [Cl(-)](o), and [Ca(2+)](o), characteristic of ischemic-traumatic brain insults. We investigated the ionic basis of the HAIR-induced injury. After HAIR exposure, reperfusion in 0 Ca(2+)/EGTA media completely protected astrocytes. Preincubation of cells in BAPTA-AM ester was also protective, indicating that the injury was triggered by Ca(2+) influx during reperfusion. Neither nimodipine, CNQX, APV, nor TTX reduced injury. Astrocyte death could be blocked by 100 microM Ni(2+) or 100 microM benzamil, suggesting involvement of Na(+)-Ca(2+) exchange. KB-R7943, which preferentially inhibits reverse Na(+)-Ca(2+) exchange, also protected astrocytes. Elevation of [K(+)](o) was not necessary for astrocyte death. However, when [Na(+)](o) was maintained at 151 mM throughout the HAIR protocol, cell death was markedly reduced. We postulate that [Na(+)](o) shifts aid reversal of Na(+)-Ca(2+) exchange by favoring cytosolic Na(+) loading. Possible means of astrocytic Na(+) accumulation are discussed.     

Bradykinin regulates the expression of claudin‐5 in brain microvascular endothelial cells via calcium‐induced calcium release

To investigate the mechanism underlying the regulation of claudin‐5, a tight junction protein that participates primarily in the constitution of the blood–brain barrier by bradykinin (BK), we established a primary culture of rat brain microvascular endothelial cells (BMECs). BMECs were treated with 10^?5 M BK, and changes in the intracellular Ca²⁺ levels were measured by using the sensitive fluorescent dye fluo‐3; the expression and distribution of claudin‐5 were investigated by immunocytochemistry and Western blot analyses. We did not detect any expression of bradykinin B2 receptors in the BMECs or freshly isolated rat brain microvessels. We found that 10^?5 M BK triggered Ca²⁺ transients in BMECs, and further investigations revealed that inositol 1,4,5‐trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs) on the endoplasmic reticulum (ER) were responsible for the Ca²⁺ fluctuation. Consequently, these intracellular Ca²⁺ changes that occur in response to BK application were identified as Ca²⁺‐induced Ca²⁺ release (CICR). Immunocytochemistry and Western blot results demonstrated that 10^?5 M BK could cause the internalization and a decrease in the expression of claudin‐5; agonists of IP₃Rs and RyRs, such as IP₃ and caffeine, enhanced the BK‐induced downregulation of claudin‐5, whereas antagonists of IP₃Rs and RyRs, such as 2‐APB and ryanodine, abrogated BK's effect on claudin‐5. In conclusion, the BK‐induced CICR in primary culture BMECs might be the mechanism by which BK modulates claudin‐5. © 2014 Wiley Periodicals, Inc.     

Differential Expression of Markers and Activities in a Group of PC12 Nerve Cell Clones

Sixteen clones, recently isolated from the PC12 nerve cell line, were analysed for a variety of markers and activities. Two endoplasmic reticulum (ER) luminal markers, the chaperone protein BiP and the major Ca²⁺ storage protein calreticulin, as well as the 40-kD rough ER membrane marker and the plus-end-directed mirotubule motor protein, kinesin, were found to be expressed at similar levels. These results suggest that the size of the ER, the function of microtubules and the capacity of the rapidly exchanging Ca²⁺ store do not change substantially among the clones. Other proteins expressed at comparable levels were synapsin I and IIa, members of a nerve cell-specific protein family known to bind synaptic vesicles to the cytoskeleton. In contrast, another ER membrane protein, calnexin, and the markers of secretory organelles were found to vary markedly. One clone (clone 27) completely lacked both chromogranin B and secretogranin II, the proteins contained within dense granules, and synaptophysin, a marker of clear vesicles. Other clones expressed these markers to variable and apparently mutually unrelated levels. Marked variability was observed also in the uptake of exogenous catecholamines, in their release both at rest and after stimulation, and in nerve growth factor-induced differentiation. These results provide indirect information about the mechanisms that regulate the expression of structures and activities in PC12 cells. Of particular interest is clone 27, which appears globally incompetent for regulated secretion and might therefore be a valuable tool for the study of this activity in a nerve cell.     

Depolarization amplitude and Ca2+-inflow control the time course of quantal releases at murine motor nerve terminals

In order to test whether the time courses of quantal releases after a depolarization pulse are affected by the depolarization amplitude, time courses were measured for small depolarization pulses that elicited close to threshold releases and for large depolarizations that elicited releases approaching saturation level. Diaphragms of young mice were excised and superfused with Bretag's solution at 18°C. Synaptic currents were elicited and recorded through a perfused macropatch pipette. Releases elicited by threshold depolarizations rose earlier than releases elicited by saturation depolarizations. The short delays in the rising phases of release after large depolarizations may be due to the shift of Ca²⁺ currents flowing during the pulse to tail currents. After its peak, release decayed with a time constant τ. For saturation depolarizations τ was about 0.3 ms, and for threshold depolarizations τ increased up to 0.8 ms. In order to differentiate between the effects of variations in Ca²⁺ inflow and in depolarization, the amplitudes of large depolarization pulses were held constant while the amount of release was depressed by halving the Ca²⁺ concentration at the terminal. The time course of the lowered releases was slightly delayed while τ remained at 0.3 ms as typical for saturation depolarizations. Double pulse facilitation unexpectedly revealed a short phase of depression of release after the pulse. This depression may contribute to the rapid decay (τ) of release after large depolarizations. The dependence of τ on depolarization amplitude indicates that the final phase of the time course of release is largely controlled by the amplitude of the preceding depolarization.