Isolated Schwannoma of the Olfactory Groove: A Case Report

Introduction Schwannoma of the olfactory groove is an extremely rare tumor that can share a differential diagnosis with meningioma or neuroblastoma. Objectives The authors present a case of giant schwannoma involving the anterior cranial fossa and ethmoid sinuses. Case Report The patient presented with a 30-month history of left nasal obstruction, anosmia, and sporadic ipsilateral bleeding. Computed tomography of the paranasal sinuses revealed expansive lesion on the left nasal cavity extending to nasopharynx up to ethmoid and sphenoid sinuses bilaterally with intraorbital and parasellar extension to the skull base. Magnetic resonance imaging scan confirmed the expansive tumor without dural penetration. Biopsy revealed no evidence of malignancy and probable neural cell. Bifrontal craniotomy was performed combined with lateral rhinotomy (Weber-Ferguson approach), and the lesion was totally removed. The tumor measured 8.0 4.3 3.7 cm and microscopically appeared as a schwannoma composed of interwoven bundles of elongated cells (Antoni A regions)mixed with less cellular regions (Antoni B). Immunohistochemical study stained intensively for vimentin and S-100. Conclusion Schwannomas of the olfactory groove are extremely rare, and the findings of origin of this tumor is still uncertain but recent studies point most probably to the meningeal branches of trigeminal nerve or anterior ethmoidal nerves. .

Airborne agricultural particulate matter induces inflammatory cytokine secretion by respiratory epithelial cells: Mechanisms of regulation by eicosanoid lipid signal mediators.

The purpose of this study was to elucidate the mechanism of the airborne poultry dust (particulate matter, PM)-induced respiratory tract inflammation, a common symptom in agricultural respiratory diseases. The study was based on the hypothesis that poultry PM would induce the release of inflammatory cytokine interleukin-8 (IL-8) by respiratory epithelial cells under the upstream regulation by cytosolic phospholipase A2 (cPLA2) activation and subsequent formation of cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites (eicosanoids). Human lung epithelial cells (A549) in culture were treated with the poultry PM (0.1-1.0 mg) for different lengths of time, following which PLA2 activity, release of eicosanoids and secretion of IL-8 in cells were determined. Poultry PM (1.0 mg/ml) caused a significant activation of PLA2 in a time-dependent manner (15-60 min), which was significantly attenuated by the calcium-chelating agents, cPLA2-specific inhibitor (AACOCF3) and antioxidant (vitamin C) in A549 cells. Poultry PM also significantly induced the release of COX- and LOX-catalyzed eicosanoids (prostaglandins, thromboxane A2 and leukotrienes B4 and C4) and upstream activation of AA LOX in the cells. Poultry PM also significantly induced release of IL-8 by the cells in a dose- and time-dependent manner, which was significantly attenuated by the calcium chelating agents, antioxidants and COX- and LOX-specific inhibitors. The current study for the first time revealed that the poultry PM-induced IL-8 release from the respiratory epithelial cells was regulated upstream by reactive oxygen species, cPLA2-, COX- and LOX-derived eicosanoid lipid signal mediators.

High susceptibility of activated lymphocytes to oxidative stress-induced cell death

The present study provides evidence that activated spleen lymphocytes from Walker 256 tumor bearing rats are more susceptible than controls to tert-butyl hydroperoxide (t-BOOH)-induced necrotic cell death in vitro. The iron chelator and antioxidant deferoxamine, the intracellular Ca2+ chelator BAPTA, the L-type Ca2+ channel antagonist nifedipine or the mitochondrial permeability transition inhibitor cyclosporin A, but not the calcineurin inhibitor FK-506, render control and activated lymphocytes equally resistant to the toxic effects of t-BOOH. Incubation of activated lymphocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. These results indicate that the higher cytosolic Ca2+ level in activated lymphocytes increases their susceptibility to oxidative stress-induced cell death in a mechanism involving the participation of mitochondrial permeability transition.

O presente estudo demonstra que linfócitos ativados de baço de ratos portadores do tumor de Walker 256 são mais susceptíveis à morte celular necrótica induzida por tert-butil hidroperóxido (t-BOOH) in vitro quando comparados aos controles. O quelante de ferro e antioxidante deferoxamina, o quelante intracelular de Ca2+ BAPTA, o antagonista de canal de Ca2+ nifedipina ou o inibidor da transição de permeabilidade mitocondrial ciclosporina-A, mas não o inibidor de calcineurina FK-506, inibiram de maneira similar a morte celular induzida por t-BOOH em linfócitos ativados e controles. Os linfócitos ativados apresentaram redução do potencial de membrana mitocondrial induzida por t-BOOH num mecanismo sensível a ciclosporina-A. Nossos resultados indicam que o aumento da concentração de Ca2+ citosólico em linfócitos ativados aumenta a susceptibilidade dos mesmos à morte celular induzida por estresse oxidativo, num mecanismo envolvendo a participação do poro de transição de permeabilidade mitocondrial.

Arachidonic acid is a chemoattractant for Dictyostelium discoideum cells.

Cyclic AMP (cAMP)is a natural chemoattractant of the social amoeba Dictyostelium discoideum. It is detected by cell surface cAMP receptors. Besides a signalling cascade involving phosphatidylinositol 3,4,5-trisphosphate (PIP3), Ca2+ signalling has been shown to have a major role in chemotaxis. Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca2+ concentration by causing the release of Ca2+ from intracellular stores and activating influx of extracellular Ca2+. Here we report that AA is a chemoattractant for D. discoideum cells differentiated for 8-9 h. Motility towards a glass capillary filled with an AA solution was dose-dependent and qualitatively comparable to cAMP-induced chemotaxis. Ca2+ played an important role in AA chemotaxis of wild-type Ax2 as ethyleneglycol-bis(b-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) added to the extracellular buffer strongly inhibited motility. In the HM1049 mutant whose iplA gene encoding a putative Ins(1,4,5)P3 -receptor had been knocked out, chemotaxis was only slightly affected by EGTA. Chemotaxis in the presence of extracellular Ca2+ was similar in both strains. Unlike cAMP, addition of AA to a cell suspension did not change cAMP or cGMP levels. A model for AA chemotaxis based on the findings in this and previous work is presented.

Smear layer removal and chelated calcium ion quantification of three irrigating solutions

The purpose of this study was to evaluate, by scanning electron microscopy (SEM), smear layer removal and quantify, by atomic absorption spectrophotometry, the amount of calcium ion present in the chelating solutions after their use. Sixteen extracted canines were instrumented using the step-back technique and were assigned to 3 groups according to the irrigating solution used: G1: 1 mL 17 percent EDTAC between each file; G2: 1 mL 17 percent CDTA; G3: 1 mL 17 percent EGTA. The solutions were collected after use. The teeth were cleaved longitudinally, evaluated under SEM and assessed for smear layer by blinded examiners and scored from 1 to 4. In order to quantify calcium ion release, the collected solutions were examined by atomic absorption spectrophotometry. Freidman's test was used for statistical analysis of SEM values and showed that canals irrigated with 17 percent EDTAC and 17 percent CDTA had significantly less smear layer throughout the canals than 17 percent EGTA (p<0.01). For analysis of the collected solutions, Tukey's test was used and showed that EDTAC and CDTA had a greater amount of calcium ions (22.8±7.54 and 60.6±20.67 æg/mL, respectively) compared to EGTA (70.5±14.2 æg/mL) (p<0.01). The association both methodologies may contribute to the understanding of how these solutions act in the root canal.

O objetivo do presente estudo foi avaliar a remoção de smear layer por meio da microscopia eletrônica de varredura (MEV) e quantificar a liberação de íons cálcio resultante da irrigação com as soluções quelantes estudadas, por meio da espectrofotometria de absorção atômica. Dezesseis caninos extraídos foram instrumentados com a técnica step-back e divididos em 3 grupos de acordo com a solução irrigadora utilizada: G1: 1 mL de EDTAC a 17 por cento entre cada lima; G2: CDTA a 17 por cento; e G3: EGTA a 17 por cento. As soluções foram coletadas após o uso. Os dentes foram secionados longitudinalmente e as raízes examinadas por MEV para verificação de smear layer nos terços por meio de escores (variando de 1 a 4), e avaliadas por três examinadores calibrados "cegos". Para quantificar a liberação de íons cálcio, as soluções coletadas foram avaliadas por espectrofotometria de absorção atômica. Com relação ao smear layer, o teste de Friedman evidenciou diferença estatisticamente significante (p<0,01) comparando-se o EGTA 17 por cento ao EDTAC 17 por cento e ao CDTA 17 por cento, sendo que os canais irrigados com estas duas soluções apresentaram menor quantidade de smear layer que aqueles irrigados com EGTA. As soluções de EDTAC 17 por cento (70,5±14,2 æg/mL Ca) e CDTA 17 por cento (60,6±20,67 æg/mL Ca) apresentaram maiores quantidades de íons cálcio (p<0,01) quando comparadas ao EGTA 17 por cento (22,8±7,54 æg/mL Ca). Desta forma, pode-se concluir que a associação destas metodologias pode contribuir para o entendimento da ação das soluções quelantes no interior dos canais radiculares.

Demineralization effect of EDTA, EGTA, CDTA and citric acid on root dentin: a comparative study / Efeito do EDTA, EGTA, CDTA e ácido cítrico na desmineralização da dentina radicular: estudo comparativo

Este trabalho teve como objetivo comparar o efeito desmineralizante do EDTA (pH 7,4), EGTA (pH 7,4), CDTA (pH 7,4), ácido cítrico (pH 1,0 e 7,4) e da solução salina (controle) sobre a dentina radicular. Todas as soluções teste foram preparadas na concentração de 1%. Quarenta e oito dentes unirradiculares recém-extraídos foram utilizados neste experimento. Após a instrumentação dos canais radiculares pela técnica "step-back", as raízes foram aleatoriamente divididas em 6 grupos experimentais (n = 8) de acordo com a solução teste utilizada na irrigação final. Em cada grupo, 30 µL da solução teste foram pipetados no interior de cada canal radicular e mantidos estáveis por 5 minutos. Decorrido esse período, 15 µL da solução foram removidos do canal e depositados em frasco contendo 5 mL de água deionizada. A concentração de Ca2+ (µg/mL) extraída dos espécimes foi determinada pela espectrometria de absorção de massa (ICP-AES); e os dados foram submetidos à análise estatística pelos testes de Kruskal-Wallis e de mediana de Mood. O ácido cítrico em pH 1,0 foi a solução mais efetiva na remoção de Ca2+ comparativamente às demais soluções-teste (p < 0,05). Nenhuma diferença estatística foi observada entre a ação do EDTA e a do EGTA. Ambos os quelantes removeram significantemente mais Ca2+ que o CDTA e o ácido cítrico em pH 7,4 (p < 0,05). Não houve diferença significante entre ácido cítrico em pH 7,4 e solução salina (p > 0,05). Os resultados deste estudo indicam que o ácido cítrico em solução de pH 1,0 apresenta-se como uma boa opção para remover a "smear layer" e facilitar o preparo biomecânico do sistema de canal radicular.

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.

Effect of different EGTA concentrations on dentin microhardness

The effect of 1 percent, 3 percent and 5 percent EGTA (ethylene glycol-bis-(b-amino-ethyl ether) N,N,N´,N´-tetra-acetic acid) on the microhardness of root dentin of the cervical third of human teeth was studied. Five newly extracted maxillary incisors were sectioned transversely at the cementoenamel junction, and the crowns were discarded. The roots were embedded in blocks of high-speed polymerized acrylic resin and cut transversely into 1-mm sections. The second section of the cervical third of the root of each tooth was sectioned and divided into four parts. Each part was placed on an acrylic disc that was used as a base for microhardness measurement. Fifty microliters of 1 percent EGTA, 3 percent EGTA, or 5 percent EGTA were applied to the dentin surface. Deionized and distilled water was used as control. Dentin microhardness was then measured with a load of 50 g for 15 s. Statistical analysis showed that the three concentrations of the chelating solution EGTA significantly reduced dentin microhardness when compared with water (ANOVA, p<0.01), and that there was a statistically significant difference among the three solutions (Tukey test, p<0.05)

The calcium chelator BAPTA affects the binding of assembly protein AP-2 to membranes

Clathrin coated vesicles are involved in receptor-mediated transport. The coat of these vesicles is constituted mostly of clathrin and the assembly proteins AP-1 or AP-2. In the present study using an in vitro binding system, we found that the interaction of AP-2 but not AP-1 with membranes diminished when the calcium chelating agent BAPTA was added. The maximal inhibitory effect was observed with 10 mM of the chelating agent. Binding of AP-2 to membranes was recovered by adding calcium in a concentration-dependent fashion. Binding was also affected when the membranes were previously treated with BAPTA and then washed. However, other chelating agents such as EDTA or EGTA, as well as the zinc chelating TPEN, did not have any effect on the binding. From these results we postulate a role for calcium in regulating the assembly-disassembly cycle of adaptors in the formation of clathrin coated vesicles.

Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.

Effects of cations on ceramide-activated protein phosphatase 2A

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.

Effects of cations on ceramide-activated protein phosphatase 2A

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.

Protective mechanism of glucose against alloxan-induced beta-cell damage: pivotal role of ATP

Glucose prevents the development of diabetes induced by alloxan. In the present study, the protective mechanism of glucose against alloxan-induced beta-cell damage was investigated using HIT-T 15 cell, a Syrian hamster transformed beta-cell line. Alloxan caused beta-cell damages with DNA fragmentation, inhibition of glucose-stimulated insulin release, and decrease of cellular ATP level, but all of these beta-cell damages by alloxan were prevented by the presence of 20 mM glucose. Oligomycin, a specific inhibitor of ATP synthase, completely abolished the protective effects of glucose against alloxan-induced cell damage. Furthermore, treatment of nuclei isolated from HIT-T15 cells with ATP significantly prevented the DNA fragmentation induced by Ca2+. The results indicate that ATP produced during glucose metabolism plays a pivotal role in the protection of glucose against alloxan-induced beta-cell damage.

Avaliaçäo da biocompatibilidade do EDTA, EGTA e ácido cítrico pela técnica de exsudaçäo de corantes vitais / Evaluation of enhanced vascular permeability of EDTA, EGTA and citric acid

As reaçöes teciduais induzidas por alguns ácidos utilizados na prática endodôntica, foram estudadas durante a fase exsudativa do processo inflamatório. Injetou-se, intravenosamente na veia caudal lateral de 32 ratos machos de linhagem Wistar, variaçäo albina, pesando 380-400 gramas, 20mg/Kg de azul de Evans 2 por cento. No tecido subcutâneo, na regiäo dorsal, foram inoculadas as drogas selecionadas para o teste: EDTDA 15 por cento pH 7,4, EGTA 15 por cento pH 7,4, ácido cítrico 15 por cento pH 1,0 e soro fisiológico (controle). Após intervalos de 30 minutos, 1, 3 e 6 horas, os animais foram sacrificados, suas peles dorsais excisadas e submetidas ao processo de remoçäo e análise do corante extravasado pela espectrofotometria de abdorçäo de luz (620nm). Os valores em µg da quantidade de corante extravasado foram submetidos à estatística pela análise de variância a 2 critérios e, posteriormente, ao teste de Tukey para comparaçöes individais. Concluiu-se que a ordem decrescente da média de potencial irritativo das substâncias avaliadas, foram: EDTA (1447,33µg), EGTA (770,59µg), ácido cítrico (329,81µg) e soro fisiológico (139,55µg) sendo observadas diferenças estatisticamente significantes (p<0,01) entre todos os grupos. Com relaçäo ao fator tempo, foi notada diferença estatisticamente significante (p<0,05) apenas entre os grupos de 3 horas (594,29µg) e 6 horas (835,89µg). Comparando com o soro fisiológico (controle), o ácido cítrico foi a substância menos irritante, independente do tempo analisado

Changes in the expression of c-myc, RB and tyrosine-phosphorylated proteins during proliferation of NIH 3T3 cells induced by hyaluronic acid

We have shown that hyaluronic acid stimulates the proliferation of quiescent NIH 3T3 cells. We have shown that treatment of 1 mg/ml hyaluronic acid results in increase of tyrosine phosphorylation of two proteins, MW 124 kDa and 60 kDa as detected by anti-tyrosine antibodies by Western blot analysis. Maximum phosphorylation occurred within 2 h after addition of 1 mg/ml hyaluronic acid. Stimulation of proliferation was also accompanied by increase in c-Myc protein, which was inhibited by amlloride, an inhibitor of Na+/H+ antiporter and EGTA and increase in the steady state level of pRb, the RB gene product. These results suggest that the intracellular signal transduction pathways that mediate the stimulatory effects of hyaluronic acid on cellular proliferation are similar to those of growth factors.

Role of Ca2+ influx in the tert-butyl hydroperoxide-induced apoptosis of HepG2 human hepatoblastoma cellse

Oxidative stress appears to be implicated in the pathogenesis of various diseases including alcoholic liver injury. In this study we investigated the mechanism of apoptosis induced by tert-butyl hydroperoxide (TBHP) in HepG2 human hepatoblastoma cells. Treatment with TBHP significantly reduced glutathione content and glutathione reductase activity, and increased glutathione peroxidase activity, indicating that TBHP induced oxidative stress in the HepG2 cells. TBHP also induced reduction of cell viability and DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, TBHP induced a sustained increase in intracellular Ca2+ concentration, which was completely prevented by the extracellular Ca2+ chelation with EGTA. TBHP also induced Mn2+ influx. These results indicate that the intracellular Ca2+ increase by TBHP is exclusively due to Ca2+ influx from the extracellular site. Treatment with either an extracellular (EGTA) or an intracellular Ca2+ chelator (BAPTA/AM) significantly suppressed the TBHP-induced apoptosis. Taken together, these results suggest that TBHP induced the apoptotic cell death in the HepG2 cells and that Ca2+ influx may play an important role in the apoptosis induced by TBHP.

Ca-independent augmentation of endplate potential during repetitive stimulation

Con objetivo de elucidar la función del Ca intracelular en la transmisión neuromuscular investigamos en preparaciones de músculo de rana los efectos del ácido 1,2-bis (o-aminofenoxi)etano-N,N,N',N'-tetraacético (BAPTA) sobre el aumento-potenciación por frecuencia (anteriormente llamdo facilitación por frecuencia) el que ha sido de utilidad para identificar-los sitios de acción de varios agentes colinérgicos. La disminución de los iones Ca del espacio intracelular por BAPTA sólo suprimió el componente dependiente de Ca del fenómeno (mo) sin modificar el factor de estimulación dependiente de frecuencia (K). La depresión causada por BAPTA en la facilitación de corto plazo del potencial de placa (EPP) fue la misma tanto en reposo como en la estimulación. El efecto del BAPTA fue parcialmente antagonizado, por el ionóforo de Ca A23187. Esto sugiere que la capacidad de "buffer" de Ca del BAPTA se mantiene durante la estimulación repetitiva de baja frecuencia. BAPTA no modificó la potenciación post-tetánica de los EPP miniatura en medio libre de Ca. Estos resultados indican que los iones Ca son esenciales para la liberación de transmisor y para la facilitación de corto plazo, pero no son responsables de todos los cambios en la liberación de transmisor