RESUMO
AIM: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS: Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION: We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.
Assuntos
Sinalização do Cálcio/fisiologia , Células Cromafins/fisiologia , Exocitose/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Feminino , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp/métodosRESUMO
Ca²âº influx across the plasma membrane upon drastic reduction of the sarcoplasmic reticulum Ca²âº content was studied in voltage clamped frog skeletal muscle fibers. Depletion was produced by the application of 30 µM cyclopiazonic acid (CPA) in Ca²âº-free, [Mg²âº] = 8 mM external salines and produced an increase in resting free myoplasmic [Ca²âº]. Once depletion was attained the external solution was changed to one containing the same concentration of the drug but with Ca²âº instead of Mg²âº. Of 27 fibers studied only nine showed a secondary increase in free myoplasmic [Ca²âº] upon readmitting Ca²âº in the external perfusate. In the presence of CPA the resting myoplasmic [Ca²âº] in Ca²âº-free external saline was 0.08 ± 0.01 µM (Mean ± SEM), and in Ca²âº-containing external saline 0.10 ± 0.02 µM when the intracellular solution contained [EGTA] = 5 mM (n = 18). In cells with lower (0.5 mM) intracellular [EGTA] resting [Ca²âº] went from 0.35 +/- 0.08 µM in Ca²âº-free external solution to 0.42 +/- 0.12 µM upon reapplication of Ca²âº(n = 9). In both cases the differences between means were not statistically significant (paired t test, p = 0.13 in high EGTA and p = 0.25 in low EGTA). In the nine fibers that showed a secondary increase of resting [Ca²âº] the holding current measured at -90 mV did not significantly change. These results suggest the Ca²âº entry secondary to store depletion is a labile mechanism in frog skeletal muscle and when present does not have an obvious electrical manifestation.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Músculo Esquelético/metabolismo , Rana catesbeiana/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Ácido Egtázico/metabolismo , Fenômenos Eletrofisiológicos , Técnicas In Vitro , Indóis/farmacologia , Manganês/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Técnicas de Patch-Clamp , Rana catesbeiana/fisiologia , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/fisiologia , Fatores de TempoRESUMO
The plasma membrane Ca2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca2+ at a rate near 1.5% of that attained at saturating concentrations of Ca2+. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca2+-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca2+ and is catalyzed by E(2)-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Animais , Membrana Celular/metabolismo , Ácido Egtázico/metabolismo , Eritrócitos/enzimologia , Hidrólise , SuínosRESUMO
The medium from cultured optic tectum of the goldfish intact or at various days after optic nerve crush, or the co-culture of this optic tectum with post-crush goldfish retinal explants plating in the absence of fetal calf serum, but in the presence of glucose, modulate its outgrowth. The a dition of taurine did not further stimulate outgrowth but rather inhibited it in the presence of optic tectum. These processes were related to calcium fluxes and taurine transport into the cells. Taurine increased the length of neurites from 5-day-old rat retinal explants in the presence of fetal calf serum. The goldfish optic tectum, either medium or in co-culture with the retina, stimulated retinal outgrowth. The study of optic nerve regeneration in the presence of defined media contributes to understanding tissue-target and interspecies interaction.
Assuntos
Meios de Cultura/química , Carpa Dourada/anatomia & histologia , Retina/metabolismo , Colículos Superiores/metabolismo , Taurina/metabolismo , Técnicas de Cultura de Tecidos , Animais , Quelantes/metabolismo , Técnicas de Cocultura , Ácido Egtázico/metabolismo , Carpa Dourada/metabolismo , Nervo Óptico/patologia , Nervo Óptico/cirurgia , RatosRESUMO
We have previously described that alpha-ketoisocaproic acid (KIC), the main metabolite accumulating in maple syrup urine disease (MSUD), increased the in vitro phosphorylation of cytoskeletal proteins in cerebral cortex of 17- and 21-day-old rats through NMDA glutamatergic receptors. In the present study we investigated the protein kinases involved in the effects of KIC on the phosphorylating system associated with the cytoskeletal fraction and provided an insight on the mechanisms involved in such effects. Results showed that 1 mM KIC increased the in vitro incorporation of 32P into intermediate filament (IF) proteins in slices of 21-day-old rats at shorter incubation times (5 min) than previously reported. Furthermore, this effect was prevented by 10 microM KN-93 and 10 microM H-89, indicating that KIC treatment increased Ca2+/calmodulin- (PKCaMII) and cAMP- (PKA) dependent protein kinases activities, respectively. Nifedipine (100 microM), a blocker of voltage-dependent calcium channels (VDCC), DL-AP5 (100 microM), a NMDA glutamate receptor antagonist and BAPTA-AM (50 microM), a potent intracellular Ca2+ chelator, were also able to prevent KIC-induced increase of in vitro phosphorylation of IF proteins. In addition, KIC treatment was able to significantly increase the intracellular cAMP levels. This data support the view that KIC increased the activity of the second messenger-dependent protein kinases PKCaMII and PKA through intracellular Ca2+ levels. Considering that hyperphosphorylation of cytoskeletal proteins is related to neurodegeneration it is presumed that the Ca2+-dependent hyperphosphorylation of IF proteins caused by KIC may be involved to the neuropathology of MSUD patients.
Assuntos
Cálcio/metabolismo , Córtex Cerebral/metabolismo , AMP Cíclico/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Cetoácidos/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/metabolismo , Quelantes/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Técnicas In Vitro , Nifedipino/metabolismo , Radioisótopos de Fósforo/metabolismo , Fosforilação , Ratos , Ratos WistarRESUMO
Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca(2+) concentration ([Ca(2+)](i)) using double-barreled, Ca(2+)-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca(2+)](i) was approximately twofold in MHS compared with MHN quiescent myoballs (232 +/- 35 vs. 112 +/- 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca(2+)](i) in both groups; however, the concentration required to cause a rise in [Ca(2+)](i) elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca(2+) buffer BAPTA reduced [Ca(2+)](i), raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca(2+)](i), and reduced the amount of Ca(2+) release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca(2+)](i) levels in these cells.
Assuntos
Cafeína/farmacologia , Cálcio/metabolismo , Cresóis/farmacologia , Ácido Egtázico/análogos & derivados , Hipertermia Maligna/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Animais , Animais Recém-Nascidos , Estimulantes do Sistema Nervoso Central/farmacologia , Quelantes/metabolismo , Ácido Egtázico/metabolismo , Eletrofisiologia , Fungicidas Industriais/farmacologia , Potenciais da Membrana/fisiologia , Microeletrodos , SuínosRESUMO
In this study, we demonstrated that ADP-induced platelet aggregation activates the binding of testosterone (T) to its receptor. It is well known that binding of ADP to its receptors induced the release of Ca2+ ions from dense bodies into the cytosol of platelets. In this work, we compared the binding of testosterone or dihydrotestosterone to their receptors using cytosol obtained from ADP-treated and non-treated platelets. These experiments were repeated using EGTA (a calcium chelator) or U73122 (a phospholipase C enzymatic activity inhibitor) to the ADP-treated platelets. In addition, we also developed a competition analysis for the androgen receptors (AR) using [3H]DHT, non-radioactive T, DHT or cyproterone acetate from ADP-treated platelets cytosol. The results from this study indicate that the cytosol obtained from non-ADP-treated platelets did not show any binding to [3H]T or [3H]DHT, whereas cytosol from ADP-treated platelets binds to the radio-labeled androgens. Furthermore cytosol from ADP plus U73122-treated platelets did not show binding to [3H]T or [3H]DHT. These data suggest that intracellular Ca2+ ions stimulates the binding of androgens to their receptors in platelets cytosol. The competition analysis shows that T and DHT have high affinities for the androgen receptors with similar IC50 values, whereas cyproterone acetate shows a lower affinity. The results from these data clearly indicate the presence of androgen receptors in platelets.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Receptores Androgênicos/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Ligação Competitiva , Western Blotting , Cálcio/química , Citosol/metabolismo , Di-Hidrotestosterona/metabolismo , Relação Dose-Resposta a Droga , Ácido Egtázico/metabolismo , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Humanos , Immunoblotting , Íons , Ligação Proteica , Pirrolidinonas/farmacologia , Esteroides/química , Testosterona/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
It has been recognised that adherence and invasion to host cells are important steps in the pathogenesis of entero-pathogenic bacteria, including Aeromonas caviae. However, the virulence factors of A. caviae remain, for the most part, poorly known. This study examined the interaction of A. caviae isolates to Caco-2 cells in different polarisation and differentiation conditions. The adherence of A. caviae may be related to accessibility of host cell basolateral receptors. Aggregative A. caviae isolates, grown at 22 degrees C, were more adherent in both non-polarised and undifferentiated Caco-2 cells and EGTA-treated polarised and differentiated Caco-2 cells. Furthermore, monolayers pre-incubated with 43-kDa outer-membrane protein (OMP) or A. caviae strains pre-incubated with rabbit IgG anti-43-kDa OMP decreased adherence of some A. caviae strains to EGTA-treated polarised and differentiated Caco-2 cells, suggesting an interaction of 43-kDa OMP with basolateral cell receptors. Bacterial cells were observed adhering to microvilli and to plasma membrane on both the apical and basal surfaces of the monolayer. Pedestal-like formation with cytoskeletal rearrangement was also observed. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. Furthermore, A. caviae were observed free in the cytosol of Caco-2 cells, suggesting escape form cytoplasmatic vacuoles.
Assuntos
Aeromonas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Diferenciação Celular/fisiologia , Animais , Aderência Bacteriana/fisiologia , Proteínas da Membrana Bacteriana Externa/química , Células CACO-2/microbiologia , Células CACO-2/ultraestrutura , Polaridade Celular , Ácido Egtázico/metabolismo , Humanos , Junções Intercelulares/metabolismo , Peso Molecular , Ligação ProteicaRESUMO
In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.
Assuntos
Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Exocitose/fisiologia , Oócitos/efeitos dos fármacos , Ouriços-do-Mar/fisiologia , Venenos de Vespas/farmacologia , Animais , Quelantes/metabolismo , Ácido Egtázico/metabolismo , Exocitose/efeitos dos fármacos , Feminino , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Fusão de Membrana/fisiologia , Oócitos/fisiologia , Peptídeos , Espermatozoides/metabolismo , Venenos de Vespas/químicaRESUMO
Virulent and avirulent clones of Leishmania mexicana amazonensis promastigotes or amastigotes were loaded with the fluorescent reagent fura 2/AM to measure intracellular free calcium ([Ca2+]i). When the cells were treated with the calcium ionophore ionomycin in the nominal absence of extracellular Ca2+, there was an increase of [Ca2+]i that was further elevated by addition of either NH4Cl, nigericin, or the vacuolar H+-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in L. mexicana amazonensis promastigotes and amastigotes is present in an acidic compartment rich in Ca2+ (acidocalcisome). Results indicated that more releasable Ca2+ is stored intracellularly in virulent amastigotes than in virulent promastigotes or avirulent cells of both stages. This higher amount of releasable Ca2+ was correlated with the presence of Ca2+ signals in the virulent amastigotes during invasion of macrophages. Ca2+ signals and invasion were reduced by preloading the parasites with intracellular Ca2+ chelators (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca2+-chelating analog (N-(2-methoxyphenyl)imidoacetic acid/AM). The gene encoding an organelle-type Ca2+-ATPase was cloned and sequenced and found overexpressed in virulent amastigotes as compared with all other forms. Together, these results demonstrate a significant link between expression of a Ca2+-ATPase, intracellular Ca2+ pool content and signaling, and virulence.