RESUMO
Fast neuromodulatory effects of 17-ß-estradiol (E2) on cytosolic calcium concentration ([Ca(2+)](i)) have been reported in many cell types, but little is known about its direct effects on vesicular neurotransmitter secretion (exocytosis). We examined the effects of E2 on depolarization-evoked [Ca(2+)](i) in PC12 cells using fluorescence measurements. Imaging of [Ca(2+)](i) with FURA-2 revealed that depolarization-evoked calcium entry is inhibited after exposure to 10 nM and 10 µM E2. Calcium entry after exposure to 50 µM E2 decreases slightly, but insignificantly. To relate E2-induced changes in [Ca(2+)](i) to functional effects, we measured exocytosis using amperometry. It was observed that E2 in some cells elicits exocytosis upon exposure. In addition, E2 inhibits depolarization-evoked exocytosis with a complex concentration dependence, with inhibition at both physiological and pharmacological concentrations. This rapid inhibition amounts to 45% at a near physiological level (10 nM E2), and 50% at a possible pharmacological concentration of 50 µM. A small percentage (22%) of cells show exocytosis during E2 exposure ("Estrogen stimulated"), thus vesicle depletion could possibly account (at least partly) for the E2-induced inhibition of depolarization-evoked exocytosis. In cells that do not exhibit E2-stimulated release ("Estrogen quiet"), the E2-induced inhibition of exocytosis is abolished by a treatment that eliminates the contribution of N-type voltage-gated calcium channels (VGCCs) to exocytosis. Overall, the data suggest that E2 can act on N-type VGCCs to affect secretion of neurotransmitters. This provides an additional mechanism for the modulation of neuronal communication and plasticity by steroids.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Estradiol/farmacologia , Exocitose/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Animais , Cálcio/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Células PC12 , RatosRESUMO
Glucocorticoids exert an opposing rapid regulation of glutamate and GABA synaptic inputs to hypothalamic magnocellular neurons via the activation of postsynaptic membrane-associated receptors and the release of retrograde messengers. Glucocorticoids suppress synaptic glutamate release via the retrograde release of endocannabinoids and facilitate synaptic GABA release via an unknown retrograde messenger. Here, we show that the glucocorticoid facilitation of GABA inputs is due to the retrograde release of neuronal nitric oxide and that glucocorticoid-induced endocannabinoid synthesis and nitric oxide synthesis are mediated by divergent G-protein signaling mechanisms. While the glucocorticoid-induced, endocannabinoid-mediated suppression of glutamate release is dependent on activation of the G(alpha)s G-protein subunit and cAMP-cAMP-dependent protein kinase activation, the nitric oxide facilitation of GABA release is mediated by G(beta)gamma signaling that leads to activation of neuronal nitric oxide synthase. Our findings indicate, therefore, that glucocorticoids exert opposing rapid actions on glutamate and GABA release by activating divergent G-protein signaling pathways that trigger the synthesis of, and glutamate and GABA synapse-specific retrograde actions of, endocannabinoids and nitric oxide, respectively. The simultaneous rapid stimulation of nitric oxide and endocannabinoid synthesis by glucocorticoids has important implications for the impact of stress on the brain as well as on neural-immune interactions in the hypothalamus.
Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Inibidores Enzimáticos/farmacologia , Glucocorticoides/farmacologia , Ácido Glutâmico/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Arginina/farmacologia , Moduladores de Receptores de Canabinoides/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dexametasona/farmacologia , Dronabinol/análogos & derivados , Dronabinol/farmacologia , Estimulação Elétrica/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Hipotálamo/citologia , Técnicas In Vitro , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Doadores de Óxido Nítrico/farmacologia , Técnicas de Patch-Clamp/métodos , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Piperidinas/farmacologia , Piranos/farmacologia , Pirazóis/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Rimonabanto , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Tionucleotídeos/farmacologia , Valina/análogos & derivados , Valina/farmacologiaRESUMO
MN9D cells have been used as a successful model to investigate dopamine pharmacology and to test the specific effects of drugs for the treatment of Parkinson's disease. However, quantitative measurements of quantal release from these cells have not been carried out. In this work, we used amperometry to investigate catecholamine release from MN9D cells. Amperometric events were observed in both undifferentiated and differentiated (butyric acid-treated) cells. An increase in quantal size and half-width was observed for differentiated cells versus undifferentiated cells; however, the number of events per cell and the amplitude remained constant. In transmission electron microscopy images, no obvious cluster of small synaptic vesicles was observed, and large dense-core vesicles were present in the cell body of undifferentiated cells; however, after differentiation, vesicles were concentrated in the cell processes. In differentiated cells, l-DOPA caused an increase in quantal size and half-width, which could be blocked by the vesicular monoamine transporter inhibitor, reserpine.
Assuntos
Catecolaminas/análise , Diferenciação Celular/fisiologia , Linhagem Celular Transformada/metabolismo , Técnicas Eletroquímicas/métodos , Inibidores da Captação Adrenérgica/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada/ultraestrutura , Dopaminérgicos/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Levodopa/farmacologia , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Cloreto de Potássio/farmacologia , Reserpina/farmacologia , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestruturaRESUMO
We have amperometrically measured dopamine release from rat pheochromocytoma cells (PC12 cells) in high osmolarity conditions with and without L-3,4-dihydroxyphenylalanine (L-DOPA) treatment. We observe an increase in the number of release events displaying a prespike feature or "foot" when the cells are stimulated in high osmolarity saline. We also see an increase in foot area and duration when cells are stimulated in high osmolarity saline, or high osmolarity saline subsequent to incubation with the dopamine precursor L-DOPA in isotonic saline, which serves to increase the vesicle size. The data suggest that membrane biophysics are an important component in defining the rate, duration and amount of neurotransmitter release via the fusion pore.
Assuntos
Estruturas da Membrana Celular/efeitos dos fármacos , Estruturas da Membrana Celular/metabolismo , Dopamina/metabolismo , Levodopa/farmacologia , Animais , Fenômenos Biofísicos , Biofísica , Exocitose/efeitos dos fármacos , Modelos Biológicos , Concentração Osmolar , Células PC12 , Porosidade/efeitos dos fármacos , RatosRESUMO
AIM: A fraction of vesicles in cells treated with hypertonic solution exhibit multiple dense cores and this is enhanced by treatment with L-3,4-dihydroxyphenylalanine (L-DOPA). These cells were examined to determine if the multicore vesicles are the product of endocytosis or homotypic fusion. METHODS: Electron microscopy was used to determine the number of multicore vesicles and amperometry was used to examine if the multicore vesicles are a competent fraction of the readily releasable pool. RESULTS: In this study, we observed that a substantial portion (15.3%) of large dense core vesicles in PC12 cells contained multiple cores in hypertonic saline loaded with L-DOPA, and amperometric measurements show a bimodal distribution of quantal sizes in treated cells. CONCLUSIONS: The results suggest that the multicored vesicles are formed from homotypic fusion of LCDVs prior to exocytosis.
Assuntos
Dopaminérgicos/farmacologia , Levodopa/farmacologia , Fusão de Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Vesículas Secretórias/efeitos dos fármacos , Animais , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Microscopia Eletrônica de Transmissão , Neurônios/ultraestrutura , Concentração Osmolar , Células PC12 , Ratos , Solução Salina Hipertônica/farmacologia , Vesículas Secretórias/ultraestruturaRESUMO
Membrane composition serves to identify intracellular compartments, signal cell death, as well as to alter a cell's electrical and physical properties. Here we use amperometry to show that supplementation with the phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and phosphatidylserine (PS) can alter several aspects of exocytosis. Changes in the amperometric peak shape derived from individual exocytosing vesicles reveal that PC slows expulsion of neurotransmitter while PE accelerates expulsion of neurotransmitter. Amperometry data reveal a reduced amount of catecholamine released per event from PC-treated cells while electron micrographs indicate the vesicles in these cells are 50% larger than controls, thus providing evidence of pharmacological changes in vesicle concentration. Addition of SM appears to affect the rate of fusion pore expansion, indicated by slower peak rise times, but does not affect decay times or quantal size. Addition of PS results in a 1.7-fold increase in the number of events elicited by high-K(+) depolarization. Electron micrographs of PS-treated cells suggest that increased vesicle recruitment underlies enhanced secretion. We did not observe any effect of phosphatidylinositol (PI) treatment. Together these data suggest that differences in membrane composition affect exocytosis and might be involved in mechanisms of cell function controlling the dynamics of communication via exocytosis.