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1.
Biofizika ; 57(2): 253-7, 2012.
Artículo en Ruso | MEDLINE | ID: mdl-22594281

RESUMEN

Formation of reactive oxygen species in rat brain synaptosomes was studied using DCFDA fluorescent dye at lowered extracellular pH. It has been shown that decrease in pH value from 7.4 to 7.0 and up to 6.0 leads to increase of fluorescence that is indicative of oxidative stress. The effect is observed regardless of whether Ca ions are present in incubation medium or no. Acidification of the incubation medium induces quenching of fluorescence of previously oxidized form of the dye in experiments without synaptosomes This evidences that increase of dye fluorescence is really associated with reactive oxygen species accumulation. Thus, it has been demonstrated that pH declined up to 7.0 in the incubation medium is sufficient to induce the formation of reactive oxygen species in synaptosomes.


Asunto(s)
Encéfalo/metabolismo , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sinaptosomas/metabolismo , Animales , Química Encefálica/fisiología , Fluoresceínas/química , Concentración de Iones de Hidrógeno , Ratas , Sinaptosomas/química
2.
Biofizika ; 57(3): 454-9, 2012.
Artículo en Ruso | MEDLINE | ID: mdl-22873069

RESUMEN

The inhibitor of Rho-kinase Y-27632 induces non-secretory exocytosis in PC12 cells. The influence of this compound on central synapses remains uninvestigated. We showed that Y-27632 at the concentration 100 jtM led to spontaneous [14C]glutamate release in synaptosomes, which was not accompanied by plasma membrane depolarization. Membrane potential was registered by fluorescent dye DiSC3(5). Y27632 induced an increase of acridine orange fluorescence, exercising no influence over fluorescence of FM2-10 dye. These results suggest that Rho-kinase inhibition decreases pH gradient of synaptic vesicles not inducing exocytosis. Dissipation of the gradient leads to leakage of neurotransmitters to cytosol pumping them out by plasma membrane transporters. Our results show the involvement of Rho-dependent branch of intracellular signaling in regulation of pH gradient in synaptic vesicles.


Asunto(s)
Amidas/farmacología , Encéfalo/efectos de los fármacos , Ácido Glutámico/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Sinaptosomas/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Benzotiazoles/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Carbocianinas/metabolismo , Exocitosis/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/efectos de los fármacos , Neurotransmisores/metabolismo , Células PC12 , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Ratas , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Sinaptosomas/metabolismo
3.
Biofizika ; 54(5): 876-80, 2009.
Artículo en Ruso | MEDLINE | ID: mdl-19894628

RESUMEN

The influence of glutamate and the agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes has been investigated using the fluorescent dye DCFDA. It was shown that glutamate at concentrations of 100 microM and 1 mM increases the synthesis of reactive oxygen species. This phenomenon was eliminated by removing calcium from incubation medium. The addition of NMDA (100 microM) and kainate (100 microM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, the activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.


Asunto(s)
Química Encefálica/efectos de los fármacos , Encéfalo/metabolismo , Radicales Libres/metabolismo , Ácido Glutámico/farmacología , Sinaptosomas/metabolismo , Animales , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Fluoresceínas/farmacología , Quinoxalinas/farmacología , Ratas
4.
Biofizika ; 52(3): 560-4, 2007.
Artículo en Ruso | MEDLINE | ID: mdl-17633549

RESUMEN

The influence of hypotonic swelling and hypertonic shrinking on cytosolic pH in synaptosomes was investigated. It was shown that decreasing the osmolarity of incubation medium to 230 mOsm leads to alkalization and increasing the osmolarity of incubation medium to 810 mOsm leads to acidification. Alkalization was inhibited by amiloride, indicating the involvement of the Na+/H+ exchanger. The acidification of cytosol upon hypertonic shrinking was insensitive, to amiloride and the inhibitor of Na+, K+, Cl- cotransport bumetanide. Thus, the Na+/H+ exchange in synaptosomes is activated by hypotonic swelling but not hypertonic shrinking, in contrast with erythrocytes and lymphocytes, which have been investigated earlier.


Asunto(s)
Encéfalo/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Sinaptosomas/fisiología , Amilorida/farmacología , Animales , Encéfalo/efectos de los fármacos , Bumetanida/farmacología , Citosol/química , Concentración de Iones de Hidrógeno , Soluciones Hipotónicas/farmacología , Transporte Iónico , Concentración Osmolar , Ratas , Solución Salina Hipertónica/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Sinaptosomas/efectos de los fármacos
5.
Biofizika ; 50(6): 1120-4, 2005.
Artículo en Ruso | MEDLINE | ID: mdl-16358793

RESUMEN

The influence of lanthanoids on exocytosis was investigated. It was shown that gadolinium increases the spontaneous release of the glutamate nonmetabolizing analogue [3H]D-aspartate. It was established using the fluorescent dye acridine orange that gadolinium and lanthanum induce exocytosis. The effect was dose-dependent and was maximum at 300 microM Gd3+. The exocytosis induced by gadolinium was calcium-independent. It is suggested that lanthanides induce a vesicular release of neurotransmitters by the mechanisms common for all polyvalent cations.


Asunto(s)
Encéfalo/metabolismo , Gadolinio/farmacología , Ácido Glutámico/metabolismo , Lantano/farmacología , Vesículas Sinápticas/metabolismo , Sinaptosomas/metabolismo , Animales , Ácido Aspártico/metabolismo , Transporte Biológico/efectos de los fármacos , Exocitosis/efectos de los fármacos , Ratas
6.
Neuroreport ; 10(8): 1763-5, 1999 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-10501571

RESUMEN

We studied the influence of plasma membrane depolarization on cAMP content in presynaptic nerve endings (synaptosomes) isolated from brain hemispheres (HS) and cerebellum (CS). Depolarization by elevated [K+]o decreased basal cAMP level in both types of synaptosomes; reduced cAMP content in HS and increased cAMP in CS in the presence of IBMX; and lowered forskolin-stimulated cAMP accumulation in both the HS and the CS. Similar results were obtained when depolarization was induced by veratrine or when [Ca2+]i was elevated by treatment of the synaptosomes with the ionophore A23187. In Ca2+-free media, depolarization was not able to affect the synaptosomal cAMP levels. These data suggest that in brain synaptosomes intracellular cAMP pathway is modulated by alterations in [Ca2+]i.


Asunto(s)
Encéfalo/metabolismo , AMP Cíclico/metabolismo , Sinaptosomas/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Adenilil Ciclasas/metabolismo , Animales , Encéfalo/ultraestructura , Calcimicina/farmacología , Calcio/metabolismo , Membrana Celular/fisiología , Cerebelo/enzimología , Cerebelo/fisiología , Electrofisiología , Técnicas In Vitro , Ionóforos/farmacología , Masculino , Potasio/metabolismo , Ratas , Ratas Wistar , Veratrina/farmacología
7.
Biofizika ; 48(5): 880-3, 2003.
Artículo en Ruso | MEDLINE | ID: mdl-14582414

RESUMEN

The effect of hypotonic and hypertonic shock on exocytosis in rat brain synaptosomes was studied using the fluorescent dye acridine orange. It was shown that an increase in medium osmolarity leads to calcium-independent exocytosis. The response of the probe was directly proportional to the amount of osmolithes added. A decrease in medium osmolarity to 230 mOsm led to an increase of acridine orange fluorescence, which is comparable with exocytosis occurring by the action of 15 mM KCl. This effect was independent of calcium concentration. It is assumed that, under hypotonic shock, part of neurotransmitters are released from the vesicular pool.


Asunto(s)
Encéfalo/metabolismo , Exocitosis , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Naranja de Acridina , Animales , Encéfalo/citología , Colorantes Fluorescentes , Concentración Osmolar , Ratas
8.
Biofizika ; 43(2): 315-8, 1998.
Artículo en Ruso | MEDLINE | ID: mdl-9591106

RESUMEN

The origin of calcium responsible for earlier observed acidosis-induced decrease in ATP content and inhibition of respiration in rat brain synaptosomes was studied. Acidosis (pH 6.0) inhibits both basal and potassium-stimulated 45Ca2+ uptake (60 mM KCl). Calcium channel blockers verapamil (100 microM) and 45Ca2+ (100 microM) have no effect on the level of ATP and respiration rate at pH 6.0. Theophylline (10 mM) releasing calcium from intracellular stores lowered ATP and O2 consumption rate at pH 7.4 but not at pH 6.0 being effective only in calcium-containing medium. Inhibitor of calcium transport in mitochondria ruthenium red (10 microM) prevented acidosis-induced ATP decrease. It is suggested that acidosis inhibits oxidative phosphorylation by releasing calcium from cytoplasmic stores with its subsequent transport into intrasynaptosomal mitochondria.


Asunto(s)
Acidosis/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa , Sinaptosomas/metabolismo , Animales , Ratas
9.
Biofizika ; 49(3): 524-8, 2004.
Artículo en Ruso | MEDLINE | ID: mdl-15327213

RESUMEN

The effect of calcium ionophore A23187 on the release of nonmetabolizable glutamate analogues [3H]D-aspartate and the exocytosis registered by fluorescent dyes in synaptosomes was investigated. It was shown that A23187 is able to induce neurotransmitter release both in calcium-containing and calcium-free medium, the effect in the latter case being more pronounced. Calcium ionophore is able to induce exocytosis registered by acridine orange and FM 2-10. The influence of A23187 on the fluorescence of acridine orange was mainly calcium-independent, whereas the change in the fluorescence of FM 2-10 was calcium-dependent. It was suggested that the calcium-independent increase in acridine orange fluorescence is related to the dissipation of pH gradient in synaptic vesicles. Probably, the calcium-independent release of D-aspartate is also associated with the dissipation of pH gradient and subsequent leakage of neurotransmitters.


Asunto(s)
Encéfalo/efectos de los fármacos , Calcimicina/farmacología , Neurotransmisores/metabolismo , Sinaptosomas/efectos de los fármacos , Animales , Encéfalo/metabolismo , Exocitosis , Fluorescencia , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Ratas , Sinaptosomas/metabolismo
10.
Biofizika ; 42(2): 408-11, 1997.
Artículo en Ruso | MEDLINE | ID: mdl-9172685

RESUMEN

Accumulation of TBA-reactive substances after a 40 min incubation of rat brain synaptosomes at 37 degrees C was analysed. A lowering of pH to 6.5-5.5 or arachidonic acid (0.1-1.0 mM) increased lipid peroxidation which was blocked by antioxidants. Acidosis (pH 6.0) and arachidonic acid used in combination had a strong synergic effect. Depolarisation of plasma membranes or intrasynaptosomal mitochondria were without influence on lipid peroxidation at neutral or acid pH. The results support a leading role of acidosis and phospholipases in stimulation of peroxidation under ischaemia.


Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Peroxidación de Lípido , Sinaptosomas/metabolismo , Animales , Antioxidantes/farmacología , Ácido Araquidónico/farmacología , Encéfalo/efectos de los fármacos , Membrana Celular/metabolismo , Calor , Concentración de Iones de Hidrógeno , Mitocondrias/metabolismo , Ratas , Sinaptosomas/efectos de los fármacos , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo
11.
Biofizika ; 42(2): 412-6, 1997.
Artículo en Ruso | MEDLINE | ID: mdl-9172686

RESUMEN

The influence of acidosis on the transmembrane potential, sodium pump and membranous systems of calcium transport was studied on isolated presynaptic nerve terminals (synaptosomes) from rat brain. It is established that acidic shift causes a decrease of membrane potential, a large inhibition of the sodium pump (by three times at pH 6.0). All the systems controlling both inward- and outward-directed calcium fluxes are partially blocked by low pH. At pH 6.0 the basal influx and calcium pump are reduced two-fold while the voltage-sensitive calcium channels and Na+/Ca2+ exchanger are inhibited by three and four to five times, respectively. We have no found any evidence of acidosis-induced net flux of calcium directed inwards.


Asunto(s)
Acidosis/fisiopatología , Encéfalo/fisiopatología , Calcio/metabolismo , Potenciales de la Membrana/fisiología , Sinaptosomas/fisiología , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Transporte Iónico , Ratas , Intercambiador de Sodio-Calcio , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Sinaptosomas/enzimología , Sinaptosomas/metabolismo
15.
Biochemistry (Mosc) ; 63(6): 662-70, 1998 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9668206

RESUMEN

Nitric oxide (NO) is known to potentiate neurotransmitter release in several types of neuronal cells. In the present study, the influence of NO on the membrane potential of isolated nerve endings (synaptosomes) from rat brain was studied. NO donors--sodium nitroprusside (SNP), S-nitroso-L-cysteine (CysNO), and hydroxylamine (HA)--induced synaptosome depolarization monitored by decreasing accumulation of 86Rb+ and the lipophilic potential-sensitive probe [3H]tetraphenylphosphonium. SNP reduced plasma membrane potential by 3-5 mV with half-maximal effect at approximately 10 microM. More potent NO donors, CysNO and HA, led to significant depolarization of the plasma membrane at 10-100 microM concentrations and also induced depolarization of mitochondria at concentrations above 1 mM. At 10 microM-10 mM concentrations, NO donors inhibited potassium channels; CysNO and HA also suppressed the activity of the sodium pump. NO-induced depolarization was not blocked by guanylate cyclase inhibitor methylene blue and the permeable cGMP analog dibutyryl-cGMP did not affect the membrane potential. The effects of NO donors were mimicked by SH-modifying reagents including 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). Non-permeable SH-reagent DTNB caused small depolarization resembling SNP action in its magnitude and kinetics. Significant decrease of potential in the presence of NEM, which permeates through the plasma membrane, was similar to that of CysNO and HA. The data suggest that in the presynaptic nerve endings, NO-induced depolarization of the plasma and mitochondrial membranes involves modification of protein SH-groups. The plasma membrane depolarization is due to the decreased potassium permeability and inhibition of the sodium pump.


Asunto(s)
Encéfalo/efectos de los fármacos , Terminaciones Nerviosas/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Animales , Encéfalo/fisiología , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Técnicas In Vitro , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Azul de Metileno/farmacología , Terminaciones Nerviosas/fisiología , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Wistar , Rubidio/metabolismo , Bloqueadores de los Canales de Sodio , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Distribución Tisular
16.
Biochemistry (Mosc) ; 63(2): 180-4, 1998 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9526111

RESUMEN

Incubation of rat brain synaptosomes at pH 6.0 in Ca2+-containing medium is associated with a decrease in the ATP content and the rate of oxygen consumption. ATP/ADP ratio decreased from 6.6 +/- 0.24 at pH 7.4 to 3.2 +/- 0.17 at pH 6.0. The content of 86Rb+ and [3H]tetraphenylphosphonium measured at pH 7.4 did not change after preincubation at pH 6.0, indicating the absence of lesion of synaptosomal plasma membranes and intrasynaptosomal mitochondria. Incubation with 1 mM EGTA in Ca2+-free medium as well as addition of 1 mM ouabain or 10 microM ruthenium red prevents the effect of acidosis. Similar results were obtained when 5 mM pyruvate was used as a mitochondrial substrate instead of glucose. It is suggested that acidosis-induced decrease in the ATP level is associated with the increase in Ca2+ concentration in the cytoplasm and its transport into mitochondria. Ouabain reverses this process due to activation of Na+/Ca2+ exchange.


Asunto(s)
Acidosis/metabolismo , Encéfalo/metabolismo , Calcio/farmacología , Metabolismo Energético/efectos de los fármacos , Sinaptosomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Técnicas In Vitro , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Rubidio/metabolismo , Sinaptosomas/efectos de los fármacos
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