RESUMEN
The endocannabinoid system is crucially involved in the regulation of brain activity and inflammation. We have investigated the localization of cannabinoid CB1 and CB2 receptors in adult rat brains before and after focal cerebral ischemia due to endothelin-induced transient occlusion of the middle cerebral artery (eMCAO). Using immunohistochemistry, both receptor subtypes were identified in cortical neurons. After eMCAO, neuronal cell death was accompanied by reduced neuronal CB1 and CB2 receptor-linked immunofluorescence. In parallel, CB1 receptor was found in activated microglia/macrophages 3 days post eMCAO and in astroglia cells at days 3 and 7. CB2 receptor labeling was identified in activated microglia/macrophages or astroglia 3 and 7d ays post ischemia, respectively. In addition, immune competent CD45-positive cells were characterized by pronounced CB2 receptor staining 3 and 7 days post eMCAO. KN38-72717, a potent and selective CB1 and CB2 receptor agonist, revealed a significant, dose-dependent and long-lasting reduction of cortical lesion sizes due to eMCAO, when applied consecutively before, during and after eMCAO. In addition, severe motor deficits of animals suffering from eMCAO were significantly improved by KN38-7271. KN38-7271 remained effective, even if its application was delayed up to 6h post eMCAO. Finally, we show that the endocannabinoid system assembles a comprehensive machinery to defend the brain against the devastating consequences of cerebral ischemia. In summary, this study underlines the therapeutic potential of CB1 and/or CB2 receptor agonists against neurodegenerative diseases or injuries involving acute or chronic imbalances of cerebral blood flow and energy consumption.
Asunto(s)
Encéfalo/metabolismo , Trastornos del Movimiento/tratamiento farmacológico , Trastornos del Movimiento/patología , Fármacos Neuroprotectores/uso terapéutico , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Encéfalo/efectos de los fármacos , Infarto Encefálico/etiología , Infarto Encefálico/prevención & control , Cannabinoides/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ectodisplasinas/metabolismo , Endocannabinoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Indanos/uso terapéutico , Infarto de la Arteria Cerebral Media/complicaciones , Antígenos Comunes de Leucocito/metabolismo , Masculino , Trastornos del Movimiento/etiología , Alcamidas Poliinsaturadas/farmacología , Desempeño Psicomotor/efectos de los fármacos , Quinoxalinas/farmacología , Quinoxalinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Ácidos Sulfónicos/uso terapéutico , Factores de TiempoRESUMEN
Atrial tachyarrhythmia (AF) alters intracellular calcium homeostasis and induces cellular hypertrophy of atrial myocytes. The impact of the calcium-dependent calcineurin pathway on the development of AF-induced atrial hypertrophy has not yet been analyzed. In this study, atrial tissue samples from patients with sinus rhythm and chronic persistent atrial fibrillation (CAF) were used to determine changes in expression and activity of calcineurin A (CnA), and its relation to CnA-regulated transcription factors NFATc1-4, and hypertrophic markers ANP, troponin I, and beta-MHC. CnA phosphatase activity and CnAbeta protein contents were significantly upregulated in patients with CAF. Calcineurin activation led to dephosphorylation, redistribution, and subsequent accumulation of NFATc3 in nuclei during CAF, and expression of hypertrophic genes was increased. CAF-dependent changes were reproduced by ex vivo pacing (2-4 Hz) of human atrial tissue slices. FK506 abolished the hypertrophic response induced by electrical-field stimulation. Atrial tachyarrhythmia causes atrial hypertrophy by activation of the CnA signal pathway, which thereby contributes to structural remodeling of human atria.
Asunto(s)
Fibrilación Atrial/metabolismo , Calcineurina/metabolismo , Cardiomegalia/metabolismo , Anciano , Apéndice Atrial/metabolismo , Calcineurina/genética , Femenino , Expresión Génica , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Transducción de Señal , Taquicardia/metabolismoRESUMEN
Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.
Asunto(s)
Calcio/metabolismo , Muerte Celular/fisiología , Clonazepam/análogos & derivados , Glucosa/deficiencia , Hipocampo/citología , Hipoxia , Sodio/metabolismo , Tiourea/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Compuestos de Boro/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Clonazepam/farmacología , Dantroleno/farmacología , Maleato de Dizocilpina/farmacología , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Fura-2/metabolismo , Hipocampo/metabolismo , Etiquetado Corte-Fin in Situ/métodos , Indoles/farmacología , Espacio Intracelular/metabolismo , Intercambio Iónico , Lidocaína/farmacología , Mibefradil/farmacología , Nimodipina/farmacología , Técnicas de Cultivo de Órganos , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Tiazepinas/farmacología , Tiourea/farmacología , Factores de TiempoRESUMEN
The protein p42(IP4), expressed mainly in brain, specifically recognizes two second messenger molecules, Ins(1,3,4,5)P(4) (IP(4)), a water soluble metabolite of IP(3) and the lipid PtdIns(3,4,5)P(3) (PIP(3)), the product of the growth factor-activated enzyme PI-3-kinase. Here, we studied whether there is short-term regulation of the expression level of p42(IP4) in limbic brain areas following acoustic and electric stimulation. The stimuli down-regulated the mRNA and protein levels within 2h in amygdala, hypothalamus and cingulate/retrospenial cortex. p42(IP4) mRNA decreased by about 50% for about 24h, but recovered to control values after 72 h. The present results are the first indication of a specific role of p42(IP4) in the short-term regulation of a behavioral response. They indicate that p42(IP4), an adapter protein in PIP(3)-dependent cellular signaling, may play an important role in the signal transduction pathways regulating plasticity in neuronal cells.
Asunto(s)
Estimulación Acústica/métodos , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Encéfalo/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Regulación hacia Abajo/fisiología , Fosfatos de Inositol/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Estimulación Eléctrica/métodos , Proteínas Activadoras de GTPasa , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Especificidad de Órganos/fisiología , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de TiempoRESUMEN
Prothrombin, protease-activated receptors (PARs) and the specific thrombin inhibitor protease nexin-1 (PN-1) are expressed in the brain. Recent studies have shown that the serine protease thrombin, depending on its concentration, plays an important role in neuronal degeneration or protection after cerebral ischemia. However, it is still uncertain whether a change in prothrombin or alterations in the expression of specific PAR-subtypes or PN-1 are associated with postischemic thrombin effects. Using semi-quantitative reverse transcription-polymerase chain reaction analysis, we show that prothrombin was up-regulated in the hippocampal formation 24 h after transient global ischemia in rats (two-vessel occlusion with hypotension), whereas the expression of PN-1 and the expression of PAR-subtypes 1-3 did not change significantly. Thus, control of the balance between the expression of prothrombin and PN-1 may reflect an important mechanism that underlies postischemic thrombin effects.
Asunto(s)
Isquemia Encefálica/metabolismo , Proteínas Portadoras/biosíntesis , Protrombina/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Trombina/biosíntesis , Regulación hacia Arriba , Precursor de Proteína beta-Amiloide , Animales , Hipocampo/metabolismo , Masculino , Nexinas de Proteasas , Protrombina/genética , Ratas , Ratas Wistar , Receptor PAR-1 , Receptor PAR-2 , Receptores de Superficie Celular , Regulación hacia Arriba/genéticaRESUMEN
A variety of extracellular serine proteases are expressed in the central nervous system or might permeate the blood-brain barrier under pathological conditions. However, their intracerebral targets and physiological functions are largely unknown. Here, we show that four distinct subtypes of protease-activated receptors (PARs) are abundantly expressed in the adult rat brain and in organotypic hippocampal slice cultures. PAR-1 expression was significant in the hippocampus, cortex and amygdala. Highest densities of PAR-2 and PAR-3 were observed in hippocampus, cortex, amygdala, thalamus, hypothalamus and striatum. Apart from the striatum, a similar localization was found for PAR-4. Within the hippocampal formation, each PAR subtype was predominantly localized in the pyramidal cell layers. Additionally, we identified PAR-2 in mossy fibers between dentate gyrus and CA3, PAR-3 in the subiculum and PAR-4 in CA3 and in mossy fibres as well as in the stratum lacunosum moleculare. After exposing hippocampal slice cultures to a severe experimental ischemia (oxygen-glucose deprivation), the expression of PARs 1-3 was up-regulated with subtype-specific kinetics. The localization of PARs in brain regions particularly vulnerable to ischemic insults as well as distinct alterations in the expression pattern after experimental ischemia support the notion of an important role of extracellular serine proteases and PARs in cerebral ischemia.
Asunto(s)
Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Receptores de Trombina/metabolismo , Regulación hacia Arriba/fisiología , Animales , Isquemia Encefálica/fisiopatología , Expresión Génica/fisiología , Hipocampo/fisiopatología , Inmunohistoquímica , Masculino , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor PAR-1 , Receptor PAR-2 , Receptores de Trombina/genéticaRESUMEN
In diseases associated with neuronal degeneration, such as Alzheimer's or cerebral ischemia, the cytosolic Ca2+ concentration ([Ca2+]cyt) is pathologically elevated. It is still unclear, however, under which conditions Ca2+ induces either apoptotic or necrotic neuronal cell death. Studying respiration and morphology of rat brain mitochondria, we found that extramitochondrial [Ca2+] above 1 M causes reversible release of cytochrome c, a key trigger of apoptosis. This event was NO-independent but required Ca2+ influx into the mitochondrial matrix. The mitochondrial permeability transition pore (PTP), widely thought to underlie cytochrome c release, was not involved. In contrast to noncerebral tissue, only relatively high [Ca2+] (is approximately equal to 200 M) opened PTP and ruptured mitochondria. Our findings might reflect a fundamental mechanism to protect postmitotic neuronal tissue against necrotic devastation and inflammation.
Asunto(s)
Encéfalo/metabolismo , Calcio/metabolismo , Grupo Citocromo c/metabolismo , Mitocondrias/metabolismo , Animales , Apoptosis/fisiología , Encéfalo/citología , Encéfalo/enzimología , Respiración de la Célula/fisiología , Ciclosporina/farmacología , Dextranos/farmacología , Inhibidores Enzimáticos/farmacología , Membranas Intracelulares/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Porinas/metabolismo , Ratas , Canales Aniónicos Dependientes del VoltajeRESUMEN
We have considered the extracellular serine protease thrombin and its receptor as endogenous mediators of neuronal protection against brain ischemia. Exposure of gerbils to prior mild ischemic insults, here two relatively short-lasting occlusions (2 min) of both common carotid arteries applied at 1-day intervals 2 days before a severe occlusion (6 min), caused a robust ischemic tolerance of hippocampal CA1 neurons. This resistance was impaired if the specific thrombin inhibitor hirudin was injected intracerebroventricularly before each short-lasting insult. Thus, efficient native neuroprotective mechanisms exist and endogenous thrombin seems to be involved therein. In vitro experiments using organotypic slice cultures of rat hippocampus revealed that thrombin can have protective but also deleterious effects on hippocampal CA1 neurons. Low concentrations of thrombin (50 pM, 0.01 unit/ml) or of a synthetic thrombin receptor agonist (10 microM) induced significant neuroprotection against experimental ischemia. In contrast, 50 nM (10 units/ml) thrombin decreased further the reduced neuronal survival that follows the deprivation of oxygen and glucose, and 500 nM even caused neuronal cell death by itself. Degenerative thrombin actions also might be relevant in vivo, because hirudin increased the number of surviving neurons when applied before a 6-min occlusion. Among the thrombin concentrations tested, 50 pM induced intracellular Ca(2+) spikes in fura-2-loaded CA1 neurons whereas higher concentrations caused a sustained Ca(2+) elevation. Thus, distinct Ca(2+) signals may define whether or not thrombin initiates protection. Taken together, in vivo and in vitro data suggest that thrombin can determine neuronal cell death or survival after brain ischemia.
Asunto(s)
Isquemia Encefálica/prevención & control , Hipocampo/metabolismo , Neuronas/metabolismo , Trombina/metabolismo , Animales , Antitrombinas/farmacología , Señalización del Calcio , Supervivencia Celular , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Gerbillinae , Hipocampo/citología , Hirudinas/farmacología , Humanos , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Trombina/metabolismo , Trombina/efectos adversos , Trombina/antagonistas & inhibidoresRESUMEN
Arachidonic acid has been shown to affect the intracellular calcium concentration in many cell types (1-5), but the target of this regulation was unclear. Here we show that two types of intracellular calcium release channel, the inositol 1,4,5-trisphosphate-gated channel (IP3R) and the ryanodine receptor (RyR) are modulated in an opposing manner by arachidonic acid and its product leukotriene B4 (LTB4). The IP3R was inhibited by arachidonic acid (Ki = 27 nM), whereas the RyR was unaffected by this compound. In contrast, 100 nM LTB4 fully activated the RyR but did not influence the IP3R. The concerted action of arachidonic acid and LTB4 could provide specific mobilization of stored calcium by terminating IP3-induced release and activating the RyR/calcium release channel by its newly identified agonist.
Asunto(s)
Ácido Araquidónico/farmacología , Canales de Calcio/efectos de los fármacos , Leucotrieno B4/farmacología , Animales , Canales de Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Perros , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/metabolismo , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
Calcium release from intracellular stores occurs through two types of channels associated with intracellular membranes, namely, the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. Recently, it has been shown that these channels are regulated by allosteric mechanisms and associated proteins. Release of intracellular calcium induces the opening of calcium-permeable channels on the plasma membrane. Current work has focused on the molecular and functional characterization of these channels which have been identified as store-operated channels or calcium release activated channels.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Activación del Canal Iónico , Proteínas Portadoras , Membrana Celular/metabolismo , Proteínas de Unión al ADN , Proteínas de Choque Térmico , Membranas Intracelulares/metabolismo , Ligandos , Modelos Biológicos , Conformación Proteica , Proteínas de Unión a TacrolimusRESUMEN
The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.
Asunto(s)
Canales de Calcio/metabolismo , Cerebelo/química , Manganeso/farmacocinética , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Canales de Calcio/química , Canales de Calcio/efectos de los fármacos , Cerebelo/efectos de los fármacos , Perros , Indicadores y Reactivos , Receptores de Inositol 1,4,5-Trifosfato , Manganeso/farmacología , Técnicas de Placa-Clamp , Permeabilidad/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/efectos de los fármacosRESUMEN
The effect of phorbol 12-myristate 13-acetate (PMA) on the expression and shedding of intercellular adhesion molecule-1 (ICAM-1) was investigated on the hematopoietic cell lines K 562 and U 937 using flow cytometry, fluorescence microscopy and ELISA technique. At low concentration of 1 nM, PMA stimulated the expression of ICAM-1 on the cell surface about 4-fold within 24 h, whereas a short-term treatment with 100 nM PMA led to the shedding of 35% of ICAM-1 from the surface of K 562 cells. The release of surface ICAM-1 was found on single cells by fluorescence microscopy to be a uniform process proceeding within 15 min. The shedding of ICAM-1 correlated with elevated levels of sICAM-1 in the supernatants of cultured cells. Also on K 562 cells stimulated by TNF-alpha, a PMA-induced release of ICAM-1 was observed in addition to the known spontaneous shedding. In contrast to the results with K 562 cells, no PMA-induced shedding of ICAM-1 was found on U 937 cells. This indicates a cell-specific process for K 562 cells. The PMA-mediated release of ICAM-1 from K 562 cells suggests that the shedding process does not only occur in parallel to the surface expression of ICAM-1, but may be controlled by particular mechanisms of down-regulation.
Asunto(s)
Molécula 1 de Adhesión Intercelular/metabolismo , Western Blotting , Línea Celular , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Cinética , Leucemia Eritroblástica Aguda , Microscopía Fluorescente , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The quenching of fura-2 fluorescence by the influx of extracellular Mn2+ was measured to indicate the flux rates through receptor-operated calcium channels in the plasma membrane of rat hepatocytes. Neomycin, an inhibitor of phospholipase C, inhibited the vasopressin-induced influx of Mn2+. Thus, the agonist-induced entry of extracellular calcium into hepatocytes is linked to a phospholipase C-generated second messenger. Microinjection of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or 3-deoxy-3-fluoro-Ins(1,4,5)P3 revealed that Ins(1,4,5)P3 rather than Ins(1,3,4,5)P4 is responsible for calcium entry. The activation of phospholipase C by vasopressin produced an influx of Mn2+ independent of the depletion of intracellular calcium stores if this depletion was delayed by the Ins(1,4,5)P3 receptor antagonist heparin or by the use of a low agonist concentration. Thapsigargin, an inhibitor of the store calcium pump, leading to an Ins(1,4,5)P3-independent emptying of stores, gave a short living signal (less than 3 min) for calcium entry. We propose that Ins(1,4,5)P3 is able to stimulate calcium entry by two pathways. (a) Ins(1,4,5)P3 activates receptor-operated calcium channels in a direct manner. The calcium entry resulting from this is followed (b) by the Ins(1,4,5)P3-induced depletion of calcium stores, producing a store-dependent entry.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Receptores de Inositol 1,4,5-Trifosfato , Transporte Iónico , Hígado/citología , Masculino , Manganeso/metabolismo , Ratas , Ratas WistarRESUMEN
The conditions of treatment of human skeletal muscle fibers from M. vastus lateralis with saponin were optimized to achieve complete permeabilization of cell membrane at intact mitochondrial oxidative phosphorylation. After 30 min of incubation with saponin all lactate dehydrogenase, 50% of creatine kinase, 30% of adenylate kinase and less than 20% of citrate synthase was released into the permeabilization medium. These skinned fibers behave similar to isolated mitochondria from human skeletal muscle: (i) the respiration with mitochondrial substrates can be stimulated by ADP, (ii) inhibited by carboxyatractyloside and (iii) it is possible to detect fluorescence changes of mitochondrial NAD(P)H on additions of substrates, uncoupler and cyanide. From a comparison of rates of respiration per cytochrome aa3 content of isolated human skeletal muscle mitochondria and saponin-skinned muscle fibers it was possible to calculate that almost 85% of mitochondria in those fibers are accessible for the investigation of oxidative phosphorylation. As shown by the investigation of biopsy samples of two patients with undefined myopathies these fibers are a suitable object for the replacement of isolated mitochondria in the diagnosis of mitochondrial myopathies and encephalomyopathies.
Asunto(s)
Mitocondrias Musculares/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Saponinas/farmacología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Niño , Humanos , Técnicas In Vitro , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Mitocondrias Musculares/metabolismo , RatasRESUMEN
Fura-2 loaded rat hepatocytes were used to determine whether the L-type channel blockers, verapamil and diltiazem, affect receptor-operated calcium channels (ROCCs). The flux through ROCCs was followed by quenching of fura-2 fluorescence due to the influx of extracellular Mn2+ induced by vasopressin. Verapamil as well as diltiazem inhibited vasopressin-stimulated Mn2+ influx in a dose-dependent manner up to 60% at concentrations of 200-400 microM. Furthermore, both inhibitors decreased significantly the frequency of phenylephrine-induced oscillation of [Ca2+]i. The experimental findings indicate that L-type channel blockers inhibit ROCCs in rat hepatocytes.