RESUMEN
INTRODUCTION: Hypericum perforatum is, with Ginkgo biloba, one of the most frequently prescribed medicinal plants in the world. Its popular name, St. John's wort (SJW), is due to the fact that its flowers, yellow, are gathered around the feast of St. John the Baptist (24th June) whereas "wort" is an old English word for plant. Of interest, SJW possesses antidepressant actions and is currently used to alleviate symptoms of mild to moderate depression. Nearly two dozens of bioactive compounds have been isolated from SJW. Hypericin, originally described as a monoamine oxidase inhibitor type A, was thought to be responsible for the antidepressant properties of SJW extracts. However, subsequent studies could not confirm this observation and hyperforin, a phloroglucinol derivative, was shown to display antidepressive properties. Indeed, the efficiency of the extracts of SJW has been reported to be dependent on the concentration of hyperforin. However, its effects on brain cells and on the mechanisms underlying its putative clinical antidepressant effect remain poorly characterized. OBJECTIVE: The aim of this review article is to propose an overview of the recent scientific publications that have provided new and relevant insights into the neurobiological actions of hyperforin. RESULTS: Hyperforin has been described as an inhibitor of the reuptake of many neurotransmitters such as dopamine, norepinephrine, serotonin or glutamate. It is thus a potent modulator of synaptic transmission. In addition, it blocks the activity of many receptors such as gamma-aminobutyric acid (GABA) and N-Methyl-D-aspartate (NMDA) receptors. More recently, hyperforin has been shown to activate TRPC6, a Ca(2+)-conducting channel of the plasma membrane, which is the only channel opened by this molecule. Interestingly, the other transient receptor potential channels of C type (TRPC) isoforms (TRPC1, TRPC3, TRPC4, TRPC5 and TRPC7) are insensitive to hyperforin. Due to this specific property, it is now used as a convenient pharmacological tool to investigate the functions of endogenous TRPC6 channels in various cell types. Chronically applied to neuronal cell line PC12, hyperforin promotes the extension of neurites via a mechanism implying TRPC6 channels. It is also known to trigger an intracellular signalling pathway that involves the cAMP-dependent protein kinase A and the transcription factor cyclic adenosine monophosphate response element binding protein (CREB). This leads to an up-regulation of the expression of the brain-derived neurotrophic factor (BDNF) receptor neurotrophic tyrosine kinase (TrkB) and TRPC6. This hyperforin-dependent cascade is controlled by Ca(2+) ions and occurs specifically in the cortex but not in the hippocampus. One key aspect of the cellular responses induced by hyperforin is its impact on the homeostasis of several cations (Na(+), Ca(2+), Zn(2+) and H(+)). In vitro experiments demonstrated that hyperforin, which changes the fluidity of membranes, elevates the intracellular concentration of these elements by promoting their influx and/or their release from internal compartments. CONCLUSION: The phloroglucinol derivative hyperforin is an important bioactive molecule of Hypericum perforatum exhibiting antidepressive properties. Although it inhibits the reuptake of many neurotransmitters, hyperforin is in fact a multi-target drug influencing the cellular homeostatic mechanisms of Ca(2+), Zn(2+), H(+) and Na(+) due to its effects on their influx and/or release from internal stores. In addition, hyperforin is a potent modulator of mitochondrial functions. In spite of recent progress in the characterization of the cellular hyperforin responses, it remains unclear what pharmacological aspects of hyperforin functions are relevant in vivo.
Asunto(s)
Antidepresivos/farmacología , Encéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Floroglucinol/análogos & derivados , Terpenos/farmacología , Antracenos , Encéfalo/fisiopatología , Hypericum/química , Neuronas/fisiología , Perileno/análogos & derivados , Perileno/farmacología , Floroglucinol/farmacologíaRESUMEN
INTRODUCTION: Many metals like iron (Fe), copper (Cu) or zinc (Zn) fulfil various essential biological functions and are thus vital for all living organisms. For instance, they play important roles in nervous tissue, participating in a wide range of processes such as neurotransmitter synthesis, myelination or synaptic transmission. STATE OF THE ART: As in other tissues, brain cells tightly control the concentration of metals but any excess or deficit can lead to deleterious responses and alter cognitive functions. Of note, certain metals such as Zn, Fe or Cu accumulate in specific brain structures over lifespan and several neurodegenerative diseases are associated with a dysregulation of the homeostatic mechanisms controlling the concentration of these cations. CONCLUSION AND PERSPECTIVES: This review will address some of the cellular and molecular processes controlling the entry and distribution of selected metals (mainly Zn and Fe) in the brain, as well as their roles in synaptic transmission, in the pathogenesis of some neurologic diseases such as Parkinson's disease and Alzheimer's disease, and their impact on cognitive functions.
Asunto(s)
Química Encefálica/fisiología , Encéfalo/fisiología , Hierro/fisiología , Oligoelementos/metabolismo , Zinc/fisiología , Animales , Humanos , Hierro/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Transmisión Sináptica/fisiología , Zinc/metabolismoRESUMEN
Cadmium is found in the environment as part of several, mainly zinc-rich, ores. It has been used in many technological applications, but biological systems generally failed to safely deal with this element. In mammalian biology, cadmium exposure jeopardizes health and mechanisms of cadmium toxicity are multifarious. Mainly because bioavailable cadmium mimics other metals that are essential to diverse biological functions, cadmium follows a Trojan horse strategy to get assimilated. Metals susceptible to cadmium deceit include calcium, zinc, and iron. The wealth of data addressing cadmium toxicity in animal cells is briefly reviewed with special emphasis on disturbance of the homeostasis of calcium, zinc, and iron. A limited number of tissues and cell types are considered as main targets for cadmium toxicity. Still, the diversity of pathways affected by cadmium exposure points to a more general threat to basic cellular functions. The poor efficiency of cellular export systems for cadmium explains the long residence time of the element in mammals. Therefore, proper disposal and educated uses of this technologically appealing, but biologically malicious, element should be favored in the future. The comprehensive knowledge of cadmium biological effects is indeed a necessary step to protect human and animal populations from environmental and anthropological exposures.
Asunto(s)
Cadmio/toxicidad , Supervivencia Celular/efectos de los fármacos , Metales/farmacología , Animales , Cinética , Modelos BiológicosRESUMEN
Presynaptic action potentials trigger the exocytosis of neurotransmitters. However, even in the absence of depolarisation-dependent Ca2+ entry nearby release sites, spontaneous vesicular release still occurs. Even though this happens at low rate, such spontaneous release may play a trophic role in maintaining the shape of dendritic structures. Like evoked responses, action potential-independent release is subject to modulation. This review describes some of the regulatory factors that rapidly and presynaptically regulate the ongoing Ca2+-independent release of neurotransmitters in the hippocampus. For instance, the electrical activity of the nerve ending, neurotransmitters, hypertonic solutions, neurotoxins, polycations, neurotrophic factors, immunoglobulins, cyclothiazide and psychotropic drugs can all modify the rate of spontaneous release. This can be achieved through various mechanisms that can be Ca2+-dependent or Ca2+-independent, protein kinase-dependent or independent. Since action potential-independent release contributes to the maintenance of dendritic structures, neuromodulators are likely to influence the density and/or length of dendritic spines, which in turn may modulate information processing in the central nervous system (CNS).
Asunto(s)
Hipocampo/metabolismo , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/metabolismo , Potenciales de Acción/fisiología , Animales , Dendritas/metabolismo , HumanosRESUMEN
Intracellular Ca(2+) ([Ca(2+)](i)) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo-3. In the absence of external Ca(2+), the cholinergic agonist carbachol (200 microM) and the sarcoendoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (0.4 microM) both transiently elevated [Ca(2+)](i). A subsequent addition of Ca(2+) into the bathing medium caused a second [Ca(2+)](i) change which was blocked by lanthanum (50 microM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca(2+) entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca(2+) entry in excitable cells.
Asunto(s)
Calcio/metabolismo , Hipocampo/citología , Neuronas/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Compuestos de Anilina/metabolismo , Animales , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Carbacol/farmacología , Células Cultivadas , Agonistas Colinérgicos/farmacología , Conductividad Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Lantano/farmacología , Neuronas/efectos de los fármacos , Potasio/farmacología , Ratas , Tetrodotoxina/farmacología , Tapsigargina/farmacología , Xantenos/metabolismoRESUMEN
Kainate receptors (KARs) are abundantly expressed in the basal ganglia, but their function in synaptic transmission has not been established. In the present study, we show that the GluR6 subunit of KARs is expressed in both substance P- and enkephalin-containing GABAergic projection neurons of the mouse striatum. Using whole-cell voltage-clamp recordings in brain slices, we demonstrate the presence of functional KARs in the dorsal striatum activated by low concentrations of the AMPA/KAR agonist domoate in wild-type but not GluR6-deficient mice. Despite the abundance of KARs, we found no evidence for synaptic activation of these receptors after single or repetitive stimulation of glutamatergic afferents. Domoate induces a transient increase in the frequency of spontaneous IPSCs of small amplitude and a sustained depression of large IPSCs evoked by minimal electrical stimulation within the striatum in wild-type mice but not in GluR6-deficient mice. This depressant effect is inhibited in presence of adenosine A(2A) receptor antagonists, ZM-241385 and SCH-58261. These data strongly suggest that, in striatal neurons, KARs depress GABAergic synaptic transmission indirectly via release of adenosine acting on A(2A) receptors.
Asunto(s)
Cuerpo Estriado/química , Cuerpo Estriado/fisiología , Regulación hacia Abajo/fisiología , Receptores de Ácido Kaínico/metabolismo , Transmisión Sináptica/fisiología , Animales , Benzodiazepinas/farmacología , Cuerpo Estriado/citología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Expresión Génica/fisiología , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacología , Ratones , Ratones Endogámicos C57BL , Fármacos Neuromusculares Despolarizantes/farmacología , Neuronas/química , Neuronas/fisiología , Quinoxalinas/farmacología , ARN Mensajero/análisis , Receptores Adrenérgicos alfa 2/fisiología , Receptores de Ácido Kaínico/genética , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , Receptor de Ácido Kaínico GluK2RESUMEN
Cultured hippocampal neurons from neonatal rats were used to investigate the effect of adenosine on the release of glutamate. Spontaneous tetrodotoxin-resistant miniature excitatory postsynaptic currents (mEPSCs) through AMPA receptor channels were recorded by means of the whole-cell patch-clamp technique. Adenosine (50 microM) reversibly reduced the frequency of mEPSCs by approximately 50-60%, but did not change their amplitudes. The protein kinase A inhibitor Rp-cyclic adenosine monophosphate (100-150 microM) did not block the adenosine-dependent reduction of the mEPSC frequency, showing that adenosine is not depressing synaptic transmission via a protein kinase A (PKA)-dependent mechanism. The D1 dopamine agonist SKF-38393 (250 microM), forskolin (5 microM) and 8Br-cAMP (2 mM), known to activate the cAMP/PKA-dependent signalling pathway, all enhanced the mEPSC frequency. A subsequent application of adenosine (50 microM) strongly reduced the potentiation produced by any one of these three drugs. It also reversed protein kinase C (PKC)-dependent stimulation of glutamate release induced by phorbol myristate acetate (100 nM). Taken together, adenosine not only inhibits the spontaneous release of glutamate independently of protein kinases A and C but also reverses the enhancement of exocytosis produced by protein kinases A and C activators.
Asunto(s)
Adenosina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ácido Glutámico/metabolismo , Hipocampo/enzimología , Proteína Quinasa C/antagonistas & inhibidores , Transmisión Sináptica/fisiología , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenosina/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Animales Recién Nacidos , Carbacol/farmacología , Carcinógenos/farmacología , Células Cultivadas , Agonistas Colinérgicos/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Agonistas de Dopamina/farmacología , Activación Enzimática/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Exocitosis/efectos de los fármacos , Hipocampo/citología , Técnicas de Placa-Clamp , Proteína Quinasa C/fisiología , Ratas , Transmisión Sináptica/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Voltage-gated ion channels and morphological differentiation were studied in rat PC12 pheochromocytoma cells after treatment with nerve growth factor (NGF) or forskolin. Ca2+ and Na+ channels were analyzed by electrophysiological techniques (using Ba2+ as charge carrier through Ca2+ channels) and by binding studies with specific ligands. With NGF, Na+ current (I(Na)) density increased in parallel with neurite extension. Ba2+ current (I(Ba)) density and Ca2+ channel numbers were both enhanced after a 2-day latency period. The tyrosine kinase inhibitor genistein blocked NGF-induced neurite extension but not the increase in I(Na) density. With forskolin, neurite outgrowth was linked to an apparent increase in I(Ba) density similar to the one induced by NGF, while no change in I(Na) was observed. Dihydropyridine-sensitive (L-type) as well as omega-conotoxin-sensitive (N-type) currents contributed to this effect. In spite of its stimulating effect on I(Ba), binding studies with radiolabeled ligands in forskolin-treated cells showed no change in N-type and an apparent loss of high affinity L-type Ca2+ channel binding. Our results suggest that induction of individual voltage-dependent channel types as well as morphological differentiation each require the activation of different signaling pathways. NGF and forskolin both enhanced current flow through voltage-dependent Ca2+ channels. However, only NGF increased channel expression while forskolin appeared to modulate channel kinetics.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Colforsina/farmacología , Factores de Crecimiento Nervioso/farmacología , Canales de Sodio/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Bario/metabolismo , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Recuento de Células , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Genisteína/farmacología , Activación del Canal Iónico , Neuritas/efectos de los fármacos , Células PC12 , Técnicas de Placa-Clamp , Ensayo de Unión Radioligante , Ratas , Sodio/metabolismo , Canales de Sodio/metabolismoRESUMEN
Tricyclic antidepressants (e.g., imipramine, desipramine) are currently used in the treatment of mood disorders such as depression. At the cellular level they inhibit the re-uptake of the exocytosed monoamines serotonin and noradrenaline. However, they also stimulate phospholipase C activity and the production of the second messenger inositol 1,4,5-trisphosphate. Since phospholipase C activation can also lead to the production of the protein kinase C activator diacylglycerol, we have undertaken experiments to see whether acutely applied desipramine could change the synaptic strength of neurons in a protein kinase C-dependent manner. Experiments performed with cultured hippocampal neurons dissociated from neonatal rats revealed that desipramine rapidly enhanced the spontaneous vesicular release of glutamate. This was observed by measuring the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. Analysis of amplitude distribution histograms indicated a presynaptic site of action. The protein kinase inhibitor staurosporine and down-regulation of protein kinase C activity greatly reduced the desipramine-dependent enhancement of the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. This presynaptic modulation requires SNARE proteins because cleavage of SNAP-25 with the botulinum neurotoxin A strongly reduced the desipramine-induced glutamate release. Thus, acute applications of desipramine stimulated the ongoing neurotransmitter release pathway, probably by activating protein kinase C. Our data indicate that tricyclic antidepressant drugs not only act on serotoninergic and/or noradrenergic cells but can also modify the activity of glutamatergic neurons.
Asunto(s)
Antidepresivos Tricíclicos/farmacología , Desipramina/farmacología , Exocitosis/efectos de los fármacos , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Terminales Presinápticos/efectos de los fármacos , Animales , Antidepresivos Tricíclicos/administración & dosificación , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Desipramina/administración & dosificación , Membranas Intracelulares/metabolismo , Terminales Presinápticos/fisiología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The present study was undertaken to better assess the role of dopamine on exocytosis. Since direct activation of adenylate cyclase (e.g., with forskolin) enhances neurotransmitter release it was of interest to see whether the activation of D1-type dopamine receptors, which are positively coupled to adenylate cyclase, could also modulate the molecular machinery underlying the fusion of synaptic vesicles and the release of neurotransmitter. To answer this question we have looked at the effect of the D1-type dopamine receptor agonist SKF-38393 on the spontaneous release of glutamate from cultured rat hippocampal neurons. SKF-38393 enhanced the frequency but not the amplitude of tetrodotoxin-resistant excitatory postsynaptic currents which argues for a presynaptic locus of D1 action. This effect was blocked by the D1-dopaminergic receptor antagonist SCH-23390 and the protein kinase A inhibitors H-7 and Rp-cAMP whereas pertussis toxin failed to affect the dopaminergic response. In addition, carbachol and Ruthenium Red also stimulated exocytosis but did not occlude the SKF-38393-induced modulation. These results indicate that SKF-38393 presynaptically enhances the release of glutamate via a pertussis toxin-insensitive and protein kinase A-dependent mechanism, which most likely involves D1-type dopamine receptors. Our results underline the importance of protein kinase A as potent modulator of synaptic transmission and suggest that high concentrations of dopamine can greatly enhance the release of glutamate in the hippocampus.
Asunto(s)
2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Agonistas de Dopamina/farmacología , Ácido Glutámico/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Receptores de Dopamina D1/agonistas , Animales , Colforsina/farmacología , Ratas , Receptores de Dopamina D1/fisiología , Tetrodotoxina/farmacologíaRESUMEN
L-type Ca2+ channels are important targets for drugs, such as dihydropyridines (DHPs), in the treatment of cardiovascular diseases. Channel expression is regulated by alternative splicing. It has been suggested that in the cardiovascular system tissue-specific expression of different L-type Ca2+ channel splice variants may underlie the observed differences in sensitivities to channel block by DHPs. We investigated the sensitivity of Ca2+ channel splice variants derived from the human alpha1C gene to the DHP isradipine. Among seven alpha1C channels we observed up to 10-fold differences in IC50 values for isradipine, as well as changes in the voltage dependence of DHP action.
Asunto(s)
Empalme Alternativo/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Isradipino/farmacología , Animales , Exones/genética , Humanos , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1-43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca2+ signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca2+ concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca2+ concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4beta-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.
Asunto(s)
Adenosina/farmacología , Hipocampo/fisiología , Proteína Quinasa C/metabolismo , Receptores Muscarínicos/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Animales Recién Nacidos , Atropina/farmacología , Calcio/metabolismo , Carbacol/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Activación Enzimática , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Neuronas/citología , Neuronas/fisiología , Antagonistas Nicotínicos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Estaurosporina/farmacología , Sinapsis/efectos de los fármacosRESUMEN
Spontaneous excitatory postsynaptic currents (sEPSC) through AMPA-type channels were recorded on cultured hippocampal pyramidal neurons by means of the whole-cell patch-clamp technique. The protein kinase C (PKC) agonist 4beta-phorbol 12-myristate 13-acetate (4beta-PMA) produced a long-lasting increase in sEPSC frequency not mimicked by the inactive analogue 4alpha-PM and blocked by the protein kinase inhibitor staurosporine. The 4beta-PMA-induced change in sEPSC frequency occurred without detectable change in [Ca2+]i. After treatment with the microtubule-disrupting agent colchicine, 4beta-PMA caused a small and transient increase in sEPSC frequency. It is concluded that colchicine affects the PKC-induced functional plasticity of nerve cells most likely by disturbing the axonal transport.
Asunto(s)
Calcio/metabolismo , Colchicina/farmacología , Hipocampo/fisiología , Proteína Quinasa C/metabolismo , Células Piramidales/fisiología , Transmisión Sináptica/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Animales Recién Nacidos , Cadmio/farmacología , Calcio/farmacología , Células Cultivadas , Potenciales Evocados/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/análogos & derivados , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
The pore-forming alpha1C subunit is the principal component of the voltage-sensitive L-type Ca2+ channel. It has a long cytoplasmic carboxyl-terminal tail playing a critical role in channel gating. The expression of alpha1C subunits is characterized by alternative splicing, which generates its multiple isoforms. cDNA cloning points to a diversity of human hippocampus alpha1C transcripts in the region of exons 40-43 that encode a part of the 662-amino acid carboxyl terminus. We compared electrophysiological properties of the well defined 2138-amino acid alpha1C,77 channel isoform with two splice variants, alpha1C,72 and alpha1C,86. They contain alterations in the carboxyl terminus due to alternative splicing of exons 40-42. The 2157-amino acid alpha1C,72 isoform contains an insertion of 19 amino acids at position 1575. The 2139-amino acid alpha1C,86 has 80 amino acids replaced in positions 1572-1651 of alpha1C,77 by a non-identical sequence of 81 amino acids. When expressed in Xenopus oocytes, all three splice variants retained high sensitivity toward dihydropyridine blockers but showed large differences in gating properties. Unlike alpha1C,77 and alpha1C,72, Ba2+ currents (IBa) through alpha1C,86 inactivated 8-10 times faster at +20 mV, and its inactivation rate was strongly voltage-dependent. Compared to alpha1C,77, the inactivation curves of IBa through alpha1C,86 and alpha1C,72 channels were shifted toward more negative voltages by 11 and 6 mV, respectively. Unlike alpha1C,77 and alpha1C,72, the alpha1C,86 channel lacks a Ca2+-dependent component of inactivation. Thus the segment 1572-1651 of the cytoplasmic tail of alpha1C is critical for the kinetics as well as for the Ca2+ and voltage dependence of L-type Ca2+ channel gating.
Asunto(s)
Canales de Calcio/química , Calcio/metabolismo , Exones , Empalme Alternativo , Aminoácidos/análisis , Electrofisiología , Humanos , Cinética , Datos de Secuencia Molecular , Plásmidos/químicaRESUMEN
Propagation of action potentials in axons and dendrites increases intracellular Na+ ([Na+]i) and Ca2+ concentrations ([Ca2+]i). While the importance of [Ca2+]i in synaptic transmission is well established, a possible functional role of [Na+]i is unclear. In cultured hippocampal cells, [Na+]i was increased by veratridine. We have then measured spontaneous excitatory postsynaptic currents (sEPSCs) and, by means of fluorescent dyes, changes in [Na+]i, in [Ca2+]i, and in the turnover of the vesicular pool of individual boutons. An elevation of [Na+]i and a concomitant rise in [Ca2+]i, led to a large increase in sEPSC frequency and in the turnover of the presynaptic vesicular pool. Extracellular Ca2+ was essential for these effects of elevated [Na+]i on synaptic transmission. They probably occur via Na+/Ca2+ exchange.
Asunto(s)
Hipocampo/citología , Sodio/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Animales , Transporte Biológico/fisiología , Calcio/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/fisiología , Dendritas/fisiología , Electrofisiología , Exocitosis/fisiología , Colorantes Fluorescentes , Potenciales de la Membrana/fisiología , Neuronas/citología , Neuronas/fisiología , Neuronas/ultraestructura , Terminales Presinápticos/fisiología , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Ratas , Ratas Sprague-Dawley , Veratridina/farmacologíaRESUMEN
We have investigated in Xenopus oocytes the effects of phorbol ester-induced protein kinase C (PKC) stimulation on dihydropyridine (DHP)-insensitive and -sensitive Ca2+ channels. DHP-insensitive Ba2+ currents (IBa) were recorded from endogenous channels in non-injected oocytes and in oocytes injected with cRNAs encoding the auxiliary rabbit alpha 2/delta and beta 1 Ca2+ channel subunits. A human alpha 1C cRNA, injected alone or in combination with cRNAs of the auxiliary subunits, was used for studying DHP-sensitive IBa. We found that DHP-insensitive IBa was increased by 4 beta-phorbol 12-myristate 13-acetate (PMA), while DHP-sensitive IBa was decreased. In both cases, the effects depended only on the co-expression of the beta 1 subunit.
Asunto(s)
Canales de Calcio/metabolismo , Proteína Quinasa C/metabolismo , Animales , Bario/metabolismo , Secuencia de Bases , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dihidropiridinas/farmacología , Conductividad Eléctrica , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Oocitos , Conejos , Relación Estructura-Actividad , Acetato de Tetradecanoilforbol/farmacología , XenopusRESUMEN
Whole-cell patch-clamp measurements were made in nerve-growth-factor (NGF)-treated PC12 cells. External application of bradykinin (BK) activated an outward and an inward current which could be separated by using KCl- or CsCl-containing pipette solutions. The slowly activating inward current could be induced by BK independently of the filling of intracellular Ca2+ stores. By using GDP-beta-S in the pipette medium, we showed that BK-induced outward and inward currents were differentially regulated through G-protein-sensitive and -insensitive mechanisms, respectively. While the outward current was inhibited by GDP-beta-S, the inward current was not affected. Our results show that occupancy of BK receptors activates different signaling pathways for the induction of outward and inward currents.
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Bradiquinina/farmacología , Canales Iónicos/efectos de los fármacos , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Células PC12 , Técnicas de Placa-Clamp , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Terpenos/farmacología , TapsigarginaRESUMEN
Voltage-dependent inhibition by 1,4-dihydropyridines is a characteristic property of L-type Ca2+ channels. Six out of 50 exons of the channel alpha 1C subunit gene are subjected to alternative splicing, thus generating channel isoform diversity. Using Xenopus oocytes as an expression system, we have found that transmembrane segment IIIS2 of human alpha 1C subunit is involved in the control of voltage dependence of dihydropyridine action. This segment is genetically regulated through alternative splicing of exons 21/22. Site-directed mutagenesis points to two amino acids in IIIS2, which determine the difference of the splice variants in their sensitivities to dihydropyridines. This finding provides new insight into molecular mechanisms of Ca2+ channel inhibition by this important class of drugs.
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Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/química , Dihidropiridinas/farmacología , Potenciales de la Membrana , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Cartilla de ADN/química , Conductividad Eléctrica , Humanos , Técnicas In Vitro , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Rat pheochromocytoma (PC12) cells were treated with nerve growth factor (NGF) for 3-4 days. They formed growth cones and extended neurites. Addition of the phosphatase inhibitor calyculin A (CL-A) caused a concentration-dependent complete retraction of neurites within 15 min. Retraction of growth cones started with the filopodia still present. The cell bodies acquired a grape-like shape opposite to the cell nucleus. These morphological changes were reversible. After washout of the inhibitor, the cell bodies recovered to normal shape within about 30-60 min while neurites started to grow again within 1 day. Okadaic acid (OA) which, compared to CL-A, is less potent as a PP-1 and equally potent as a PP-2A class inhibitor, caused neurite retraction only when added at more than a thousand-fold higher concentration than CL-A. Ca2+ levels within neurites and cell bodies remained stable and low during neurite retraction as measured with fura-2. However, cells treated with CL-A showed reduced activity of voltage-gated Ca2+ channels. The results suggest that the observed reversible changes in cell morphology occur at a constant low Ca2+ level and are most likely due to the inhibition of PP-1 class phosphatases.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Éteres Cíclicos/farmacología , Fura-2 , Toxinas Marinas , Neuritas/metabolismo , Neuritas/ultraestructura , Ácido Ocadaico , Células PC12 , RatasRESUMEN
We have studied the effects of protein kinase C (PKC) activators 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA) and 1-oleoyl-2-acetylglycerol (OAG) and of phosphatase inhibitors (okadaic acid and calyculin A) on voltage-activated Ca2+ and K+ channels in nerve-growth-factor-(NGF)-differentiated pheochromocytoma (PC12) cells. Whole-cell Ba2+ and K+ currents were recorded at room temperature with the patch-clamp technique. By using omega-conotoxin (CgTX) and isradipine, two specific Ca2+ channel blockers, we found three types of Ba2+ currents (IBa): (1) a omega-CgTX-sensitive IBa; (2) an isradipine-sensitive IBa; and (3) a omega-CgTX plus isradipine-resistant IBa. The external application of 4 beta-PMA or OAG down-modulated the isradipine-sensitive IBa whereas the two other IBa were not affected. 4 beta-PMA-induced inhibition of IBa was prevented by staurosporine (a protein kinase inhibitor) and PKC (19-31) (a specific PKC inhibitor). The delayed rectifier K+ current (IK) was unaffected by PKC activators. Both okadaic acid and calyculin A affected the components of the IBa in different manners. The presence of okadaic acid decreased the isradipine-sensitive IBa more than the omega-CgTX-sensitive IBa. The omega-CgTX plus isradipine-resistant IBa was not affected. Calyculin A down-modulated all three components of IBa to a similar degree. Our results suggest a differential modulation of voltage-activated Ca2+ and K+ channels by the PKC signalling pathway in NGF-differentiated PC12 cells.