RESUMEN
Calcium buffering by mitochondria plays a relevant physiological function in the regulation of Ca(2+) and exocytotic signals in mature chromaffin cells (CCs) from various adult mammals. Whether a similar or different role of mitochondrial Ca(2+) buffering is present in immature CCs at early life has not been explored. Here we present a comparative study in rat embryonic CCs and rat mother CCs, of various physiological parameters that are known to be affected by mitochondrial Ca(2+) buffering during cell activation. We found that the clearance of cytosolic Ca(2+) transients ([Ca(2+)]c) elicited by high K(+) was 7-fold faster in embryo CCs compared to mother CCs. This strongly suggests that at embryonic life, the mitochondria play a more significant role in the clearance of [Ca(2+)]c loads compared to adult life. Consistent with this view are the following results concerning the transient suppression of mitochondrial Ca(2+) buffering by protonophore FCCP, in embryonic CCs compared to mother CCs: (i) faster and greater inactivation of inward calcium currents, (ii) higher K(+)-elicited [Ca(2+)]c transients with 25-fold faster clearance, (iii) higher increase of basal catecholamine release and (iv) higher potentiation of K(+)-evoked secretion. These pronounced differences could be explained by two additional features (embryo versus mother CCs): (a) slower recovery of mitochondrial resting membrane potential after the application of a transient FCCP pulse and (b) greater relative density of the mitochondria in the cytosol. This tighter control by the mitochondria of Ca(2+) and exocytotic signals may be relevant to secure a healthy catecholamine secretory response at early life.
Asunto(s)
Señalización del Calcio , Células Cromafines/metabolismo , Exocitosis , Médula Suprarrenal/citología , Médula Suprarrenal/embriología , Médula Suprarrenal/metabolismo , Animales , Catecolaminas/farmacología , Células Cultivadas , Células Cromafines/efectos de los fármacos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Ratas , Ratas WistarRESUMEN
Mitochondria regulate cellular Ca(2+) oscillations, taking up Ca(2+) through its uniporter and releasing it through the mitochondrial sodium/calcium exchanger. The role of mitochondria in the regulation of Ca(2+) cycle has received much attention recently, as it is a central stage in neuronal survival and death processes. Over the last decades, the 4,1-benzothiazepine CGP37157 has been the only available blocker of the mitochondrial sodium/calcium exchanger, although it targets several other calcium transporters. We report the synthesis of 4,1-benzothiazepine derivatives with the goal of enhancing mitochondrial sodium/calcium exchanger blockade and selectivity, and the evaluation of their cytoprotective effect. The compound 4c presented an interesting neuroprotective profile in addition to an important blockade of the mitochondrial sodium/calcium exchanger. The use of this benzothiazepine could help to understand the physiological functions of the mitochondrial sodium/calcium exchanger. In addition, we hypothesize that a moderate blockade of the mitochondrial sodium/calcium exchanger would provide enhanced neuroprotection in neurons.
Asunto(s)
Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Tiazepinas/farmacología , Animales , Calcio/metabolismo , Bovinos , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cromafines/efectos de los fármacos , Células Cromafines/fisiología , Citoprotección , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Sodio/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/metabolismo , Tiazepinas/síntesis química , Tiazepinas/químicaRESUMEN
Adrenal chromaffin cells (CCs) express high-voltage activated calcium channels (high-VACCs) of the L, N and PQ subtypes; in addition, T-type low-VACCs are also expressed during embryo and neonatal life. Effects of the more frequently used T channel blockers NNC 55-0396 (NNC), mibefradil, and Ni2+ on the whole-cell Ba2+ current (IBa), the K+-elicited [Ca2+]c transients and catecholamine secretion have been studied in adult bovine CCs (BCCs) and rat embryo CCs (RECCs). NNC, mibefradil, and Ni2+ blocked BCC IBa with IC50 of 1.8, 4.9 and 70 µM, while IC50 to block IBa in RECCs were 2.1, 4.4 and 41 µM. Pronounced blockade of K+-elicited [Ca2+]c transients and secretion was also elicited by the three agents. However, the hypoxia-induced secretion (HIS) of catecholamine in RECCs was blocked substantially (75%) with thresholds concentrations of NCC (IC20 to block IBa); this was not the case for mibefradil and Ni2+ that required higher concentrations to block the HIS response. Thus, out of the three compounds, NNC seemed to be an adequate pharmacological tool to discern the contribution of T channels to the HIS response, without a contamination with high-VACC blockade.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Catecolaminas/metabolismo , Células Cromafines/efectos de los fármacos , Exocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Bario/metabolismo , Bencimidazoles/farmacología , Canales de Calcio/metabolismo , Bovinos , Hipoxia de la Célula/efectos de los fármacos , Células Cromafines/citología , Células Cromafines/metabolismo , Ciclopropanos/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Potenciales de la Membrana/efectos de los fármacos , Mibefradil/farmacología , Naftalenos/farmacología , Níquel/farmacología , Potasio/farmacología , RatasRESUMEN
The concept of stimulus-secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca(2+)) entry and this generates a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers the exocytotic release of catecholamines. The control of the [Ca(2+)](c) signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca(2+) from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca(2+) extrusion mechanisms, such as the plasma membrane Ca(2+)-stimulated ATPase, and the Na(+)/Ca(2+) exchanger. Computation of the rates of Ca(2+) fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca(2+) transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca(2+)](c) transients occurring at different cell compartments, and generated by redistribution and release of Ca(2+) by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo-endocytotic activities with energy production and protein synthesis.
Asunto(s)
Médula Suprarrenal/fisiología , Señalización del Calcio , Células Cromafines/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Orgánulos/metabolismo , Acetilcolina/metabolismo , Médula Suprarrenal/patología , Animales , Células Cromafines/ultraestructura , Citosol/metabolismo , Endocitosis , Epinefrina/metabolismo , Exocitosis , Humanos , Modelos Animales , Norepinefrina/metabolismo , Orgánulos/ultraestructuraRESUMEN
Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca2+ entry and neuronal Ca2+ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K+-evoked [Ca2+]c transients with an IC50 of 5.31 µM. At 10 µM, the compound decreased the amplitude and area of the Ca2+ transient elicited by challenging single fura-2-loaded cells with high K+, by 40% and 80%, respectively. This concentration also caused a blockade of K+-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca2+ currents (IC50=6.52 µM). At 10 µM, ITH/IQM also inhibited the Ca2+-dependent outward K+ current, leaving untouched the voltage-dependent component of IK. The inward Na+ current was unaffected. Inhibition of depolarisation-elicited Ca2+ entry, [Ca2+]c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.
Asunto(s)
Encéfalo/metabolismo , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Células Cromafines/metabolismo , Ácido Glutámico , Neuronas/metabolismo , Compuestos de Anilina/análisis , Animales , Encéfalo/patología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Bloqueadores de los Canales de Calcio/farmacología , Catecolaminas/metabolismo , Bovinos , Células Cromafines/citología , Células Cromafines/efectos de los fármacos , Exocitosis/efectos de los fármacos , Fura-2/análisis , Ácido Glutámico/análogos & derivados , Ácido Glutámico/farmacología , Transporte Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Técnicas de Placa-Clamp , Potasio/farmacología , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Xantenos/análisisRESUMEN
Studies on the bulk catecholamine release from fetal and neonatal rat adrenals, adrenal slices, or isolated chromaffin cells stimulated with high K(+), hypoxia, hypercapnia, or acidosis are available. However, a study analyzing the kinetics of quantal secretion is lacking. We report here such a study in which we compare the quantal release of catecholamines from immature rat embryo chromaffin cells (ECCs) and their mothers' (MCCs). Cell challenging with a strong depolarizing stimulus (75 mM K(+)) caused spike bursts having the following characteristics. ECCs released more multispike events and wave envelopes than MCCs. This, together with narrower single-spike events, a faster decay, and a threefold smaller quantal size suggest a faster secretory machinery in ECCs. Furthermore, with a milder stimulus (25 mM K(+)) enhanced Ca(2+) entry by L-type Ca(2+) channel activator BAY K 8644 did not change the kinetic parameters of single spikes in ECCs; in contrast, augmentation of Ca(2+) entry increased spike amplitude and width, quantal size, and decay time in MCCs. This suggests that in mature MCCs, the last exocytotic steps are more tightly regulated than in immature ECCs. Finally, we found that quantal secretion was fully controlled by L-type voltage-dependent Ca(2+) channels (VDCCs) in ECCs, whereas both L- and non-L VDCCs (N and PQ) contributed equally to secretion control in MCCs. Our results have the following physiological, pharmacological, and clinical relevance: 1) they may help to better understand the regulation of adrenal catecholamine release in response to stress during fetal life and delivery; 2) if clinically used, L-type Ca(2+) channel blockers may augment the incidence of sudden infant death syndrome (SIDS); and 3) so-called Ca(2+) promotors or activators of Ca(2+) entry through L-type VDCCs may be useful to secure a healthy catecholamine surge upon violent stress during fetal life, at birth, or to prevent the SIDS in neonates at risk.