RESUMEN
The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 µM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.
Asunto(s)
Encéfalo/irrigación sanguínea , Calcio/metabolismo , Células Endoteliales/efectos de los fármacos , Histamina/farmacología , Microvasos/citología , Óxido Nítrico/metabolismo , Línea Celular , Células Endoteliales/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , NADP/análogos & derivados , NADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca2+ from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+ using fluorescent Ca2+ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+ release from intracellular Ca2+ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.
Asunto(s)
Ácido Araquidónico/farmacología , Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Espermátides/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Cinética , Masculino , Microscopía Confocal , NADP/análogos & derivados , NADP/metabolismo , Fosfolipasas A2/metabolismo , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Salicilatos/farmacología , Sesterterpenos/farmacología , Espermátides/citología , Espermátides/metabolismo , Testículo/citología , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
NAADP (nicotinic acid adenine dinucleotide phosphate) has been proposed as a second messenger for glutamate in neuronal and glial cells via the activation of the lysosomal Ca2+ channels TPC1 and TPC2. However, the activities of glutamate that are mediated by NAADP remain unclear. In this study, we evaluated the effect of glutamate on autophagy in astrocytes at physiological, non-toxic concentration. We found that glutamate induces autophagy at similar extent as NAADP. By contrast, the NAADP antagonist NED-19 or SiRNA-mediated inhibition of TPC1/2 decreases autophagy induced by glutamate, confirming a role for NAADP in this pathway. The involvement of TPC1/2 in glutamate-induced autophagy was also confirmed in SHSY5Y neuroblastoma cells. Finally, we show that glutamate leads to a NAADP-dependent activation of AMPK, which is required for autophagy induction, while mTOR activity is not affected by this treatment. Taken together, our results indicate that glutamate stimulates autophagy via NAADP/TPC/AMPK axis, providing new insights of how Ca2+ signalling glutamate-mediated can control the cell metabolism in the central nervous system.
Asunto(s)
Astrocitos/metabolismo , Autofagia/fisiología , Canales de Calcio/metabolismo , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Western Blotting , Señalización del Calcio/fisiología , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Confocal , NADP/análogos & derivados , NADP/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Egg activation at fertilization is an excellent process for studying calcium regulation. Nicotinic acid adenine dinucleotide-phosphate (NAADP), a potent calcium messenger, is able to trigger calcium release, likely through two-pore channels (TPCs). Concomitantly, a family of ectocellular enzymes, the ADP-ribosyl cyclases (ARCs), has emerged as being able to change their enzymatic mode from one of nucleotide cyclization in formation of cADPR to a base-exchange reaction in the generation of NAADP. Using sea star oocytes we gain insights into the functions of endogenously expressed TPCs and ARCs in the context of the global calcium signals at fertilization. Three TPCs and one ARC were found in the sea star (Patiria miniata) that were localized in the cortex of the oocytes and eggs. PmTPCs were localized in specialized secretory organelles called cortical granules, and PmARCs accumulated in a different, unknown, set of vesicles, closely apposed to the cortical granules in the egg cortex. Using morpholino knockdown of PmTPCs and PmARC in the oocytes, we found that both calcium regulators are essential for early embryo development, and that knockdown of PmTPCs leads to aberrant construction of the fertilization envelope at fertilization and changes in cortical granule pH. The calcium signals at fertilization are not significantly altered when individual PmTPCs are silenced, but the timing and shape of the cortical flash and calcium wave are slightly changed when the expression of all three PmTPCs is perturbed concomitantly, suggesting a cooperative activity among TPC isoforms in eliciting calcium signals that may influence localized physiological activities.
Asunto(s)
ADP-Ribosil Ciclasa/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Embrión no Mamífero/metabolismo , Fertilización/fisiología , Oocitos/metabolismo , Estrellas de Mar/fisiología , ADP-Ribosil Ciclasa/genética , Animales , Canales de Calcio/genética , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Morfolinos/genética , NADP/análogos & derivados , NADP/metabolismo , Estrellas de Mar/metabolismoRESUMEN
Nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as an important modulator of Ca(2+) release from the endo-lysosomal system in a variety of cells by a new and ubiquitous class of endo-lysosomal ion channels known as the two-pore channels (TPCs). However, the role of TPCs in NAADP action in smooth muscle is not known. In the present work, we investigated the effects of NAADP in gastric smooth muscle cells and its ability to release Ca(2+) by TPCs. We show that Ca(2+) signals mediated by NAADP were inhibited by disrupting Ca(2+) handling by either acidic organelles (using bafilomycin A1) or the Endoplasmic Reticulum (using thapsigargin, ryanodine or 2-APB). Transcripts for endogenous TPC1 and TPC2 were readily detected and recombinant TPCs localized to the endosomes and/or lysosomes. Overexpression of wild-type TPCs but not pore mutants enhanced NAADP-mediated cytosolic Ca(2+) signals. Desensitizing the NAADP pathway inhibited Ca(2+)-responses to extracellular stimulation with carbachol but not ATP. Taken together, these results indicate that NAADP likely induces Ca(2+) release from the endolysosomal system through TPCs which is subsequently amplified via the ER in an agonist-specific manner. Thus, we suggest a second messenger role for NAADP in smooth muscle cells.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , NADP/análogos & derivados , Animales , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , NADP/farmacología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estómago/citologíaRESUMEN
Ca(2+) signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca(2+) signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca(2+) and pH. Ca(2+) fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca(2+)] increases in human sperm even in the absence of extracellular Ca(2+). Using LysoTracker, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker, suggesting that these stores are the targets of NAADP action.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , NADP/análogos & derivados , Espermatozoides/fisiología , Células Cultivadas , Humanos , Masculino , NADP/metabolismoRESUMEN
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing messenger that in many cells releases Ca(2+) from the endolysosomal system. Recent studies have shown that NAADP-induced Ca(2+) mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca(2+) signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca(2+) signals mediated by TPCs regulate autophagy.
Asunto(s)
Astrocitos/metabolismo , Autofagia/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , NADP/análogos & derivados , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Astrocitos/citología , Beclina-1 , Canales de Calcio/genética , Canales de Calcio/metabolismo , Células Cultivadas , Humanos , NADP/genética , NADP/metabolismo , RatasRESUMEN
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent activator of intracellular Ca2+ release in several vertebrate and invertebrate systems. The role of the NAADP system in physiological processes is being extensively investigated at the present time. The NAADP receptor and its associated Ca2+ pool have been hypothesized to be important in several physiological processes including fertilization, T cell activation, and pancreatic secretion. However, whether NAADP is a new second messenger or a tool for the discovery of a new Ca2+ channel is still an unanswered question. Research developed over the last two years has provided some important clues to whether NAADP is or not a physiological cellular messenger. In this short review, I will discuss some of these new findings that are helping us to find an answer to the important question: Is NAADP a second messenger or not?
Asunto(s)
Calcio/metabolismo , NADP/análogos & derivados , Sistemas de Mensajero Secundario/fisiología , Animales , Calcio/fisiología , Señalización del Calcio/fisiología , Humanos , Líquido Intracelular/fisiología , NADP/metabolismo , NADP/fisiologíaRESUMEN
Cells possess multiple intracellular Ca2+-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca2+ release. In the present study the mechanism of interaction between three intracellular Ca2+ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP3)-regulated Ca2+ stores, is explored. The data indicate that the NAADP Ca2+ pool could be used to sensitize the cADPR system. In contrast, the IP3 pool was not affected by the Ca2+ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca2+ release, promoted by Ca2+ released from the NAADP pool, is mediated by the mechanism of Ca2+-induced Ca2+ release. These data raise the possibility that the NAADP Ca2+ store may have a role as a regulator of the cellular sensitivity to cADPR.