RESUMO
Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 µM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 µM DCEBIO), but not by an IKCa channel opener (1 µM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 µM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.
Assuntos
Melatonina , Glândula Pineal , Canais de Potássio Cálcio-Ativados , Animais , Apamina/farmacologia , Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Melatonina/metabolismo , Glândula Pineal/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Pirazóis/farmacologia , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
Domain of Unknown Function 506 proteins are ubiquitous in plants. The phosphorus (P) stress-inducible REPRESSOR OF EXCESSIVE ROOT HAIR GROWTH1 (AtRXR1) gene encodes the first characterized DUF506. AtRXR1 inhibits root hair elongation by interacting with RabD2c GTPase. However, functions of other P-responsive DUF506 genes are still missing. Here, we selected two additional P-inducible DUF506 genes for further investigation. The expression of both genes was induced by auxin. Under P-stress, At3g07350 gene expressed ubiquitously in seedlings, whereas At1g62420 (AtRXR3) expression was strongest in roots. AtRXR3 overexpressors and knockouts had shorter and longer root hairs, respectively. A functional AtRXR3-green fluorescent protein fusion localized to root epidermal cells. Chromatin immunoprecipitation and quantitative reverse-transcriptase-polymerase chain reaction revealed that AtRXR3 was transcriptionally activated by RSL4. Bimolecular fluorescence complementation and calmodulin (CaM)-binding assays showed that AtRXR3 interacted with CaM in the presence of Ca2+ . Moreover, cytosolic Ca2+ ([Ca2+ ]cyt ) oscillations in root hairs of rxr3 mutants exhibited elevated frequencies and dampened amplitudes compared to those of wild type. Thus, AtRXR3 is another DUF506 protein that attenuates P-limitation-induced root hair growth through mechanisms that involve RSL4 and interaction with CaM to modulate tip-focused [Ca2+ ]cyt oscillations.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sinalização do Cálcio , Calmodulina/genética , Calmodulina/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/metabolismoRESUMO
Spermatozoa can be recovered in half of patients with non-obstructive azoospermia (NOA) via testicular sperm extraction (TESE) or microTESE. Intracytoplasmic sperm injection (ICSI) with the recovered spermatozoa has been established at IVF clinics to help these patients. Those who fail to achieve spermatozoa in testicular samples usually turn to donor spermatozoa or adoption. Instead of spermatozoa, only round spermatids are present in testicular biopsy in some NOA patients, a form of globozoospermia. Of these men, those who are unwilling to use donor spermatozoa still have the option to have their own biological child. In recent years, round spermatid injection (ROSI) has been developed as a potential option, with about 100 healthy babies born. However, the outcomes have so far been poor, with low pregnancy rates. One reason for this could be oocyte activation deficiency (OAD). Different from regular ICSI, round spermatids after ROSI do not induce calcium oscillation, which is critical for later oocyte activation and embryo development. Therefore, optimal assisted oocyte activation (AOA) stimulation is needed to mimic the physiological events. So far, a number of methods have been examined, including vigorous cytoplasm aspiration, calcium chloride injection, calcium ionophore treatment and electroporation. Over 100 healthy babies have been born, with no developmental or physiological abnormalities compared with regular children or those born from IVF and ICSI procedures, although some studies have found epigenetic modification. More recent studies have shown that electroporation for AOA has more credits than those tested so far. The overall positive outcome of ROSI is still poor and unstable, so it has not become a routine procedure in IVF clinics. Success rates would be improved with further optimization of AOA to enable patients to have their own genetic offspring.
Assuntos
Sêmen , Espermátides , Azoospermia , Feminino , Humanos , Masculino , Oócitos , Gravidez , Injeções de Esperma Intracitoplásmicas/métodos , EspermatozoidesRESUMO
The transmembrane docking of endoplasmic reticulum (ER) Ca2+-sensing STIM proteins with plasma membrane (PM) Orai Ca2+ channels is a critical but poorly understood step in Ca2+ signal generation. STIM1 protein dimers unfold to expose a discrete STIM-Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER-PM junctions. We reveal that each monomer within the SOAR dimer interacts independently with single Orai1 subunits to mediate cross-linking between Orai1 channels. Superresolution imaging and mobility measured by fluorescence recovery after photobleaching reveal that SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. A concatenated SOAR1 heterodimer containing one monomer point mutated at its critical Orai1 binding residue (F394H), although fully activating Orai channels, is completely defective in cross-linking Orai1 channels. Importantly, the naturally occurring STIM2 variant, STIM2.1, has an eight-amino acid insert in its SOAR unit that renders it functionally identical to the F394H mutant in SOAR1. Contrary to earlier predictions, the SOAR1-SOAR2.1 heterodimer fully activates Orai1 channels but prevents cross-linking and clustering of channels. Interestingly, combined expression of full-length STIM1 with STIM2.1 in a 5:1 ratio causes suppression of sustained agonist-induced Ca2+ oscillations and protects cells from Ca2+ overload, resulting from high agonist-induced Ca2+ release. Thus, STIM2.1 exerts a powerful regulatory effect on signal generation likely through preventing Orai1 channel cross-linking. Overall, STIM-mediated cross-linking of Orai1 channels is a hitherto unrecognized functional paradigm that likely provides an organizational microenvironment within ER-PM junctions with important functional impact on Ca2+ signal generation.
Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/química , Molécula 2 de Interação Estromal/metabolismo , Cálcio/metabolismo , Dimerização , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Domínios Proteicos , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genéticaRESUMO
Calcium (Ca2+) plays an important role in regulating the differentiation and function of osteoclasts. Calcium oscillations (Ca oscillations) are well-known phenomena in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption via calcineurin. Many modifiers are involved in the fine-tuning of Ca oscillations in osteoclasts. In addition to macrophage colony-stimulating factors (M-CSF; CSF-1) and RANKL, costimulatory signaling by immunoreceptor tyrosine-based activation motif-harboring adaptors is important for Ca oscillation generation and osteoclast differentiation. DNAX-activating protein of 12 kD is always necessary for osteoclastogenesis. In contrast, Fc receptor gamma (FcRγ) works as a key controller of osteoclastogenesis especially in inflammatory situation. FcRγ has a cofactor in fine-tuning of Ca oscillations. Some calcium channels and transporters are also necessary for Ca oscillations. Transient receptor potential (TRP) channels are well-known environmental sensors, and TRP vanilloid channels play an important role in osteoclastogenesis. Lysosomes, mitochondria, and endoplasmic reticulum (ER) are typical organelles for intracellular Ca2+ storage. Ryanodine receptor, inositol trisphosphate receptor, and sarco/endoplasmic reticulum Ca2+ ATPase on the ER modulate Ca oscillations. Research on Ca oscillations in osteoclasts has still many problems. Surprisingly, there is no objective definition of Ca oscillations. Causality between Ca oscillations and osteoclast differentiation and/or function remains to be examined.
Assuntos
Reabsorção Óssea/patologia , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Diferenciação Celular , Osteoclastos/citologia , Osteogênese , Animais , Reabsorção Óssea/metabolismo , Humanos , Osteoclastos/metabolismoRESUMO
Mitofusin 2 (Mfn2) is a mitochondrial outer membrane protein that participates in tethering mitochondria to the ER. Mitochondria-ER tethering has been demonstrated to play important roles in many cellular activities by regulating homeostasis of metabolites and calcium. Intracellular calcium signaling is crucial for the differentiation of osteoclasts, the bone-resorbing cells. In this study, we investigated whether Mfn2 plays a role in osteoclastogenesis by receptor activator of nuclear factor kappa B (RANKL) in primary cells. We found that RANKL increased Mfn2 expression during osteoclast formation from mouse bone marrow-derived macrophages (BMMs). When Mfn2 expression was suppressed in BMMs by using a siRNA-mediated gene knock-down system, osteoclast differentiation and activity of mature osteoclasts were reduced. Mfn2 knock-down also decreased the RANKL-mediated induction of NFATc1, the key transcription factor for osteoclast gene expression, without affecting c-Fos level. This effect on NFATc1 was associated with decreased calcium oscillation and calcineurin activity in Mfn2-deficient osteoclasts. Taken together, our results indicate that Mfn2 positively contributes to RANKL-induced osteoclast differentiation by regulating the calcium-calcieurin-NFATc1 axis, raising the importance of a previously under-recognized role of mitochondria in osteoclastogenesis.
Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteogênese , Transdução de Sinais , Animais , Sinalização do Cálcio , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismoRESUMO
Astrocytes in various brain regions exhibit spontaneous intracellular calcium elevations both in vitro and in vivo; however, neither the temporal pattern underlying this activity nor its function has been fully evaluated. Here, we utilized a long-term optical imaging technique to analyze the calcium activity of more than 4000 astrocytes in acute hippocampal slices as well as in the neocortex and hippocampus of head-restrained mice. Although astrocytic calcium activity was largely sparse and irregular, we observed a subset of cells in which the fluctuating calcium oscillations repeated at a regular interval of â¼30 s. These intermittent oscillations i) depended on type 2 inositol 1,4,5-trisphosphate receptors; ii) consisted of a complex reverberatory interaction between the soma and processes of individual astrocytes; iii) did not synchronize with those of other astrocytes; iv) did not require neuronal firing; v) were modulated through cAMP-protein kinase A signaling; vi) were facilitated under pathological conditions, such as energy deprivation and epileptiform hyperexcitation; and vii) were associated with enhanced hypertrophy in astrocytic processes, an early hallmark of reactive gliosis, which is observed in ischemia and epilepsy. Therefore, calcium oscillations appear to be associated with a pathological state in astrocytes.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Animais , Animais Recém-Nascidos , Hipocampo/fisiologia , Camundongos , Camundongos Knockout , Neocórtex/metabolismo , Neurônios/fisiologiaRESUMO
The neurotransmitters dopamine and serotonin control the peristaltic movement of the gut that consists of propagating waves of rhythmic contraction and relaxation. While intestinal cells of Cajal (ICC) serve as a pacemaker in the gut, the effect of dopamine on the pacemaker activity of ICC remains unknown. Here, we report that together with serotonin receptors, the dopamine receptor D2 contributes to maintaining [Ca²âº]i oscillations in ileum ICC. When the antagonist for the D2 receptor was applied to the cell cluster or the tissue culture prepared from muscle layers of the mouse small intestine, the amplitude of [Ca²âº]i oscillations in ICC declined after a transient increase. On the other hand, treatment with the D2 receptor agonist decreased the frequency of [Ca²âº]i oscillations in ICC. These results suggest that basal level activity of the D2 receptor is crucial for maintaining [Ca²âº]i oscillations in ICC. The decrease in the [Ca²âº]i oscillation amplitude upon the D2 receptor antagonist treatment was abrogated by antagonizing the serotonin receptor 5HT2, indicating an inhibitory effect of the 5HT2 receptor on the [Ca²âº]i oscillations. Together with the finding that treatment with the antagonist for the serotonin receptor 5HT3 completely eliminated [Ca²âº]i oscillations in ICC, our results show that dopamine and serotonin receptors cooperatively regulate pacemaker activity of ICC.
Assuntos
Células Intersticiais de Cajal , Animais , Cálcio , Dopamina , Camundongos , Marca-Passo Artificial , Receptores de SerotoninaRESUMO
Trichomes ('hair cells') on Arabidopsis thaliana stem and leaf surfaces provide a range of benefits arising from their shape and disposition. These include tempting herbivores to sample constitutive toxins before they reach the bulk of the tissue. We asked whether, in addition, small mechanical disturbances such as an insect can make elicit signals that might help the plant respond to herbivory. We imaged, pressed and brushed trichomes in several ways, most notably with confocal microscopy of trichomes transgenically provided with apoplastic pH reporter apo-pHusion and cytosolic Ca2+ reporter cameleon. In parallel, we modelled trichome wall mechanics with finite element analysis. The stimulated trichome focuses force on a pliant zone and the adjoining podium of the stalk. A buckling instability can further focus force on a skirt of cells surrounding the podium, eliciting oscillations of cytosolic Ca2+ and shifts in apoplastic pH. These observations represent active physiological response. Modelling establishes that the effectiveness of force focusing and buckling is due to the peculiar tapering wall structure of the trichome. Hypothetically, these active mechanosensing functions enhance toxin synthesis above constitutive levels, probably via a priming process, thus minimizing the costly accumulation of toxins in the absence of herbivore attack but assuring rapid build-up when needed.
Assuntos
Arabidopsis/metabolismo , Mecanotransdução Celular , Tricomas/metabolismo , Arabidopsis/fisiologia , Sinalização do Cálcio , Compartimento Celular , Parede Celular/metabolismo , Concentração de Íons de Hidrogênio , Tricomas/fisiologiaRESUMO
The pineal glands regulate circadian rhythm through the synthesis and secretion of melatonin. The stimulation of nicotinic acetylcholine receptor due to parasympathetic nerve activity causes an increase in intracellular Ca(2+) concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca(2+) oscillations that are evoked by membrane depolarization followed by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs). These Ca(2+) oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca(2+)-activated K(+) (BKCa) channel conductance on the regulation of Ca(2+) oscillation and melatonin production in rat pinealocytes. Spontaneous Ca(2+) oscillations were markedly enhanced by BKCa channel blockers (1 µM paxilline or 100 nM iberiotoxin). Nicotine (100 µM)-induced Ca(2+) oscillations were also augmented by paxilline. In contrast, spontaneous Ca(2+) oscillations were abolished by BKCa channel opener [3 µM 12,14-dichlorodehydroabietic acid (diCl-DHAA)]. Under whole cell voltage-clamp configurations, depolarization-elicited outward currents were significantly activated by diCl-DHAA and blocked by paxilline. Expression analyses revealed that the α and ß3 subunits of BKCa channel were highly expressed in rat pinealocytes. Importantly, the activity of BKCa channels modulated melatonin secretion from whole pineal gland of the rat. Taken together, BKCa channel activation attenuates these Ca(2+) oscillations due to depolarization-synchronized Ca(2+) influx through VDCCs and results in a recovery of reduced melatonin secretion during parasympathetic nerve activity. BKCa channels may play a physiological role for melatonin production via a negative-feedback mechanism.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Melatonina/metabolismo , Nicotina/farmacologia , Glândula Pineal/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Masculino , Glândula Pineal/citologia , Glândula Pineal/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Brief changes in the cytosolic and intra-organellar Ca2+ concentration serve as specific signals for various physiological processes. In mesothelial cells lining the surface of internal organs and the walls of body cavities, a re-entry in the cell cycle (G0-G1 transition) evoked by serum re-administration induces long-lasting Ca2+ oscillations with a slowly decreasing frequency. Individual mesothelial cells show a wide range of different oscillatory patterns within a single, supposedly homogenous cell population. Changes in the cytoplasmic Ca2+ concentration (ccyt) show baseline oscillatory patterns i.e., discrete Ca2+ transients starting from a constant basal ccyt level. The ER Ca2+ concentration (cER) displays a sawtooth wave at a semi-depleted ER state; the minimum level is reached just briefly after the maximal value for ccyt. These oscillations depend on plasmalemmal Ca2+ influx and on the inositol trisphosphate concentration [InsP3]; the Ca2+ influx is a crucial determinant of the oscillation frequency. Partial blocking of SERCA pumps modifies the oscillation frequency in both directions, i.e. increasing it in some cells and lowering it in others. Current mathematical models for Ca2+ oscillations mostly fail to reproduce two experimentally observed phenomena: the broad range of interspike intervals and constant basal ccyt levels between two Ca2+ spikes. Here we developed a new model based on--and fitted to--Ca2+ recordings of ccyt and cER recorded in primary mouse mesothelial cells. The model allowed for explaining many features of experimentally observed Ca2+ oscillations. We consider this model to be suitable to simulate various types of InsP3 receptor-based baseline Ca2+ oscillations.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Epitélio/metabolismo , Modelos Biológicos , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peritônio/citologia , Peritônio/metabolismo , Cultura Primária de CélulasRESUMO
UNLABELLED: Xanthotoxin (XAT) is extracted from the seeds of Ammi majus. Here, we reported that XAT has an inhibitory effect on osteoclastogenesis in vitro through the suppression of both receptor activator of nuclear factor-κB ligand (RANKL)-induced ROS generation and Ca(2+) oscillations. In vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, and restores bone strength in ovariectomized mice. INTRODUCTION: Excessive osteoclast formation and the resultant increase in bone resorption activity are key pathogenic factors of osteoporosis. In the present study, we have investigated the effects of XAT, a natural furanocoumarin, on the RANKL-mediated osteoclastogenesis in vitro and on ovariectomy-mediated bone loss in vivo. METHODS: Cytotoxicity of XAT was evaluated using bone marrow macrophages (BMMs). Osteoclast differentiation, formation, and fusion were assessed using the tartrate-resistant acid phosphatase (TRAP) stain, the actin cytoskeleton and focal adhesion (FAK) stain, and the fusion assay, respectively. Osteoclastic bone resorption was evaluated using the pit formation assay. Reactive oxygen species (ROS) generation and removal were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). Ca(2+) oscillations and their downstream signaling targets were then detected. The ovariectomized (OVX) mouse model was adopted for our in vivo studies. RESULTS: In vitro assays revealed that XAT inhibited the differentiation, formation, fusion, and bone resorption activity of osteoclasts. The inhibitory effect of XAT on osteoclastogenesis was associated with decreased intracellular ROS generation. XAT treatment also suppressed RANKL-induced Ca(2+) oscillations and the activation of the resultant downstream calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT downregulated the key osteoclastogenic factors nuclear factor of activated T cells c1 (NFATc1) and c-FOS. Our in vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice. CONCLUSION: Our findings indicate that XAT is protective against ovariectomy-mediated bone loss through the inhibition of RANKL-mediated osteoclastogenesis. Therefore, XAT may be considered to be a new therapeutic candidate for treating osteoporosis.
Assuntos
Reabsorção Óssea , Metoxaleno/farmacologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular , Feminino , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The possible association of intracellular Ca2+ with metastasis in human cancer cells is poorly understood. We have studied Ca2+ signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca2+ was measured using a membrane-permeant fluorescent Ca2+-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca2+ oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca2+ level could be induced by applying a high-K+ solution. Various parameters of the oscillations depended on extracellular Ca2+ and voltage-gated Na+ channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na+ channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca2+ oscillations. It is concluded that the functional voltage-gated Na+ channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca2+ activity. Possible mechanisms and consequences of the Ca2+ oscillations are discussed.
Assuntos
Neoplasias da Mama/patologia , Sinalização do Cálcio , Espaço Intracelular/metabolismo , Neoplasias da Próstata/patologia , Canais de Sódio Disparados por Voltagem/metabolismo , Espaço Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Masculino , Metástase NeoplásicaRESUMO
Functional positive cooperative activation of the extracellular calcium ([Ca(2+)]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca(2+)]o or amino acids elicits intracellular Ca(2+) ([Ca(2+)]i) oscillations. Here, we report the central role of predicted Ca(2+)-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca(2+)-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca(2+)]o. Next, we identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca(2+)]o and L-Phe in eliciting CaSR-mediated [Ca(2+)]i oscillations. The heterocommunication between Ca(2+) and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca(2+)]o signaling by positively impacting multiple [Ca(2+)]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca(2+)]o and amino acids into intracellular signaling events.
Assuntos
Cálcio/farmacologia , Receptores de Detecção de Cálcio/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Fenilalanina , Análise de Componente Principal , Estrutura Terciária de Proteína , Receptores de Detecção de Cálcio/química , Alinhamento de Sequência , TermodinâmicaRESUMO
Calcium (Ca(2+)) oscillations play a central role in varieties of cellular processes including fertilization and immune response, but controversy over the regulation mechanisms still exists. It has been known that nitric oxide (NO) dependently regulates Ca(2+) signaling in most physiopathological processes. Previous study indicated that eNOS translocation during some pathological process influences intracellular Ca(2+) homeostasis. In this study, we investigated the role and mechanism of NO on Ca(2+) release by overexpressing eNOS in cytoplasm (Cyto-eNOS) and endoplasmic reticulum (ER-eNOS) of HeLa cells. We found that the properties of Ca(2+) release were altered by the overexpression of eNOS. The amplitude and frequency of extracellular ATP (eATP)-induced Ca(2+) oscillation were enhanced in both Cyto-eNOS and ER-eNOS cells, respectively. Especially, the enhancement of the amplitude and frequency of the Ca(2+) oscillation was much more significant in the ER-eNOS cells than that of Cyto-eNOS cells. Further study indicated that this effect was abrogated by NO inhibitor, L-NAME, suggesting it was not an artificial result induced by ER stress. Furthermore, an up-regulated phosphorylation of phospholamban (PLB) was observed and the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) function was activated followed by the significant increase in the ER Ca(2+) load. Taken together, we revealed a novel regulatory mechanism of Ca(2+) oscillation.
Assuntos
Trifosfato de Adenosina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Citoplasma/metabolismo , Óxido Nítrico/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Citoplasma/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
Evidence increasingly shows that astrocytes play a pivotal role in brain physiology and pathology via calcium dependent processes, thus the characterization of the calcium dynamics in astrocytes is of growing importance. We have previously reported that the epidermal growth factor and basic fibroblast growth factor up-regulate the oscillation of the calcium releases that are induced by stimuli, including glutamate in cultured astrocytes. This calcium oscillation is assumed to involve protein kinase C (PKC), which is activated together with the calcium releases as a consequence of inositol phospholipid hydrolysis. In the present study, this issue has been investigated pharmacologically by using astrocytes cultured with and without the growth factors. The pharmacological activation of PKC largely reduced the glutamate-induced oscillatory and non-oscillatory calcium increases. Meanwhile, PKC inhibitors increased the total amounts of both calcium increases without affecting the peak amplitudes and converted the calcium oscillations to non-oscillatory sustained calcium increases by abolishing the falling phases of the repetitive calcium increases. Furthermore, the pharmacological effects were consistent between both glutamate- and histamine-induced calcium oscillations. These results suggest that PKC up-regulates the removal of cytosolic calcium in astrocytes, and this up-regulation is essential for calcium oscillation in astrocytes cultured with growth factors.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Proteína Quinase C/farmacologia , Proteína Quinase C/fisiologia , Animais , Células Cultivadas , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/farmacologia , Histamina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Ratos Wistar , Regulação para Cima/efeitos dos fármacosRESUMO
The pineal gland regulates circadian rhythm through the synthesis and secretion of melatonin. The rise of intracellular Ca(2+) concentration ([Ca(2+)]i) following nicotinic acetylcholine receptor (nAChR) stimulation due to parasympathetic nerve activity downregulates melatonin production. Important characteristics and roles of Ca(2+) mobilization due to nAChR stimulation remain to be clarified. We report here that spontaneous Ca(2+) oscillations can be observed in â¼15% of the pinealocytes in slice preparations from rat pineal glands when this dissociation procedure is done within 6 h from a dark-to-light change. The frequency and half-life of [Ca(2+)]i rise were 0.86 min(-1) and 19 s, respectively. Similar spontaneous Ca(2+) oscillations were recorded in 17% of rat pinealocytes that were primary cultured for several days. Simultaneous measurement of [Ca(2+)]i and membrane potential revealed that spontaneous Ca(2+) oscillations were triggered by periodic membrane depolarizations. Spontaneous Ca(2+) oscillations in cultured pinealocytes were abolished by extracellular Ca(2+) removal or application of nifedipine, a blocker of voltage-dependent Ca(2+) channel (VDCC). In contrast, blockers of intracellular Ca(2+)-release channels, 2-aminoethoxydiphenylborate and ryanodine, have no effect. Our results also reveal that, in 23% quiescent pinealocytes, Ca(2+) oscillations were observed following the withdrawal of nicotine. Norepinephrine-induced melatonin secretion from whole pineal glands was significantly decreased by the coapplication of acetylcholine (ACh). This inhibitory effect of ACh was attenuated by nifedipine. In conclusion, both spontaneous and evoked Ca(2+) oscillations are due to membrane depolarization following activation of VDCCs. This consists of VDCC α1F subunit, and the associated Ca(2+) influx can strongly regulate melatonin secretion in pineal glands.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Nicotina/farmacologia , Glândula Pineal/efeitos dos fármacos , Glândula Pineal/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Relação Dose-Resposta a Droga , Masculino , Técnicas de Cultura de Órgãos , Glândula Pineal/citologia , Ratos , Ratos WistarRESUMO
Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (-70%, P<0.05) without inducing necrosis. GCDCA increased the S1P levels in hepatocytes (P<0.05). GCDCA induced [Ca(2+)] oscillations in hepatocytes and co-treatment with the [Ca(2+)] chelator BAPTA repressed GCDCA-induced apoptosis. Ski-II inhibited the GCDCA-induced intracellular [Ca(2+)] oscillations. Transcripts of all five S1P receptors were detected in hepatocytes, of which S1PR1 and S1PR2 appear most dominant. Inhibition of S1PR1, but not S1PR2, reduced GCDCA-induced apoptosis by 20%. Exogenous S1P also significantly reduced GCDCA-induced apoptosis (-50%, P<0.05), however, in contrast to the GCDCA-induced (intracellular) SphK1 pathway, this was dependent on S1PR2 and not S1PR1. Our results indicate that SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca(2+)] signaling and activation of S1PR1.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , Hepatócitos/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Western Blotting , Caspase 3/metabolismo , Células Cultivadas , Detergentes/farmacologia , Fármacos Gastrointestinais/farmacologia , Ácido Glicoquenodesoxicólico/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Tiazóis/farmacologiaRESUMO
Cytokines released from microglia mediate defensive responses in the brain, but the underlying mechanisms are obscure. One proposed process is that nucleotide leakage or release from surrounding cells is sensed by metabotropic (P2Y) and ionotropic (P2X) purinergic receptors, which may trigger long-term intracellular Ca(2+) flux and tumor necrosis factor α (TNF-α) release. Indeed, 3h of exposure to ATP was required to evoke TNF-α release from a murine microglial cell line (MG5). A Ca(2+) chelator, ethylene glycol tetraacetic acid (EGTA), reduced ATP-induced TNF-α release, suggesting that intracellular Ca(2+) is important in this response. Therefore, Ca(2+) sensor genes (YC3.6) were transfected into MG5 cells to investigate the Ca(2+) dynamics underlying ATP-induced TNF-α release. The results demonstrated ATP-induced biphasic Ca(2+) mobilization mediated by P2Y (~5min) and P2X7 receptors (5-30min). Moreover, Ca(2+) spiking activity in cell processes progressively increased with a reduction in P2X7 receptor-mediated Ca(2+) elevation during 3-h ATP stimulation. Increased Ca(2+) spiking activity paralleled the reduction in thapsigargin-sensitive internal Ca(2+) stores, dendrite extension, and expression of macrophage scavenger receptors with collagenous structure. The Ca(2+) spiking activity was enhanced by a P2X7 receptor antagonist (A438079), but inhibited by a store-operated channel antagonist (SKF96365) or by co-transfection of small interference ribonucleic acid (siRNA) targeted on the channel component (Orai1). Furthermore, ATP-induced TNF-α release was enhanced by A438079 but was inhibited by SKF96365. Because store-operated channels (Stim1/Orai1) were expressed both in MG5 and primary microglial cultures, we suggest that P2X7 receptor signaling inhibits store-operated channels during ATP stimulation, and disinhibition of this process gates TNF-α release from microglial cells.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Microglia/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Trifosfato de Adenosina/farmacologia , Adenoviridae/efeitos dos fármacos , Adenoviridae/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Perfilação da Expressão Gênica , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Modelos Biológicos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tetrazóis/farmacologia , TransfecçãoRESUMO
Background: Increased osteoclast activity constitutes the primary etiology of excessive bone erosion in postmenopausal osteoporosis. ERp57, otherwise referred to as protein disulfide isomerase A3 (PDIA3), plays a crucial role in the regulation of intracellular calcium signaling. This is documented to exert a profound impact on osteoclast differentiation and functionality. Methods: To ascertain the potential role of ERp57 in disease progression, prevention, and treatment, network pharmacology and bioinformatics analyses were conducted in relation to postmenopausal osteoporosis and ERp57 inhibitor (Loc14). Then, subsequent experimental verifications were employed in vitro on osteoclast and osteoblast, and in vivo on ovariectomy (OVX) mice models. Results: Multiple enrichment analyses suggested that the "calcium signaling pathway" may constitute a potential avenue for therapeutic intervention by Loc14 in the treatment of postmenopausal osteoporosis. In vitro experiments demonstrated inhibition of ERp57 could block osteoclast differentiation and function by interfering with the expression of osteoclast marker genes (Traf6, Nfatc1, and Ctsk). Further mechanisms studies based on calcium imaging, qPCR, and WB established that ERp57 inhibitor (Loc14) could obstruct calcium oscillation in osteoclast precursor cells (OPCs) by limiting the entry sources of cytosolic Ca2+ and interfering with calmodulin/calcineurin/Nfatc1 pathway. Evidence from Micro-CT scanning and double calcein labeling confirmed that the application of Loc14 in vivo could alleviate bone loss and partially reversed the osteogenic impairment caused by OVX in mice. Conclusions: Our findings proved the suppressive effects of Loc14 on osteoclastogenesis via attenuating calcium oscillation and associated singling pathways, providing ERp57 as a potential therapeutic target for postmenopausal osteoporosis.