RESUMEN
Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1-/- and Epac2-/- mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.
Asunto(s)
Canales de Calcio/genética , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Enfermedades Renales/genética , Natriuresis/genética , Sodio/orina , Canales Catiónicos TRPV/genética , Animales , Biomarcadores/orina , Canales de Calcio/biosíntesis , Células Cultivadas , Modelos Animales de Enfermedad , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/genética , Canales Catiónicos TRPV/biosíntesisRESUMEN
BACKGROUND: Intracerebral hemorrhage (ICH), a common cerebrovascular disease, seriously threatens human health and has severe secondary injuries, while existing treatment methods have many limitations. α2δ-1 is a subunit of voltage-gated Ca2+ channels (VGCCs) and can act on glutamate receptor N-methyl-D-aspartate receptors (NMDARs) to relieve neuropathic pain. METHODS: We first performed ICH modeling on WT mice and Cacna2d1 knockout (KO) mice. The expression levels of GluN1 and α2δ-1 were measured by Western blot and q-PCR, and the interaction between the two proteins was evaluated by co-precipitation. The neuronal apoptosis was detected by the TUNEL assay, and the expression levels of inflammatory factors were assessed by ELISA. The nerve functions of mice were evaluated using behavioral experiments including corner turn test and forelimb use asymmetry. Cerebral hematoma was indicated by brain water content and lesion volume. RESULTS: ICH up-regulated the expression levels of α2δ-1 and GluN1. KO of Cacna2d1 significantly reduced the ICH-induced apoptosis. The treatment of gabapentin on α2δ-1 also significantly reduced the occurrence of apoptosis. KO of Cacna2d1 also reduced the ICH-induced levels of inflammatory factors. Furthermore, neural functions were also significantly improved. CONCLUSION: Cacna2d1 KO alleviates cerebral hematoma in ICH mice, exhibits a significant regulating effect on its secondary injuries such as neuronal apoptosis and inflammation, and restores the nerve functions of ICH mice. Loss of Cacna2d1 can provide useful therapeutic clues for ICH treatment.
Asunto(s)
Lesiones Encefálicas/metabolismo , Canales de Calcio/biosíntesis , Hemorragia Cerebral/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Receptores de N-Metil-D-Aspartato/biosíntesis , Animales , Lesiones Encefálicas/patología , Canales de Calcio/deficiencia , Canales de Calcio/genética , Hemorragia Cerebral/patología , Hemorragia Cerebral/prevención & control , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM+ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM+ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206+ and peritubular MHC II+ macrophages, with higher levels in CD206+ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2+ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM+ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206+MHC II+ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2+ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM+ mice. The changes in testis are distinct from the described alterations in other organs of AROM+, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM+ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM+ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.
Asunto(s)
Canales de Calcio/fisiología , Macrófagos/metabolismo , Orquitis/metabolismo , Canales Catiónicos TRPV/fisiología , Testículo/metabolismo , Glándulas Suprarrenales/metabolismo , Factores de Edad , Animales , Aromatasa/genética , Encéfalo/metabolismo , Canales de Calcio/biosíntesis , Canales de Calcio/genética , Modelos Animales de Enfermedad , Genotipo , Infertilidad Masculina/metabolismo , Lectinas Tipo C/análisis , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/análisis , Ratones , Ratones Transgénicos , NADPH Oxidasa 2/biosíntesis , NADPH Oxidasa 2/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Superficie Celular/análisis , Espermatogénesis , Canales Catiónicos TRPV/biosíntesis , Canales Catiónicos TRPV/genética , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Microglia proliferation is critical for proper development and function of the central nervous system (CNS), while dysregulation of proliferation contributes to pathology. We recently reported that male inducible nitric oxide synthase knockout (iNOS-/-) mice displayed significantly more proliferating microglia in their postnatal cortex than age-matched wildtype (WT) male mice. Moreover, nitric oxide (NO) signaling in mouse microglia greatly upregulates calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. Considering that TRPV2 activity restricts astrocytic proliferation within glioma tissues, we investigated the roles of iNOS/NO signaling and TRPV2 expression in the regulation of microglial proliferation in vitro using assays of calcium imaging, immunocytochemistry, western blot, and polymerase chain reaction. Results showed that non-dividing microglia exhibited substantially higher expression of TRPV2 on the plasma membrane and significantly larger calcium influx through TRPV2 channels in comparison to dividing microglia. Additionally, non-dividing WT microglia exhibited significantly more NO production than dividing WT microglia. Furthermore, the NO-donor NOC18 increased the nuclear translocation of nuclear factor of activated T-cells cytoplasmic 2 (NFATC2) and the mRNA of the cyclin-dependent kinase inhibitor p21 and decreased the percentage of dividing WT and iNOS-/- microglia in culture. Importantly, the presence of the TRPV2 inhibitor tranilast abolished these effects of NOC18. Together, results from this study indicated that iNOS/NO signaling inhibits microglial proliferation through TRPV2-mediated calcium influx, nuclear translocation of the transcription factor NFATC2, and p21 expression. [Figure: see text].
Asunto(s)
Canales de Calcio/biosíntesis , Señalización del Calcio/fisiología , Microglía/metabolismo , Factores de Transcripción NFATC/biosíntesis , Óxido Nítrico/biosíntesis , Canales Catiónicos TRPV/biosíntesis , Quinasas p21 Activadas/biosíntesis , Animales , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Línea Celular , Proliferación Celular/fisiología , Células Cultivadas , Femenino , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción NFATC/genética , Óxido Nítrico/genética , Canales Catiónicos TRPV/genética , Transcripción Genética/fisiología , Quinasas p21 Activadas/genéticaRESUMEN
Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.
Asunto(s)
Canales de Calcio/biosíntesis , Trastorno del Sistema de Conducción Cardíaco/genética , Trastorno del Sistema de Conducción Cardíaco/fisiopatología , Regulación de la Expresión Génica , Distrofia Miotónica/complicaciones , Animales , Canales de Calcio/genética , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Humanos , RatonesRESUMEN
Microglia phagocytosis is critical for central nervous system development, and dysregulation of phagocytosis may contribute to a variety of neurological disorders. During initial stages of phagocytosis, microglia display increased nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) activity and amplified calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. The present study investigated the regulatory role of iNOS/NO signaling in microglial phagocytosis and TRPV2 channel activation using phagocytosis assay, calcium imaging, patch clamp electrophysiology, immunocytochemistry, and immunoblot assays. Results showed that primary microglia from iNOS-knockout (iNOS-/- ) mice exhibited substantial deficits in phagocytic capacity and TRPV2 channel activity relative to wild-type (WT) controls. Specifically, iNOS-/- microglia displayed a lower level of TRPV2 protein localized on the plasma membrane (PM) without any significant change in the mRNA levels of Fc-gamma receptors and TRPV2. In addition, iNOS-/- microglia, unlike their WT controls, failed to elicit a calcium influx in response to application of the TRPV2-agonist 2-aminoethoxydiphenyl borate (2APB). Importantly, the phagocytic capacity and the PM expression and activity of TRPV2 in iNOS-/- microglia were largely corrected by pretreatment with NO-donors. Accordingly, the 2APB-evoked calcium influx and the PM expression of TRPV2 in WT microglia were significantly decreased by selective inhibition of iNOS, protein kinase-G (PKG), or phosphoinositide-3-kinase (PI3K), respectively. Together, results from this study indicated that iNOS/NO signaling upregulates microglial phagocytosis and increases TRPV2 trafficking to the PM via PKG/PI3K dependent pathway(s).
Asunto(s)
Canales de Calcio/biosíntesis , Membrana Celular/metabolismo , Microglía/metabolismo , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico/metabolismo , Fagocitosis/fisiología , Canales Catiónicos TRPV/biosíntesis , Animales , Canales de Calcio/genética , Membrana Celular/genética , Células Cultivadas , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Canales Catiónicos TRPV/genética , Regulación hacia Arriba/fisiologíaRESUMEN
The purpose of this study was to investigate the effect of clomiphene citrate and human chorionic gonadotropin (HCG) on the structural changes, as well as the evaluation of the expression of cation channel sperm-associated protein 1 (CatSper1), cation channel sperm-associated protein 2 (CatSper2), luteinizing hormone/choriogonadotropin receptor (LHCGR), and steroidogenic factor 1 (SF1) genes in testicular tissue of rats. All rats divided into five groups as follows; G1 as the control group that received normal saline, G2 received olive oil, G3 received 100 IU/kg HCG, G4 received 5 mg/kg clomiphene citrate, and G5 received 5 mg/kg clomiphene citrate and 100 IU/kg HCG. At the end of the experiment period, Day 56, blood samples were taken and the serum was isolated. Then, histomorphometric analysis, hormonal assess, and real-time polymerase chain reaction to measure the expression of CatSper1, CatSper2, LHCGR, and SF1 genes were performed. The results showed that the concentrations of testosterone, follicle-stimulating hormone, and luteinizing hormone were decreased in the G4 group, whereas these parameters were increased in the G3 group. A comparison of the sperm quality indicated a significant reduction in the quality of sperm cells in the G4 group compared with other groups. The quality of sperm was significantly enhanced in the G3 and G5 groups in comparison with the G1 group. Also, our findings demonstrated that the expression of CatSper1, CatSper2, LHCGR, and SF1 genes were significantly elevated in the G3 group when compared with other experimental groups. According to the obtained results, it seems that clomiphene citrate reduces the process of spermatogenesis and the detrimental impacts of this compound would be neutralized by the administration of HCG.
Asunto(s)
Canales de Calcio/biosíntesis , Gonadotropina Coriónica/efectos adversos , Clomifeno/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Receptores de HL/biosíntesis , Proteínas de Plasma Seminal/biosíntesis , Factor Esteroidogénico 1/biosíntesis , Testículo/metabolismo , Animales , Gonadotropina Coriónica/farmacología , Clomifeno/farmacología , Humanos , Masculino , Ratas , Ratas Wistar , Espermatogénesis/efectos de los fármacos , Testículo/patologíaRESUMEN
The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca2+) signaling. Ca2+ is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca2+ signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca2+ signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca2+ stores was decreased in patients with COPD and that the Ca2+ influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca2+ release, significant in smoker subjects, and to a decrease in Ca2+ influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca2+ signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca2+ influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca2+ signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.
Asunto(s)
Bronquios/citología , Canales de Calcio/fisiología , Calcio/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Mucosa Respiratoria/metabolismo , Fumar/metabolismo , Adulto , Anciano , Benzamidas/farmacología , Bronquios/metabolismo , Canales de Calcio/biosíntesis , Canales de Calcio/genética , Señalización del Calcio , Células Cultivadas , Cilios/efectos de los fármacos , Cilios/fisiología , Citocinas/metabolismo , Retículo Endoplásmico/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-8/biosíntesis , Masculino , Persona de Mediana Edad , Mucina 5AC/biosíntesis , Moco/metabolismo , Pirazoles/farmacología , Mucosa Respiratoria/patología , Transducción de Señal/fisiología , Humo , FumadoresRESUMEN
Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α2δ-2 can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ-2 is essential for the trans-synaptic mechanism. The novel function of α2δ-2 identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α2δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α2δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α2δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.
Asunto(s)
Axones/metabolismo , Canales de Calcio/biosíntesis , Terminales Presinápticos/metabolismo , Receptores de GABA-A/metabolismo , Potenciales Sinápticos/fisiología , Animales , Axones/química , Encéfalo/citología , Encéfalo/fisiología , Canales de Calcio/análisis , Células Cultivadas , Técnicas de Cocultivo , Femenino , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Terminales Presinápticos/química , Subunidades de Proteína/análisis , Subunidades de Proteína/biosíntesis , Receptores de GABA-A/análisisRESUMEN
CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+- permeable channel, a protein in the flagellum essential for sperm hyperactivation. Previous studies have shown that the murine Catsper1 gene promoter is regulated by different Sox proteins. Likewise, it is acknowledged that the human CATSPER1 gene promoter sequence is enriched in potential interaction sites for the sex-determining region Y gene (SRY), which suggest a novel regulatory transcriptional mechanism for CatSper1 channel expression. Therefore, in this work, we sought to determine whether the human CATSPER1 gene expression is regulated by the SRY transcription factor. To this end, a series of deletions and mutations were introduced in the wild- type CATSPER1 gene promoter to eliminate the SRY sites, and the different constructs were tested for their ability to activate transcription in human embryonic kidney and murine spermatogonial germ cell lines (HEK-293 and GC1-spg, respectively) using luciferase assays. In addition, by using a strategy that combines electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) we investigated whether the CATSPER1 gene expression is regulated by the SRY transcription factor both in vitro and in vivo. Our results show that the transcriptional factor SRY specifically binds to different sites in the promoter sequence and has the ability to control CATSPER1 gene transcription.
Asunto(s)
Canales de Calcio/biosíntesis , Regulación de la Expresión Génica , Elementos de Respuesta , Proteína de la Región Y Determinante del Sexo/metabolismo , Espermatogonias/metabolismo , Transcripción Genética , Animales , Canales de Calcio/genética , Células HEK293 , Humanos , Masculino , Ratones , Proteína de la Región Y Determinante del Sexo/genética , Espermatogonias/citologíaRESUMEN
PURPOSE: This study aimed to explore whether bone marrow- (BM-) derived endothelial progenitor cells (EPCs) contributing to monocrotaline- (MCT-) induced pulmonary arterial hypertension (PAH) in rats via modulating store-operated Ca2+ channels (SOC). METHODS: Sprague Dawley (SD) rats were assigned into MCT group (n = 30) and control group (n = 20). Rats in MCT group were subcutaneously administered with 60 mg/kg MCT solution, and rats in control group were injected with equal amount of vehicle. After 3 weeks of treatment, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) of two groups were measured, and BM-derived EPCs were isolated. Immunochemistry identification and vasculogenesis detection of EPCs were then performed. [Ca2+]cyt measurement was performed to detect store-operated calcium entry (SOCE) in two groups, followed by determination of Orai and canonical transient receptor potential (TRPC) channels expression. RESULTS: After 3 weeks of treatment, there were significant increases in RVSP and RVHI in MCT group compared with control group, indicating that MCT successfully induced PAH in rats. Moreover, the SOCE ([Ca2+]cyt rise) in BM-derived EPCs of MCT group was lower than that of control group. Furthermore, the expression levels of Orai3, TRPC1, TRPC3, and TRPC6 in BM-derived EPCs were decreased in MCT group in comparison with control group. CONCLUSIONS: The SOC activities were inhibited in BM-derived EPCs of MCT-treated rats. These results may be associated with the depressed expression of Orai3, TRPC1, TRPC3, and TRPC6, which are major mediators of SOC.
Asunto(s)
Células de la Médula Ósea/metabolismo , Canales de Calcio/biosíntesis , Células Progenitoras Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipertensión Pulmonar , Monocrotalina/toxicidad , Animales , Células de la Médula Ósea/patología , Células Progenitoras Endoteliales/patología , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Ratas , Ratas Sprague-DawleyRESUMEN
In recent years estradiol has emerged as a potential regulator of transient receptor potential vanilloid (TRPV) cationic channels in the peripheral tissues and sensory neurons, however, its analogous role in the CNS is poorly understood. TRPV channels modulate Ca2+ signalling, neurotransmission and behaviour, and expression of these ion channels and estrogen receptors show a great degree of overlap in different brain regions. Herein, we probe if Trpv1-6 genes contain estrogen receptor-binding sites and if their expression in different brain regions is modulated during estrous cycle. Bioinformatics analysis of the mouse Trpv1-6 gene sequences showed presence of putative functional estrogen response element in their promoter regions. Using qRT-PCR, Trpv1-6 mRNA expression was observed in the olfactory bulb, cortex, hypothalamus, hippocampus, brainstem, and cerebellum of mouse. In these regions, compared to estrus, metestrus, and diestrus, reduced levels of Trpv1 and Trpv5 but elevated Trpv2 and Trpv6 mRNA levels were observed during proestrus. Lower levels of Trpv3 and Trpv4 mRNAs were seen during estrus but higher expression of Trpv3 during metestrus and diestrus, and Trpv4 during proestrus was observed. Estradiol seems to regulate Trpv1/Trpv5 and Trpv2/Trpv6 mRNA expression in opposite manner. Except Trpv4 mRNA expression in the hippocampus and Trpv6 expression in the olfactory bulb, hippocampus and brainstem, expression of other members of TRPV subfamily in distinct brain regions of male mice was comparable to those in metestrus and diestrus mice. We suggest that the circulating levels of estradiol during the estrous cycle may differentially regulate the activity of TRPV1-6 ion channels in the brain.
Asunto(s)
Ciclo Estral/metabolismo , Canales Catiónicos TRPV/biosíntesis , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Mapeo Encefálico/métodos , Canales de Calcio/biosíntesis , Canales de Calcio/genética , Canales de Calcio/metabolismo , Estradiol/farmacología , Estrógenos/metabolismo , Ciclo Estral/genética , Femenino , Expresión Génica , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
Voltage-gated Ca2+ channels (Cav) are essential for pancreatic beta cell function as they mediate Ca2+ influx, which leads to insulin exocytosis. The ß3 subunit of Cav (Cavß3) has been suggested to regulate cytosolic Ca2+ ([Ca2+]i) oscillation frequency and insulin secretion under physiological conditions, but its role in diabetes is unclear. Here, we report that islets from diabetic mice show Cavß3 overexpression, altered [Ca2+]i dynamics, and impaired insulin secretion upon glucose stimulation. Consequently, in high-fat diet (HFD)-induced diabetes, Cavß3-deficient (Cavß3-/-) mice showed improved islet function and enhanced glucose tolerance. Normalization of Cavß3 expression in ob/ob islets by an antisense oligonucleotide rescued the altered [Ca2+]i dynamics and impaired insulin secretion. Importantly, transplantation of Cavß3-/- islets into the anterior chamber of the eye improved glucose tolerance in HFD-fed mice. Cavß3 overexpression in human islets also impaired insulin secretion. We thus suggest that Cavß3 may serve as a druggable target for diabetes treatment.
Asunto(s)
Canales de Calcio/genética , Diabetes Mellitus Experimental/terapia , Islotes Pancreáticos/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/biosíntesis , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Humanos , Secreción de Insulina , Trasplante de Islotes Pancreáticos , Masculino , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/genéticaRESUMEN
BACKGROUND: The CATSPER1 gene encodes a CATSPER channel protein that selectively permeates Ca2+ ions, and CATSPER expression in sperm is essential for flagellum hyperactivation and, thus, male fertility. Little is known regarding the transcriptional regulation of CATSPER1, but previous studies have performed in silico analyses of transcription factor binding sites, including three CRE sites designated 0-2, in which CRE0 is located near the transcription start site. OBJETIVES: We investigate if overexpression of CREB-A and CREMτ transcription factors might regulate CATSPER1 expression. MATERIAL AND METHODS: In this study, the transcriptional regulation of the CATSPER1 gene by CREB-A and CREMτ transcriptions factors was determined by dual-luciferase assays in HEK293 and GC1-spg cells, and important CRE sites were mutated and analyzed for transcriptional regulation. RESULTS: The deletion of the CRE1 site dramatically increased the transcriptional activity of the CATSPER1 promoter in HEK293 and GC1-spg cells. In HEK293 cells, the CREB-A transcription factor positively regulated CATSPER1 gene expression, while the presence of CREB-A and CREMτ factors synergistically enhanced promoter activity in these cells. In contrast, deletion of CRE0 prevented any transcriptional activity of the CATSPER1 promoter in GC1-spg spermatogonial cells, but expression of either CREB-A or CREMτ restored such transcriptional activity. CONCLUSIONS: The human CATSPER1 promoter is positively regulated in vitro by CREB-A in HEK293 and GC1-spg cells. Both lines showed differential transcriptional regulation, which was defined by the factors and coactivators present in each cell line as well as the context in which the CRE sites were found in the promoter.
Asunto(s)
Canales de Calcio/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Sitios de Unión/genética , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular Tumoral , Células HEK293 , Humanos , Masculino , Unión Proteica , Eliminación de Secuencia/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
BACKGROUND: The mechanism of podocyte apoptosis is not fully understood. In addition, the role of the inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (Grp75)/voltage-dependent anion channel 1 (VDAC1)/mitochondrial calcium uniporter (MCU) calcium regulation axis, which is located at sites of endoplasmic reticulum (ER) mitochondria coupling, in the mechanism of podocyte apoptosis is unclear. This study aimed to understand the roles of this axis in podocyte apoptosis and explore potential targets for podocyte protection. METHODS: The expression of IP3R, Grp75, VDAC1, and MCU and mitochondrial Ca2+ were analyzed during Adriamycin- or angiotensin II-induced apoptosis in cultured mouse podocytes. The interaction between IP3R, Grp75, and VDAC1 was investigated using co-immunoprecipitation experiments. The effects of IP3R, Grp75, and MCU agonists and antagonists on mitochondrial Ca2+ and apoptosis were investigated in cultured podocytes. The podocyte-protective effects of an MCU inhibitor were further investigated in rats with Adriamycin-induced nephropathy. RESULTS: Increased expression of IP3R, Grp75, VDAC1 and MCU, enhanced interaction among the IP3R-Grp75-VDAC1 complex, mitochondrial Ca2+ overload, and increased active caspase-3 levels were confirmed during Adriamycin- or angiotensin II-induced mouse podocyte apoptosis. Agonists of this axis facilitated mitochondrial Ca2+ overload and podocyte apoptosis, whereas specific antagonists against IP3R, Grp75, or MCU prevented mitochondrial Ca2+ overload and podocyte apoptosis. A specific MCU inhibitor prevented Adriamycin-induced proteinuria and podocyte foot process effacement in rats. CONCLUSIONS: This study identified a novel pathway in which the IP3R-Grp75-VDAC1-MCU calcium regulation axis mediated podocyte apoptosis by facilitating mitochondrial Ca2+ overload. Antagonists that inhibit Ca2+ transfer from ER to mitochondria protected mouse podocytes from apoptosis. An MCU inhibitor protected podocytes and decreased proteinuria in rats with Adriamycin-induced nephropathy. Therefore, antagonists to this pathway have promise as novel podocyte-protective drugs.
Asunto(s)
Calcio/fisiología , Doxorrubicina/toxicidad , Enfermedades Renales/metabolismo , Compuestos Macrocíclicos/farmacología , Oxazoles/farmacología , Podocitos/metabolismo , Proteinuria/metabolismo , Adenosilhomocisteinasa/antagonistas & inhibidores , Adenosilhomocisteinasa/biosíntesis , Animales , Antibióticos Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Canales de Calcio/biosíntesis , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/biosíntesis , Enfermedades Renales/inducido químicamente , Enfermedades Renales/tratamiento farmacológico , Compuestos Macrocíclicos/uso terapéutico , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/biosíntesis , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxazoles/uso terapéutico , Podocitos/efectos de los fármacos , Proteinuria/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Canal Aniónico 1 Dependiente del Voltaje/antagonistas & inhibidores , Canal Aniónico 1 Dependiente del Voltaje/biosíntesisRESUMEN
BACKGROUND: HF incurs high disease burden, and the effectiveness of known HF treatments is unsatisfactory. Therefore, seeking novel therapeutic target of HF is important. The present study aimed to investigate the role of the mitochondrial calcium uniporter (MCU) and its relationship with autophagy in overload-induced heart failure (HF). METHODS AND RESULTS: In both early-stage and end-stage of pressure overload-induced HF, MCU appeared up-regulated along with heart enlargement, increased microtubule-associated proteins 1A/1B light chain 3B (LC3B) II/I ratio and autophagosome content, damaged cardiac function, and ventricular asynchrony. However, sequestosome-1 (SQSTM1/p62) level decreased indicating blockaded autophagic flux. Seven-week administration of MCU inhibitor ruthenium red improved cardiac function and mitigated its pathological change. MCU inhibition maintained mitochondrial integrity, increased LC3B II/I ratio, up-regulated Parkin and Pink1, and down-regulated SQSTM1/p62. MCU inhibition also alleviated ventricular asynchrony of HF, and this might be related to connexin-43 up-regulation. In vitro study validated intervention on MCU leading to elevation of autophagy and mitophagy. MCU inhibition could partly prevent from excessive cellular enlargement induced by isoprenaline. CONCLUSIONS: In summary, MCU inhibition played an important role in pressure overload-induced heart failure through autophagy and mitophagy enhancement, and intervention on MCU offered cardioprotective effects. To our knowledge, the role of MCU in HF and its relationship with autophagy and mitophagy are firstly disclosed. Moreover, our study suggests that MCU inhibition could be explored as a novel therapeutic concept in HF.
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Autofagia/fisiología , Canales de Calcio/biosíntesis , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/prevención & control , Rojo de Rutenio/farmacología , Animales , Autofagia/efectos de los fármacos , Células Cultivadas , Insuficiencia Cardíaca/diagnóstico por imagen , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Rojo de Rutenio/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Spinocerebellar ataxia (SCA) type 6 is an autosomal dominant disease affecting cerebellar degeneration. Clinically, it is characterized by pure cerebellar dysfunction, slowly progressive unsteadiness of gait and stance, slurred speech, and abnormal eye movements with late onset. Pathological findings of SCA6 include a diffuse loss of Purkinje cells, predominantly in the cerebellar vermis. Genetically, SCA6 is caused by expansion of a trinucleotide CAG repeat in the last exon of longest isoform CACNA1A gene on chromosome 19p13.1-p13.2. Normal alleles have 4-18 repeats, while alleles causing disease contain 19-33 repeats. Due to presence of a novel internal ribosomal entry site (IRES) with the mRNA, CACNA1A encodes two structurally unrelated proteins with distinct functions within an overlapping open reading frame (ORF) of the same mRNA: (1) α1A subunit of P/Q-type voltage gated calcium channel; (2) α1ACT, a newly recognized transcription factor, with polyglutamine repeat at C-terminal end. Understanding the function of α1ACT in physiological and pathological conditions may elucidate the pathogenesis of SCA6. More importantly, the IRES, as the translational control element of α1ACT, provides a potential therapeutic target for the treatment of SCA6.
Asunto(s)
Canales de Calcio , Cromosomas Humanos Par 19 , Exones , Células de Purkinje , Ataxias Espinocerebelosas , Expansión de Repetición de Trinucleótido , Animales , Canales de Calcio/biosíntesis , Canales de Calcio/genética , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 19/metabolismo , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Células de Purkinje/metabolismo , Células de Purkinje/patología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/patologíaRESUMEN
Calcium is important for physiological functioning in many tissues and is essential in mucus secretion and muscle contraction. Intracellular concentrations of calcium are regulated by calcium-related proteins, such as transient receptor potential cation channel subfamily V member 4 (TRPV 4), TRPV6, Calbindin-D9k (CaBP-9k), sodium-calcium exchanger (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1). In this study, the relationship between secretion of pulmonary mucus and calcium regulation was investigated. To confirm the effect of steroid hormones, immature mice were injected with estrogen (E2) or progesterone (P4), and mature mice were injected with dexamethasone (DEX). Subsequently, the location and expression of TRPV4, TRPV6, CaBP-9k, NCX1, and PMCA1 in lung tissue were examined. Periodic acid-Schiff staining was performed to investigate functional aspects of the protein expression. There were no significant differences in calcium-related gene expression in E2- and P4-treated mice, but TRPV4, NCX1, and PMCA1 were increased in DEX-treated mice and were recovered by RU486 treatment. DEX induces the expression of calcium-related proteins through the glucocorticoid receptor-mediated pathway and may involve decreased mucin secretion in the bronchiole. TRPV4, TRPV6, CaBP-9k, NCX1, and PMCA1 were specifically expressed in Clara and alveolar type 2 cells of mouse lung. CC10, a marker of Clara cells, was decreased by DEX. In addition, mucin secretion, which is a functional aspect of this cell, was also decreased by DEX treatment. Control of calcium-related gene expression may affect the control of mucus secretion in the lung. Such a control mechanism can form the basis of studies into diseases such as inflammation attributable to mucus secretion abnormalities, coughing, and respiratory disorders and distress.
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Canales de Calcio/biosíntesis , Dexametasona/farmacología , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Pulmón/metabolismo , Mucinas/biosíntesis , ATPasas Transportadoras de Calcio de la Membrana Plasmática/biosíntesis , Progesterona/farmacología , Animales , Femenino , Pulmón/citología , Masculino , RatonesRESUMEN
The transcriptional regulation of voltage-gated Ca2+ (CaV ) channels is an emerging research area that promises to improve our understanding of how many relevant physiological events are shaped in the central nervous system, the skeletal muscle and other tissues. Interestingly, a picture of how transcription of CaV channel subunit genes is controlled is evolving with the identification of the promoter regions required for tissue-specific expression and the identification of transcription factors that control their expression. These promoters share several characteristics that include multiple transcriptional start sites, lack of a TATA box and the presence of elements conferring tissue-selective expression. Likewise, changes in CaV channel expression occur throughout development, following ischaemia, seizures or chronic drug administration. This review focuses on insights achieved regarding the control of CaV channel gene expression. To further understand the complexities of expression and to increase the possibilities of detecting CaV channel alterations causing human disease, a deeper knowledge on the structure of the 5' upstream regions of the genes encoding these remarkable proteins will be necessary.
Asunto(s)
Canales de Calcio/biosíntesis , Regulación de la Expresión Génica/fisiología , Animales , Canales de Calcio/genética , HumanosRESUMEN
OBJECTIVES: Thrombospondins, which are known to interact with the α2 δ subunit of voltage-sensitive calcium channels to stimulate the formation of excitatory synapses, have recently been implicated in the process of epileptogenesis. No studies have been so far performed on thrombospondins in models of absence epilepsy. We examined whether expression of the gene encoding for thrombospondin-1 was altered in the brain of WAG/Rij rats, which model absence epilepsy in humans. In addition, we examined the frequency of genetic variants of THBS1 in a large cohort of children affected by idiopathic/genetic generalized epilepsies (IGE/GGEs). METHODS: We measured the transcripts of thrombospondin-1 and α2 δ subunit, and protein levels of α2 δ, Rab3A, and the vesicular glutamate transporter, VGLUT1, in the somatosensory cortex and ventrobasal thalamus of presymptomatic and symptomatic WAG/Rij rats and in two control strains by real-time polymerase chain reaction (PCR) and immunoblotting. We examined the genetic variants of THBS1 and CACNA2D1 in two independent cohorts of patients affected by IGE/GGE recruited through the Genetic Commission of the Italian League Against Epilepsy (LICE) and the EuroEPINOMICS-CoGIE Consortium. RESULTS: Thrombospondin-1 messenger RNA (mRNA) levels were largely reduced in the ventrobasal thalamus of both presymptomatic and symptomatic WAG/Rij rats, whereas levels in the somatosensory cortex were unchanged. VGLUT1 protein levels were also reduced in the ventrobasal thalamus of WAG/Rij rats. Genetic variants of THBS1 were significantly more frequent in patients affected by IGE/GGE than in nonepileptic controls, whereas the frequency of CACNA2D1 was unchanged. SIGNIFICANCE: These findings suggest that thrombospondin-1 may have a role in the pathogenesis of IGE/GGEs.