RESUMO
Although radiotherapy is the most effective treatment modality for brain tumors, it always injures the central nervous system, leading to potential sequelae such as cognitive dysfunction. Radiation induces molecular, cellular, and functional changes in neuronal and glial cells. The hippocampus plays a critical role in learning and memory; therefore, concerns about radiation-induced injury are widespread. Multiple studies have focused on this complex problem, but the results have not been fully elucidated. Naked mole rat brains were irradiated with 60Co at a dose of 10 Gy. On 7 days, 14 days, and 28 days after irradiation, hippocampi in the control groups were obtained for next-generation sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were subsequently performed. Venn diagrams revealed 580 differentially expressed genes (DEGs) that were common at different times after irradiation. GO and KEGG analyses revealed that the 580 common DEGs were enriched in molecular transducer activity. In particular, CACNA1B mediated regulatory effects after irradiation. CACNA1B expression increased significantly after irradiation. Downregulation of CACNA1B led to a reduction in apoptosis and reactive oxygen species levels in hippocampal neurons. This was due to the interaction between CACNA1B and Nrf2, which disturbed the normal nuclear localization of Nrf2. In addition, CACNA1B downregulation led to a decrease in the cognitive functions of naked mole rats. These findings reveal the pivotal role of CACNA1B in regulating radiation-induced brain injury and will lead to the development of a novel strategy to prevent brain injury after irradiation.
Assuntos
Lesões Encefálicas , Fator 2 Relacionado a NF-E2 , Apoptose , Lesões Encefálicas/metabolismo , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/farmacologia , Hipocampo/metabolismo , Neurônios/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
BACKGROUND: As a peptide originally discovered from Conus achates by mass spectrometry and cDNA sequencing, Ac6.4 contains 25 amino acid residues and three disulfide bridges. Our previous study found that this peptide possesses 80% similarity to MVIIA by BLAST and that MVIIA is a potent and selective blocker of N-type voltage-sensitive calcium channels in neurons. OBJECTIVE: To recognize the target protein and analgesic activity of Ac6.4 from Conus achates. METHODS: In the present study, we synthesized Ac6.4, expressed the Trx-Ac6.4 fusion protein, tested Ac6.4 for its inhibitory activity against Cav2.2 in CHO cells and investigated Ac6.4 and Trx-Ac6.4 for their analgesic activities in mice. RESULTS: Data revealed that Ac6.4 had strong inhibitory activity against Cav2.2 (IC50 = 43.6 nM). After intracranial administration of Ac6.4 (5, 10, 20 µg/kg) and Trx-Ac6.4 (20, 40, 80 µg/kg), significant analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent. CONCLUSION: This study expands our knowledge of the peptide Ac6.4 and provides new possibilities for developing Cav2.2 inhibitors and analgesic drugs.
Assuntos
Caramujo Conus , Camundongos , Animais , Cricetinae , Caramujo Conus/química , Caramujo Conus/metabolismo , Cricetulus , Analgésicos/farmacologia , Analgésicos/química , Peptídeos/química , Canais de Cálcio Tipo N/metabolismoRESUMO
Distant metastasis is the main cause of mortality in breast cancer patients. Using the breast cancer genomic data from The Cancer Genome Atlas (TCGA), we identified brain specific Cav2.2 as a critical regulator of metastasis. Cav2.2 expression is significantly upregulated in breast cancer and its higher expression is inversely correlated with survival suggesting a previously unappreciated role of Cav2.2 in breast cancer. Cav2.2 is required for breast cancer migration, invasion, and metastasis. Interestingly, Cav2.2 promotes invadopodia formation and extracellular matrix (ECM) degradation through the stabilization of invadopodia component cortactin in a proteosome-dependent manner. Moreover, deubiquitinating enzyme USP43 mediated the functions of Cav2.2 in cortactin stabilization, invadopodia formation, ECM degradation, and metastasis. Interestingly, Cav2.2 upregulates USP43 expression through NFAT2 dephosphorylation and nuclear localization. Our study uncovered a novel pathway that regulates cortactin expression and invadopodia formation in breast cancer metastasis.
Assuntos
Neoplasias da Mama , Canais de Cálcio Tipo N , Enzimas Desubiquitinantes , Fatores de Transcrição NFATC , Podossomos , Neoplasias da Mama/patologia , Canais de Cálcio Tipo N/metabolismo , Linhagem Celular Tumoral , Cortactina/genética , Cortactina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Fatores de Transcrição NFATC/metabolismo , Invasividade Neoplásica , Podossomos/metabolismoRESUMO
Transcription factor 21 (TCF21) directly binds and regulates SF1 mRNA expression in tumor and normal adrenocortical cells, and both are involved in the development and steroidogenesis of the adrenal cortex. TCF21 is a tumor suppressor gene and its expression is reduced in malignant tumors. In adrenocortical tumors, it is less expressed in adrenocortical carcinomas (ACC) than in adrenocortical adenomas (ACA) and normal tissues. However, a comprehensive analysis to identify TCF21 targets has not yet been conducted in any type of cancer. In this study, we performed Chromatin Immunoprecipitation and Sequencing (ChIP-Seq) in an adrenocortical carcinoma cell line (NCI-H295R) overexpressing TCF21, with the aim of identifying TCF21 new targets. The five most frequently identified sequences corresponded to the PRDM7, CNTNAP2, CACNA1B, PTPRN2, and KCNE1B genes. Validation experiments showed that, in NCI-H295R cells, TCF21 negatively regulates the expression of the CACNA1B gene. Recently, it was observed that the N-type calcium channel v2.2 (Cav2.2) encoded by the CACNA1B gene is important in Angiotensin II signal transduction for corticosteroid biosynthesis in NCI-H295R adrenocortical carcinoma cells. Indeed, TCF21 inhibits CACNA1B and Cav2.2 expression in NCI-H295R. In addition, in a cohort of 55 adult patients with adrenocortical tumors, CACNA1B expression was higher in ACC than ACA and was related to poor disease-free survival in ACC patients. These results suggest a mechanism of steroidogenesis control by TCF21 in adrenocortical tumor cells, in addition to the control observed through SF1 inhibition. Importantly, steroid production could impair tumor immunogenicity, contributing to the immune resistance described in adrenal cancer.
Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Canais de Cálcio Tipo N/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Adulto , Linhagem Celular Tumoral , Humanos , Transdução de SinaisRESUMO
CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.
Assuntos
Canais de Cálcio Tipo R , Proteínas de Transporte de Cátions , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cisteína , Células HEK293 , Humanos , Masculino , Camundongos , NociceptividadeRESUMO
The differentiation of adipose tissue-derived stem cells (ASCs) to neuronal cells is greatly promoted by valproic acid (VPA), and is synergistically enhanced by the following treatment with neuronal induction medium (NIM) containing cAMP-elevating agents. In the present study, we investigated the synergism between VPA and NIM in neuronal differentiation of ASCs, assessed by the expression of neurofilament medium polypeptide (NeFM), with respect to Ca2+ entry. VPA (2 mM) treatment for 3 days followed by NIM for 2 h synergistically increased the incidence of neuronal cells differentiated from ASCs to an extent more than VPA alone treatment for 6 days, shortening the time required for the differentiation. VPA increased intracellular Ca2+ and the mRNAs of voltage-gated Ca2+ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca2+ channels were evoked electrophysiologically at high voltage potential in ASCs treated with VPA. NIM reduced the mRNAs of NeFM and Cacna1b in VPA-promoted neuronal differentiation of ASCs. It was concluded that functional N-type voltage-gated Ca2+ channels (Cav2.2) are selectively expressed in VPA-promoted neuronal differentiation of ASCs. NIM seems to enhance the mRNA translation of molecules required for the differentiation. Neuronal cells obtained from ASCs by this protocol will be used as a cell source for regenerative therapy of neurological disorders associated with altered Cav2.2 activity.
Assuntos
Tecido Adiposo/citologia , Canais de Cálcio Tipo N/metabolismo , Diferenciação Celular , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco/citologia , Ácido Valproico/farmacologia , Animais , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura , Masculino , Neurônios/efeitos dos fármacos , Ratos Wistar , Células-Tronco/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.
Assuntos
Canais de Cálcio Tipo N/efeitos dos fármacos , Conotoxinas/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de GABA-B/efeitos dos fármacos , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacologia , Animais , Canais de Cálcio Tipo N/metabolismo , Conotoxinas/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas , Receptores de GABA-B/metabolismoRESUMO
PURPOSE: This study mainly aimed to explore the influences of Calcium Voltage-Gated Channel Subunit Alpha1 B (CACNA1B) on the development of breast cancer and the related mechanism. MATERIALS AND METHODS: The information of patients with breast cancer from TCGA database was used for analyses of CACNA1B expression and its prognostic value. Loss- and gain- of functions of CACNA1B were conducted in MCF7 and Bcap-37 cells, respectively. CCK-8, colony formation and transwell assays were applied for evaluating the cell viability and motility. Western blot was used for protein expression detection. RESULTS: We revealed that highly expressed CACNA1B in breast cancer tissues was related to poor prognosis according to the data gained from TCGA database. The outcomes of functional assays showed that depletion of CACNA1B restrained MCF7 cell growth, invasion and migration and high-expression of CACNA1B fortified the growth, invasion and migration in Bcap-37 cells. Finally, we manifested that silencing CACNA1B obviously raised the protein expression level of E-cadherin and reduced the protein levels of Cyclin D1, N-cadherin and Snail in MCF7 cells, whilst, over-expression of CACNA1B reduced the level of E-cadherin and increased the expression of Cyclin D1, N-cadherin and Snail in Bcap-37 cells. CONCLUSIONS: These results identified CACNA1B as a forwarder of the growth, invasion and migration in breast cancer cells.
Assuntos
Neoplasias da Mama , Canais de Cálcio Tipo N/metabolismo , Ciclina D1 , Transição Epitelial-Mesenquimal , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclina D1/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7RESUMO
Peptides isolated from spider venoms are of pharmacological interest due to their neurotoxic activity, acting on voltage-dependent ion channels present in different types of human body tissues. Three peptide toxins titled as Ap2, Ap3 and Ap5 were purified by RP-HPLC from Acanthoscurria paulensis venom. They were partially sequenced by MALDI In-source Decay method and their sequences were completed and confirmed by transcriptome analysis of the venom gland. The Ap2, Ap3 and Ap5 peptides have, respectively, 42, 41 and 46 amino acid residues, and experimental molecular masses of 4886.3, 4883.7 and 5454.7 Da, with the Ap2 peptide presenting an amidated C-terminus. Amongst the assayed channels - NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1 and CaV2.2 - Ap2, Ap3 and Ap5 inhibited 20-30 % of CaV2.1 current at 1 µM concentration. Ap3 also inhibited sodium current in NaV1.1, Nav1.5 and Nav1.7 channels by 6.6 ± 1.91 % (p = 0.0276), 4.2 ± 1.09 % (p = 0.0185) and 16.05 ± 2.75 % (p = 0.0282), respectively. Considering that Ap2, Ap3 and Ap5 belong to the 'U'-unknown family of spider toxins, which has few descriptions of biological activity, the present work contributes to the knowledge of these peptides and demonstrates this potential as channel modulators.
Assuntos
Agatoxinas/isolamento & purificação , Agatoxinas/farmacologia , Venenos de Aranha/química , Agatoxinas/química , Animais , Células CHO , Canais de Cálcio Tipo N/metabolismo , Cricetulus , Células HEK293 , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Aranhas , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1ß in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca2+ currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.
Assuntos
Anti-Inflamatórios/farmacologia , Ácido Clodrônico/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Frequência Cardíaca/efeitos dos fármacos , Coração/inervação , Macrófagos/efeitos dos fármacos , Doenças Neuroinflamatórias/prevenção & controle , Gânglio Estrelado/efeitos dos fármacos , Taquicardia Ventricular/prevenção & controle , Fibrilação Ventricular/prevenção & controle , Potenciais de Ação , Animais , Canais de Cálcio Tipo N/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Lipossomos , Macrófagos/metabolismo , Masculino , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/fisiopatologia , Ratos Sprague-Dawley , Gânglio Estrelado/metabolismo , Gânglio Estrelado/fisiopatologia , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Fibrilação Ventricular/etiologia , Fibrilação Ventricular/metabolismo , Fibrilação Ventricular/fisiopatologiaRESUMO
OBJECTIVES: Neuropathic pain (NP) is a chronic inflammation of the sciatic nerve, associated with complex pathophysiological events like neuronal ectopic discharge with changes in neurotransmitters, growth factors, receptors/ion channels including N-methyl-d-aspartate receptors, Transient receptor cation channels, Voltage-gated calcium channels. All these events eventually lead to inflammation and apoptosis of the sciatic nerve in NP. Icariin (ICA), a natural flavonoid is well known for its anti-inflammatory potential. Hence, the present study is designed to evaluate its anti-inflammatory potential against neuropathic pain using in silico and in vivo studies. METHODS: In silico studies were conducted using targets of N-methyl-D-aspartate receptor subtype-2B (NR2B), The capsaicin receptor transient receptor cation channel subfamily-V member-1 (TRPV1), N-type voltage-gated calcium (CaV2.2) channels. In in vivo studies, after partial sciatic nerve ligation surgery to animals, received their respective treatment for 21 days, further TNF-α, IL-6, Bax (proapoptotic) and Bcl-2 (antiapoptotic) expressions were estimated. KEY FINDINGS: ICA decreased the expressions of TNF-α, IL-6, Bax and increased expression of Bcl-2. In silico studies revealed a good energy binding score towards NR2B, TRPV1 receptors and CaV2.2 ion Channel. CONCLUSIONS: ICA could be a promising agent in alleviating neuropathic pain by inhibiting NR2B, TRPV1 receptors and Cav2.2 channels, which induces anti-apoptotic potential and inhibits inflammation.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Flavonoides/farmacologia , Neuralgia , Receptores de N-Metil-D-Aspartato/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Proteínas Reguladoras de Apoptose/análise , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Ratos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologiaRESUMO
Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61ß. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.
Assuntos
Genoma , Neuralgia/genética , Receptores sigma/metabolismo , Animais , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Retículo Endoplasmático/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Tecido Nervoso/lesões , Tecido Nervoso/patologia , Membrana Nuclear/metabolismo , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores sigma/agonistas , Receptores sigma/genética , Canais de Translocação SEC/metabolismo , Transcrição Gênica , Receptor Sigma-1RESUMO
OBJECTIVE: Patients exposed to chronic sustained hypoxia frequently develop cardiovascular disease risk factors to ultimately succumb to adverse cardiovascular events. In this context, the present study intends to assess the role of cilnidipine (Cil), a unique calcium channel blocker that blocks both L-type and N-type calcium channels, on cardiovascular pathophysiology in face of chronic sustained hypoxia exposure. MATERIALS AND METHODS: The study involved Wistar strain albino rats. The group-wise allocation of the experimental animals is as follows - Group 1, control (21% O2); Group 2, chronic hypoxia (CH) (10% O2, 90% N); Group 3, Cil + 21% O2; and Group 4, CH (10% O2, 90% N) + Cil (CH + Cil). Cardiovascular hemodynamics, heart rate variability, and endothelial functions (serum nitric oxide [NO], serum endothelial nitric oxide synthase [NOS3], and serum vascular endothelial growth factor [VEGF]) were assessed. Cardiovascular remodeling was studied by histopathological examination of the ventricular tissues, coronary artery (intramyocardial), and elastic and muscular arteries. Normalized wall index of the coronary artery was also calculated. RESULTS AND CONCLUSION: The results demonstrated altered cardiovascular hemodynamics, disturbed cardiovascular autonomic balance, increased levels of VEGF and NOS3, and decreased bioavailability of NO on exposure to chronic sustained hypoxia. The histopathological examination further pointed toward cardiovascular remodeling. Treatment with Cil ameliorated the cardiovascular remodeling and endothelial dysfunction induced by CH exposure, which may be due to its blocking actions on L/N-type of calcium channels, indicating the possible therapeutic role of Cil against CH-induced cardiovascular pathophysiology.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Oxigênio/metabolismo , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/efeitos dos fármacos , Canais de Cálcio Tipo N/metabolismo , Masculino , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/sangue , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
The pivotal role played by ion-channel dysregulations in the pathogenesis of epilepsy has always garnered much attention. Since mutation of ion-channel proteins CACNA1A and GABRD have been associated with epilepsy, it is important to determine the post-traumatic epilepsy-associated changes in expression levels of these ion channel proteins. Additionally, curcumin is already known for its antiepileptic and neuroprotective potential in FeCl3-induced model of post-traumatic epilepsy. Thus, we investigated FeCl3-induced epilepsy mediated differential expression of CACNA1A and GABRD in the cortical region of the rat brain. Furthermore, we investigated the effect of curcumin on the expression of both proteins. For this, epilepsy was induced by intracortical FeCl3 injection (5 µl of 100 mM). Additionally, curcumin (conc. 1000 ppm; 75 mg/kg of b.wt.; for 14 and 28 days) was administered, mixed with normal food pellets. Results obtained from EEG-MUA and Morris water maze assay demonstrate the progression of epilepsy after FeCl3 injection. Additionally, western blotting and histological studies show the downregulation of CACNA1A and GABRD during epileptogenesis. It was observed that epilepsy-associated decline in learning and memory of animals might be linked with the dysregulation of both proteins. Results also demonstrated that curcumin administration ameliorated epilepsy-associated change in expression of both CACNA1A and GABRD proteins. In conclusion, the neuroprotective effect of curcumin against iron-induced epilepsy might be accompanied by the alleviated upregulation of these channel proteins.
Assuntos
Canais de Cálcio Tipo N/biossíntese , Curcumina/farmacologia , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Fármacos Neuroprotetores/farmacologia , Receptores de GABA-A/biossíntese , Animais , Antineoplásicos/farmacologia , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Cloretos/administração & dosagem , Modelos Animais de Doenças , Eletroencefalografia/métodos , Epilepsia/induzido quimicamente , Epilepsia/patologia , Compostos Férricos/administração & dosagem , Masculino , Teste do Labirinto Aquático de Morris , Ratos , Ratos Wistar , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMO
Hypertension and coronary events are becoming more prevalent in aging societies, and myocardial infarction usually occurs in calcium channel blocker (CCB)-treated hypertensive patients. We herein compared the effects of cilnidipine, an L/N-type CCB and amlodipine, an L-type CCB, on post-infarct left ventricular (LV) remodelling in spontaneously hypertensive rats (SHRs). Male SHRs were subjected to 30 minutes of left coronary artery occlusion followed by reperfusion (MI group). The administration of cilnidipine (10 mg/kg/d; MI + Cil group) or amlodipine (10 mg/kg/d; MI + Aml group) was initiated one week before surgery and continued for five weeks. Both CCBs decreased blood pressure. Four weeks after surgery, cilnidipine, but not amlodipine, attenuated LV dilatation, fractional shortening impairments, end-diastolic pressure elevations, and tau elongation. In the non-infarct region, myocyte hypertrophy and brain natriuretic peptide (BNP) mRNA levels were similarly attenuated by both CCBs. On the other hand, interstitial fibrosis, the mRNA expression of collagen type III and transforming growth factor (TGF) ß and immunohistological TGF ß protein expression in the non-infarct region were reduced more in the MI + Cil group than in the MI + Aml group. Additionally, elevated angiotensin-converting enzyme activity and interstitial noradrenaline concentrations in the non-infarct region were reduced by cilnidipine. These results suggest that cilnidipine reduced cardiac noradrenaline concentrations and inhibited the renin-angiotensin system, which attenuated post-infarct remodelling more than amlodipine in hypertensive rats.
Assuntos
Anlodipino/farmacologia , Anti-Hipertensivos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo N/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Infarto do Miocárdio/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/metabolismo , Modelos Animais de Doenças , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Norepinefrina/metabolismo , Ratos Endogâmicos SHR , Sistema Renina-Angiotensina/efeitos dos fármacosRESUMO
Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteômica , Receptores Adrenérgicos beta/metabolismo , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo N/metabolismo , Microambiente Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Masculino , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Fosforilação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transdução de Sinais , Proteínas ras/química , Proteínas ras/metabolismoRESUMO
Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (CaV2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. In this study, we tested whether this strategy is effective in treating established pain. We constructed AAV6-EGFP-CBD3A6K (AAV6-CBD3A6K) expressing a fluorescent CBD3A6K (replacing A to K at position 6 of CBD3 peptide), which is an optimized variant of the parental CBD3 peptide that is a more potent blocker of CaV2.2. Delivery of AAV6-CBD3A6K into lumbar (L) 4 and 5 dorsal root ganglia (DRG) of rats 2 weeks following tibial nerve injury (TNI) induced transgene expression in neurons of these DRG and their axonal projections, accompanied by attenuation of pain behavior. We additionally observed that the increased CaV2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.
Assuntos
Aptâmeros de Peptídeos/genética , Canais de Cálcio Tipo N/metabolismo , Dependovirus/genética , Terapia Genética/métodos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Neuralgia/terapia , Animais , Aptâmeros de Peptídeos/química , Aptâmeros de Peptídeos/metabolismo , Bloqueadores dos Canais de Cálcio/química , Dependovirus/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Ratos , Ratos Sprague-DawleyRESUMO
ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.
Assuntos
Artérias/metabolismo , Canais de Cálcio Tipo N/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Sistema Nervoso Simpático/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Artérias/fisiologia , Masculino , Norepinefrina/metabolismo , Terminações Pré-Sinápticas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , VasoconstriçãoRESUMO
The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo T/metabolismo , Infecções por HIV/complicações , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Paclitaxel/toxicidade , Triterpenos/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/toxicidade , Células CHO , Cricetulus , Diprenorfina/farmacocinética , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Triterpenos Pentacíclicos , Traumatismos dos Nervos Periféricos/induzido quimicamente , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/etiologia , Traumatismos dos Nervos Periféricos/virologia , Ratos , Ratos Sprague-Dawley , Trítio/farmacocinética , Ácido BetulínicoRESUMO
One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well.