RESUMO
Cav3.2 channels play an important role in the afferent nociceptive pathway, which is responsible for both physiological and pathological pain transmission. Cav3.2 channels are upregulated during neuropathic pain or peripheral inflammation in part due to an increased association with the deubiquitinase USP5. In this study, we investigated nine naturally occurring flavonoid derivatives which we tested for their abilities to inhibit transiently expressed Cav3.2 channels and their interactions with USP5. Icariside II (ICA-II), one of the flavonols studied, inhibited the biochemical interactions between USP5 and Cav3.2 and concomitantly and effectively blocked Cav3.2 channels. Molecular docking analysis predicts that ICA-II binds to the cUBP domain and the Cav3.2 interaction region. In addition, ICA-II was predicted to interact with residues in close proximity to the Cav3.2 channel's fenestrations, thus accounting for the observed blocking activity. In mice with inflammatory and neuropathic pain, ICA-II inhibited both phases of the formalin-induced nocifensive responses and abolished thermal hyperalgesia induced by injection of complete Freund's adjuvant (CFA) into the hind paw. Furthermore, ICA-II produced significant and long-lasting thermal anti-hyperalgesia in female mice, whereas Cav3.2 null mice were resistant to the action of ICA-II. Altogether, our data show that ICA-II has analgesic activity via an action on Cav3.2 channels.
Assuntos
Canais de Cálcio Tipo T , Neuralgia , Feminino , Camundongos , Animais , Canais de Cálcio Tipo T/metabolismo , Simulação de Acoplamento Molecular , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Hiperalgesia/metabolismo , Flavonoides , Flavonóis , Camundongos Knockout , Proteases Específicas de Ubiquitina/metabolismoRESUMO
BACKGROUND AND PURPOSE: Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. EXPERIMENTAL APPROACH: Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. KEY RESULTS: Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. CONCLUSION AND IMPLICATIONS: These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.
Assuntos
Canais de Cálcio Tipo T , Canais de Sódio Disparados por Voltagem , Camundongos , Ratos , Animais , Canais de Cálcio Tipo T/metabolismo , Ratos Sprague-Dawley , Sódio/metabolismo , Cálcio/metabolismo , Gânglios Espinais/metabolismo , Dor Pós-Operatória/tratamento farmacológico , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Cav3.2 calcium channels are important mediators of nociceptive signaling in the primary afferent pain pathway, and their expression is increased in various rodent models of chronic pain. Previous work from our laboratory has shown that this is in part mediated by an aberrant expression of deubiquitinase USP5, which associates with these channels and increases their stability. Here, we report on a novel bioactive rhodanine compound (II-1), which was identified in compound library screens. II-1 inhibits biochemical interactions between USP5 and the Cav3.2 domain III-IV linker in a dose-dependent manner, without affecting the enzymatic activity of USP5. Molecular docking analysis reveals two potential binding pockets at the USP5-Cav3.2 interface that are distinct from the binding site of the deubiquitinase inhibitor WP1130 (a.k.a. degrasyn). With an understanding of the ability of some rhodanines to produce false positives in high-throughput screening, we have conducted several orthogonal assays to confirm the validity of this hit, including in vivo experiments. Intrathecal delivery of II-1 inhibited both phases of formalin-induced nocifensive behaviors in mice, as well as abolished thermal hyperalgesia induced by the delivery of complete Freund's adjuvant (CFA) to the hind paw. The latter effects were abolished in Cav3.2 null mice, thus confirming that Cav3.2 is required for the action of II-1. II-1 also mediated a robust inhibition of mechanical allodynia induced by injury to the sciatic nerve. Altogether, our data uncover a novel class of analgesicsâwell suited to rapid structure-activity relationship studiesâthat target the Cav3.2/USP5 interface.
Assuntos
Analgésicos , Canais de Cálcio Tipo T , Neuralgia , Proteases Específicas de Ubiquitina , Analgésicos/farmacologia , Animais , Bloqueadores dos Canais de Cálcio , Canais de Cálcio Tipo T/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Neuralgia/metabolismo , Relação Estrutura-Atividade , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismoRESUMO
OBJECTIVES: This study aimed to identify the effect of trimetazidine (TMZ), an antianginal drug, on detrusor smooth muscle (DSM) contractility and its possible mechanisms of action. METHODS: We performed in-vitro contractility studies on isolated mouse DSM strips and investigated the effect of TMZ on Ca2+ levels in fura-2-loaded A7r5 cells. KEY FINDINGS: TMZ (300 or 1000 µM) inhibited carbachol (CCh)- and KCl-induced contractions and produced a concentration-dependent (10-1000 µM) relaxation in KCl-precontracted DSM strips. TMZ-induced relaxation was markedly decreased by BaCl2, an inward-rectifying K+ channel blocker, but was not altered by preincubation with tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. TMZ (300 or 1000 µM) reduced both the CaCl2-induced contraction of depolarized DSM strips under Ca2+-free conditions and the CCh-induced contraction of DSM strips preincubated with nifedipine in Ca2+-containing Krebs solution. Furthermore, TMZ (1000 µM) significantly decreased the Ca2+ levels in fura-2-loaded A7r5 cells. CONCLUSIONS: TMZ decreased DSM contractility and caused a concentration-dependent relaxation of the tissue possibly through its actions on Ca2+ transients and K+ channels. Our results provide preclinical evidence that TMZ would be a potential candidate to treat disorders related to the overactivity of the bladder.
Assuntos
Reposicionamento de Medicamentos/métodos , Trimetazidina/farmacologia , Bexiga Urinária Hiperativa , Bexiga Urinária , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Canais Iônicos/metabolismo , Camundongos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologia , Bexiga Urinária Hiperativa/tratamento farmacológico , Bexiga Urinária Hiperativa/fisiopatologia , Vasodilatadores/farmacologiaRESUMO
Catharanthus roseus is a well-known traditional herbal medicine for the treatment of cancer, hypertension, scald, and sore in China. Phytochemical investigation on the twigs and leaves of this species led to the isolation of two new monoterpene indole alkaloids, catharanosines A (1) and B (2), and six known analogues (3-8). Structures of 1 and 2 were established by 1H-, 13C- and 2D-NMR, and HREIMS data. The absolute configuration of 1 was confirmed by single-crystal X-ray diffraction analysis. Compound 2 represented an unprecedented aspidosperma-type alkaloid with a 2-piperidinyl moiety at C-10. Compounds 6-8 exhibited remarkable Cav3.1 low voltage-gated calcium channel (LVGCC) inhibitory activity with IC50 values of 11.83 ± 1.02, 14.3 ± 1.20, and 14.54 ± 0.99 µM, respectively.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/química , Catharanthus/química , Alcaloides Indólicos/farmacologia , Monoterpenos/farmacologia , Extratos Vegetais/farmacologia , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio Tipo T/metabolismo , Relação Dose-Resposta a Droga , Alcaloides Indólicos/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Monoterpenos/química , Extratos Vegetais/química , Relação Estrutura-AtividadeRESUMO
Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Dor/metabolismo , Receptores da Bombesina/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo T/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Adjuvante de Freund/farmacologia , Gânglios Espinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurocinina B/análogos & derivados , Neurocinina B/metabolismo , Dor/fisiopatologia , Receptores da Bombesina/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismoRESUMO
RATIONALE: The abuse of psychostimulants has adverse consequences on the physiology of the central nervous system. In Argentina, and other South American countries, coca paste or "PACO" (cocaine and caffeine are its major components) is massively consumed with deleterious clinical consequences for the health and well-being of the general population. A scant number of studies have addressed the consequences of stimulant combination of cocaine and caffeine on the physiology of the somatosensory thalamocortical (ThCo) system. OBJECTIVES: Our aim was to study ion conductances that have important implications regulating sleep-wake states 24-h after an acute or chronic binge-like administration of a cocaine and caffeine mixture following previously analyzed pasta base samples ("PACO"-like binge") using mice. METHODS: We randomly injected (i.p.) male C57BL/6JFcen mice with a binge-like psychostimulants regimen during either 1 day (acute) or 1 day on/1 day off during 13 days for a total of 7 binges (chronic). Single-cell patch-clamp recordings of VB neurons were performed in thalamocortical slices 24 h after the last psychostimulant injection. We also recorded EEG/EMG from mice 24 h after being systemically treated with chronic administration of cocaine + caffeine versus saline, vehicle. RESULTS: Our results showed notorious changes in the intrinsic properties of the VB nucleus neurons that persist after 24-h of either acute or chronic binge administrations of combined cocaine and caffeine ("PACO"-like binge). Functional dysregulation of HCN (hyperpolarization-activated cyclic nucleotide-gated) and T-type VGC (voltage-gated calcium) channels was described 24-h after acute/chronic "PACO"-like administrations. Furthermore, intracellular basal [Ca2+] disturbances resulted a key factor that modulated the availability and the activation of T-type channels, altering T-type "window currents." As a result, all these changes ultimately shaped the low-threshold spikes (LTS)-associated Ca2+ transients, regulated the membrane excitability, and altered sleep-wake transitions. CONCLUSION: Our results suggest that deleterious consequences of stimulants cocaine and caffeine combination on the thalamocortical physiology as a whole might be related to potential neurotoxic effects of soaring intracellular [Ca2+].
Assuntos
Cafeína/efeitos adversos , Canais de Cálcio Tipo T/metabolismo , Estimulantes do Sistema Nervoso Central/efeitos adversos , Cocaína/efeitos adversos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Neurônios/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Cafeína/administração & dosagem , Estimulantes do Sistema Nervoso Central/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cocaína/administração & dosagem , Sinergismo Farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Distribuição Aleatória , Transtornos da Transição Sono-Vigília/induzido quimicamente , América do Sul , Tálamo/efeitos dos fármacos , Tálamo/metabolismoRESUMO
The TATA-box binding protein associated factor 1 (TAF1) is part of the TFIID complex that plays a key role during the initiation of transcription. Variants of TAF1 are associated with neurodevelopmental disorders. Previously, we found that CRISPR/Cas9 based editing of the TAF1 gene disrupts the morphology of the cerebral cortex and blunts the expression as well as the function of the CaV3.1 (T-type) voltage gated calcium channel. Here, we tested the efficacy of SAK3 (ethyl 8'-methyl-2', 4-dioxo-2-(piperidin-1-yl)-2'H-spiro [cyclopentane-1, 3'-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate), a T-type calcium channel enhancer, in an animal model of TAF1 intellectual disability (ID) syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21, the rat pups were given SAK3 (0.25 mg/kg, p.o.) or vehicle for 14 days (i.e. till post-natal day 35) and then subjected to behavioral, morphological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued locomotion abnormalities associated with TAF1 gene editing. SAK3 treatment prevented the loss of cortical neurons and GFAP-positive astrocytes observed after TAF1 gene editing. In addition, SAK3 protected cells from apoptosis. SAK3 also restored the Brain-derived neurotrophic factor/protein kinase B/Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3ß) signaling axis in TAF1 edited animals. Finally, SAK3 normalized the levels of three GSK3ß substrates - CaV3.1, FOXP2, and CRMP2. We conclude that the T-type calcium channel enhancer SAK3 is beneficial against the deleterious effects of TAF1 gene-editing, in part, by stimulating the BDNF/AKT/GSK3ß signaling pathway.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Modelos Animais de Doenças , Histona Acetiltransferases/deficiência , Imidazóis/administração & dosagem , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/metabolismo , Compostos de Espiro/administração & dosagem , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fator de Transcrição TFIID/deficiência , Animais , Animais Recém-Nascidos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Histona Acetiltransferases/genética , Injeções Intraventriculares , Deficiência Intelectual/genética , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Gravidez , Ratos , Ratos Sprague-Dawley , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genéticaRESUMO
Absence seizures result from 3 to 5 Hz generalized thalamocortical oscillations that depend on highly regulated inhibitory neurotransmission in the thalamus. Efficient reuptake of the inhibitory neurotransmitter GABA is essential, and reuptake failure worsens human seizures. Here, we show that blocking GABA transporters (GATs) in acute rat brain slices containing key parts of the thalamocortical seizure network modulates epileptiform activity. As expected, we found that blocking either GAT1 or GAT3 prolonged oscillations. However, blocking both GATs unexpectedly suppressed oscillations. Integrating experimental observations into single-neuron and network-level computational models shows how a non-linear dependence of T-type calcium channel gating on GABAB receptor activity regulates network oscillations. Receptor activity that is either too brief or too protracted fails to sufficiently open T-type channels necessary for sustaining oscillations. Only within a narrow range does prolonging GABAB receptor activity promote channel opening and intensify oscillations. These results have implications for therapeutics that modulate inhibition kinetics.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Modelos Neurológicos , Neurônios/fisiologia , Tálamo/fisiologia , Animais , Células Cultivadas , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/metabolismo , Convulsões/metabolismoRESUMO
A hybrid of dearomatized isoprenylated acylphloroglucinol (DIAP) and monoterpenoid, hypatone A (1), together with its biosynthetic analogues 2-4 is characterized from Hypericum patulum. Structurally, 1 possesses an unprecedented spiro[bicyclo[3.2.1]octane-6,1'-cyclohexan]-2',4',6'-trione core as elucidated by extensive spectroscopic and X-ray crystallographic analyses. Biological studies reveal that compounds 1 and 2-4 produce opposite effects on Cav3.1 low voltage-gated Ca2+ channel, with 1 and 4, respectively, being the most potent Cav3.1 agonist and antagonist from natural products. Further studies suggest that compound 1 and its biogenetical precursor, 2, have the same binding site on Cav3.1 and that the rigid cagelike moiety at C-5 and C-6 is a key structural feature responsible for 1 being an agonist. Furthermore, 1 can normalize the pathological gating of a mutant Cav3.1 channel found in spinocerebellar ataxia 42 (SCA42), a hereditary neurodegenerative disorder with no available therapy. Collectively, our findings provide valuable tools for future studies on Cav3.1 physiology and pathophysiology, as well as afford possible leads for developing new drugs against SCA42, epilepsy, and pain.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Monoterpenos/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/farmacologia , Animais , Agonistas dos Canais de Cálcio/isolamento & purificação , Canais de Cálcio Tipo T/genética , Células HEK293 , Humanos , Hypericum/química , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Monoterpenos/isolamento & purificação , Mutação , Floroglucinol/isolamento & purificaçãoRESUMO
Preconditioning is a well-documented strategy that induces hepatic protection, renal protection, cardioprotection, and neuroprotection but its mechanism still remains to be elucidated. Hence, the present study investigated the protective mechanism underlying pain attenuating effects of vincristine-preconditioning in chemotherapeutic agent-induced neuropathic pain. Neuropathic pain was induced by administration of vincristine (50 µg/kg, i.p.) for 10 days in rats. Vincristine-preconditioning was induced by administration of vincristine (2, 5, and 10 µg/kg, i.p) for 5 days before administration of pain-inducing dose of vincristine (50 µg/kg, i.p.). Vincristine-preconditioning (10 µg/kg, i.p) for 5 days significantly reduced vincristine (50 µg/kg, i.p.) induced pain-related behaviors including paw cold allodynia, mechanical hyperalgesia, and heat hyperalgesia. However, vincristine (2 and 5 µg/kg, i.p) did not significantly ameliorate the vincristine (50 µg/kg, i.p.) induced neuropathic pain in rats. Furthermore, to explore the involvement of calcium channels in pain attenuating mechanism of vincristine-preconditioning, T-type calcium channel blocker, ethosuximide (100 and 200 mg/kg, i.p.) and L-type calcium channel blocker, amlodipine (5 and 10 mg/kg, i.p.) were used. Pretreatment with T-type calcium channel blocker, ethosuximide significantly abolished vincristine-preconditioning-induced protective effect. However, pretreatment with L-type calcium channel blocker, amlodipine did not alter vincristine-preconditioning-induced pain-related behaviors. This indicates that vincristine-preconditioning has protective effect on pain-related parameters due to opening of calcium channels, particularly T-type calcium channels that lead to entry of small magnitude of intracellular calcium through these channels and prevent the deleterious effects of high-dose vincristine.
Assuntos
Antineoplásicos/efeitos adversos , Canais de Cálcio Tipo T/metabolismo , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Vincristina/farmacologia , Anlodipino/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Etossuximida/farmacocinética , Feminino , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Masculino , Neuralgia/metabolismo , Ratos , Ratos WistarRESUMO
Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.
Assuntos
Ataxia/genética , Canais de Cálcio Tipo N/genética , Córtex Cerebral/metabolismo , Epilepsia Tipo Ausência/genética , Neurônios/metabolismo , Tálamo/metabolismo , Potenciais de Ação , Fatores Etários , Animais , Ataxia/metabolismo , Ataxia/fisiopatologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Epilepsia Tipo Ausência/metabolismo , Epilepsia Tipo Ausência/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Inibidores/genética , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Núcleos Talâmicos/citologia , Tálamo/fisiopatologiaRESUMO
The mechanisms by which ghrelin controls electrical activity in the hypothalamus are not fully understood. One unexplored target of ghrelin is CaV3, responsible for transient calcium currents (T-currents) that control neuronal firing. We investigated the effect of ghrelin on CaV3 subtypes and how this modulation impacts on neuronal activity. We performed whole-cell patch-clamp recordings in primary mouse hypothalamic cultures to explore the effect of ghrelin on T-currents. We also recorded calcium currents from transiently transfected tsA201 cells to study the sensitivity of each CaV3 subtype to GHSR activation. Finally, we ran a computational model combining the well-known reduction of potassium current by ghrelin with the CaV3 biophysical parameter modifications induced by ghrelin to predict the impact on neuronal electrical behavior. We found that ghrelin inhibits native NiCl2 sensitive current currents in hypothalamic neurons. We determined that CaV3.3 is the only CaV3 subtype sensitive to ghrelin. The modulation of CaV3.3 by ghrelin comprises a reduction in maximum conductance, a shift to hyperpolarized voltages of the I-V and steady-state inactivation curves, and an acceleration of activation and inactivation kinetics. Our model-based prediction indicates that the inhibition of CaV3.3 would attenuate the stimulation of firing originating from the inhibition of potassium currents by ghrelin. In summary, we discovered a new target of ghrelin in neurons: the CaV3.3. This mechanism would imply a negative feed-forward regulation of the neuronal activation exerted by ghrelin. Our work expands the knowledge of the wide range of actions of GHSR, a receptor potentially targeted by therapeutics for several diseases.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Grelina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Hipotálamo/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
T-type calcium channels are low voltage activated calcium channels that are widely expressed in various brain regions including stress-responsive regions. These channels regulate the diverse functions of the central nervous system, and modulation of these channels is shown to modulate the anxiety. Studies have described that modulation of T-type calcium channels may either aggravate or ameliorate anxiety-related behavior, suggesting the dual role of these channels. The studies employing animals with overexpression of T-type calcium channels reported their anxiety-inducing role. Therefore, the blockade of these channels using various pharmacological agents such as ethosuximide, plant extracts of linalool or rosemary, and corticotropin-releasing factor (CRF) is reported to ameliorate anxiety. On the contrary, knockout of the gene encoding these channels predisposes the rodents to anxiety-related disorders, suggesting the anxiety-attenuating role of these channels. It may be possible that these channels in normal or basal state attenuate anxiety, whereas activation of these channels in stressful condition may produce anxiety. The present review describes the dual role of T-type calcium channels in anxiety-related behavior in both preclinical and clinical studies.
Assuntos
Ansiedade/metabolismo , Canais de Cálcio Tipo T/metabolismo , Animais , Ansiedade/tratamento farmacológico , Cálcio/metabolismo , Etossuximida/farmacologiaRESUMO
BACKGROUND: Administration of amplitude modulated 27·12â¯MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2â¯T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Magnetoterapia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/patologia , Magnetoterapia/métodos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Especificidade de Órgãos , RNA Interferente Pequeno/genética , Radiometria , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Brain metastases are a major cause of death in patients with metastatic breast cancer. While surgical resection and radiation therapy are effective treatment modalities, the majority of patients will succumb from disease progression. We have developed a novel therapy for brain metastases that delivers athermal radiofrequency electromagnetic fields that are amplitude-modulated at breast cancer specific frequencies (BCF). METHODS: 27.12â¯MHz amplitude-modulated BCF were administered to a patient with a breast cancer brain metastasis by placing a spoon-shaped antenna on the anterior part of the tongue for three one-hour treatments every day. In preclinical models, a BCF dose, equivalent to that delivered to the patient's brain, was administered to animals implanted with either brain metastasis patient derived xenografts (PDXs) or brain-tropic cell lines. We also examined the efficacy of combining radiation therapy with BCF treatment. Additionally, the mechanistic underpinnings associated with cancer inhibition was identified using an agnostic approach. FINDINGS: Animal studies demonstrated a significant decrease in growth and metastases of brain-tropic cell lines. Moreover, BCF treatment of PDXs established from patients with brain metastases showed strong suppression of their growth ability. Importantly, BCF treatment led to significant and durable regression of brain metastasis of a patient with triple negative breast cancer. The tumour inhibitory effect was mediated by Ca2+ influx in cancer cells through CACNA1H T-type voltage-gated calcium channels, which, acting as the cellular antenna for BCF, activated CAMKII/p38 MAPK signalling and inhibited cancer stem cells through suppression of ß-catenin/HMGA2 signalling. Furthermore, BCF treatment downregulated exosomal miR-1246 level, which in turn decreased angiogenesis in brain environment. Therefore, targeted growth inhibition of breast cancer metastases was achieved through CACNA1H. INTERPRETATION: We demonstrate that BCF, as a single agent or in combination with radiation, is a novel treatment approach to the treatment of brain metastases. This paradigm shifting modality warrants further clinical trials for this unmet medical need.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canais de Cálcio Tipo T/metabolismo , Cálcio/metabolismo , Magnetoterapia , Animais , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Campos Eletromagnéticos , Feminino , Perfilação da Expressão Gênica , Proteína HMGA2 , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Magnetoterapia/métodos , Camundongos , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismoAssuntos
Eletricidade , Neoplasias/terapia , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/metabolismo , Terapia por Estimulação Elétrica , Humanos , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Regeneração , CicatrizaçãoRESUMO
We investigated locus coeruleus (LC) modulation of thalamic feature selectivity through reverse correlation analysis of single-unit recordings from different stages of the rat vibrissa pathway. LC activation increased feature selectivity, drastically improving thalamic information transmission. We found that this improvement was dependent on both local activation of α-adrenergic receptors and modulation of T-type calcium channels in the thalamus and was not due to LC modulation of trigeminothalamic feedforward or corticothalamic feedback inputs. Tonic spikes with LC stimulation carried three times the information as did tonic spikes without LC stimulation. Modeling confirmed norepinephrine regulation of intrathalamic circuit dynamics led to the improved information transmission. Behavioral data demonstrated that LC activation increased the perceptual performance of animals performing tactile discrimination tasks through LC-norepinephrine optimization of thalamic sensory processing. These results suggest a new subdimension within the tonic mode in which brain state can optimize thalamic sensory processing through modulation of intrathalamic circuit dynamics.
Assuntos
Rede Nervosa/fisiologia , Norepinefrina/metabolismo , Tálamo/fisiologia , Vibrissas/fisiologia , Animais , Comportamento Animal/fisiologia , Canais de Cálcio Tipo T/metabolismo , Discriminação Psicológica/fisiologia , Estimulação Elétrica , Feminino , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Percepção do Tato/fisiologiaRESUMO
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
Learning the spatial organization of the environment is essential for most animals' survival. This requires the animal to derive allocentric spatial information from egocentric sensory and motor experience. The neural mechanisms underlying this transformation are mostly unknown. We addressed this problem in electric fish, which can precisely navigate in complete darkness and whose brain circuitry is relatively simple. We conducted the first neural recordings in the preglomerular complex, the thalamic region exclusively connecting the optic tectum with the spatial learning circuits in the dorsolateral pallium. While tectal topographic information was mostly eliminated in preglomerular neurons, the time-intervals between object encounters were precisely encoded. We show that this reliable temporal information, combined with a speed signal, can permit accurate estimation of the distance between encounters, a necessary component of path-integration that enables computing allocentric spatial relations. Our results suggest that similar mechanisms are involved in sequential spatial learning in all vertebrates.