RESUMO
In this study we used ivabradine (IVA), a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, to identify its effect on spike-wave discharges (SWDs); and aimed to determine the role of IVA on the effects of T-type calcium channel blocker NNC 55-0396, GABAA receptor agonist muscimol and antagonist bicuculline in male WAG/Rij rats. After tripolar electrodes for electrocorticogram (ECoG) recordings were placed on the WAG/Rij rats' skulls, 5, 10, and 20 mg/kg IVA were intraperitoneally administered for 7 consecutive days and ECoG recordings were obtained on days 0th, 3rd, 6th, and 7th for three hours before and after injections. While acute injection of 5, 10, and 20 mg/kg IVA did not affect the total number and the mean duration of SWDs, subacute administration (7 days) of IVA decreased the SWDs parameters 24 hours after the 7th injection. Interestingly, when IVA was administered again 24 hours after the 6th IVA injection, it increased the SWDs parameters. Western-blot analyses showed that HCN1 and HCN2 expressions decreased and HCN4 increased in the 5-month-old WAG/Rij rats compared to the 1-month-old WAG/Rij and 5-month-old native Wistar rats, while subacute IVA administration increased the levels of HCN1 and HCN2 channels, except HCN4. Subacute administration of IVA reduced the antiepileptic activity of NNC, while the proepileptic activity of muscimol and the antiepileptic activity of bicuculline were abolished. It might be suggested that subacute IVA administration reduces absence seizures by changing the HCN channel expressions in WAG/Rij rats, and this affects the T-type calcium channels and GABAA receptors.
Assuntos
Canais de Cálcio Tipo T , Epilepsia Tipo Ausência , Ratos , Animais , Masculino , Epilepsia Tipo Ausência/tratamento farmacológico , Epilepsia Tipo Ausência/metabolismo , Ratos Wistar , Receptores de GABA-A , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Eletroencefalografia , Anticonvulsivantes/uso terapêutico , Muscimol , Bicuculina , Bloqueadores dos Canais de Cálcio/farmacologia , Ácido gama-Aminobutírico , Modelos Animais de DoençasRESUMO
Voltage-gated calcium channels (VGCCs), particularly T-type calcium channels (TTCCs), are crucial for various physiological processes and have been implicated in pain, epilepsy, and cancer. Despite the clinical trials of TTCC blockers like Z944 and MK8998, none are currently available on the market. This study investigates the efficacy of Lycopodium alkaloids, particularly as natural product-based TTCC blockers. We synthesized eighteen derivatives from α-obscurine, a lycodine-type alkaloid, and identified five derivatives with significant Cav3.1 blockade activity. The most potent derivative, compound 7, exhibited an IC50 value of 0.19±0.03â µM and was further analyzed through molecular docking, revealing key interactions with Cav3.1. These findings provide a foundation for the structural optimization of Cav3.1 calcium channel blockers and present compound 7 as a promising lead compound for drug development and a tool for chemical biology research.
Assuntos
Alcaloides , Bloqueadores dos Canais de Cálcio , Humanos , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/química , Simulação de Acoplamento Molecular , Alcaloides/farmacologia , Alcaloides/química , Dor , CálcioRESUMO
Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.
Assuntos
Canais de Cálcio Tipo T , Modelos Animais de Doenças , Hiperalgesia , Dor Pós-Operatória , Venenos de Escorpião , Animais , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo T/química , Camundongos , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/metabolismo , Cálcio/metabolismo , Masculino , Humanos , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/químicaRESUMO
CaV3.3 is the third member of the low-voltage-activated calcium channel family and the last to be recognized as disease gene. Previously, CACNA1I, the gene encoding CaV3.3, had been described as schizophrenia risk gene. More recently, de novo missense mutations in CACNA1I were identified in patients with variable degrees of neurodevelopmental disease with and without epilepsy. Their functional characterization indicated gain-of-function effects resulting in increased calcium load and hyperexcitability of neurons expressing CaV3.3. The amino acids mutated in the CaV3.3 disease variants are located in the vicinity of the channel's activation gate and thus are classified as gate-modifying channelopathy mutations. A persistent calcium leak during rest and prolonged calcium spikes due to increased voltage sensitivity of activation and slowed kinetics of channel inactivation, respectively, may be causal for the neurodevelopmental defects. The prominent expression of CaV3.3 in thalamic reticular nucleus neurons and its essential role in generating the rhythmic thalamocortical network activity are consistent with a role of the mutated channels in the etiology of epileptic seizures and thus suggest T-type channel blockers as a viable treatment option.
Assuntos
Canais de Cálcio Tipo T , Canalopatias , Humanos , Canalopatias/genética , Cálcio/metabolismo , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/metabolismo , MutaçãoRESUMO
T-type calcium (CaV3) channels are involved in cardiac automaticity, development, and excitation-contraction coupling in normal cardiac myocytes. Their functional role becomes more pronounced in the process of pathological cardiac hypertrophy and heart failure. Currently, no CaV3 channel inhibitors are used in clinical settings. To identify novel T-type calcium channel ligands, purpurealidin analogs were electrophysiologically investigated. These compounds are alkaloids produced as secondary metabolites by marine sponges, and they exhibit a broad range of biological activities. In this study, we identified the inhibitory effect of purpurealidin I (1) on the rat CaV3.1 channel and conducted structure-activity relationship studies by characterizing the interaction of 119 purpurealidin analogs. Next, the mechanism of action of the four most potent analogs was investigated. Analogs 74, 76, 79, and 99 showed a potent inhibition on the CaV3.1 channel with IC50's at approximately 3 µM. No shift of the activation curve could be observed, suggesting that these compounds act like a pore blocker obstructing the ion flow by binding in the pore region of the CaV3.1 channel. A selectivity screening showed that these analogs are also active on hERG channels. Collectively, a new class of CaV3 channel inhibitors has been discovered and the structure-function studies provide new insights into the synthetic design of drugs and the mechanism of interaction with T-type CaV channels.
Assuntos
Poríferos , Ratos , Animais , Miócitos Cardíacos/metabolismoRESUMO
T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.
Assuntos
Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Ativação do Canal Iônico/genética , Transtornos do Neurodesenvolvimento/genética , Adulto , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Simulação por Computador , Feminino , Mutação com Ganho de Função , Predisposição Genética para Doença/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Modelos Neurológicos , Mutação de Sentido Incorreto , Neurônios/metabolismo , Linhagem , Conformação ProteicaRESUMO
General anesthetics mainly act by modulating synaptic inhibition on the one hand (the potentiation of GABA transmission) or synaptic excitation on the other (the inhibition of NMDA receptors), but they can also have effects on numerous other proteins, receptors, and channels. The effects of general anesthetics on ion channels have been the subject of research since the publication of reports of direct actions of these drugs on ion channel proteins. In particular, there is considerable interest in T-type voltage-gated calcium channels that are abundantly expressed in the thalamus, where they control patterns of cellular excitability and thalamocortical oscillations during awake and sleep states. Here, we summarized and discussed our recent studies focused on the CaV3.1 isoform of T-channels in the nonspecific thalamus (intralaminar and midline nuclei), which acts as a key hub through which natural sleep and general anesthesia are initiated. We used mouse genetics and in vivo and ex vivo electrophysiology to study the role of thalamic T-channels in hypnosis induced by a standard general anesthetic, isoflurane, as well as novel neuroactive steroids. From the results of this study, we conclude that CaV3.1 channels contribute to thalamocortical oscillations during anesthetic-induced hypnosis, particularly the slow-frequency range of δ oscillations (0.5-4 Hz), by generating "window current" that contributes to the resting membrane potential. We posit that the role of the thalamic CaV3.1 isoform of T-channels in the effects of various classes of general anesthetics warrants consideration.
Assuntos
Anestésicos Gerais/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Neurônios/metabolismo , Animais , Humanos , Potenciais da Membrana , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/fisiologiaRESUMO
A hyperexcitable state and spontaneous activity of nociceptors have been suggested to play a critical role in the development of chronic neuropathic pain following spinal cord injury (SCI). In male rats, we employed the action potential-clamp technique to determine the underlying ionic mechanisms responsible for driving SCI-nociceptors to a hyperexcitable state and for triggering their spontaneous activity. We found that the increased activity of low voltage activated T-type calcium channels induced by the injury sustains the bulk (â¼60-70%) of the inward current active at subthreshold voltages during the interspike interval in SCI-nociceptors, with a modest contribution (â¼10-15%) from tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In current-clamp recordings, inhibition of T-type calcium channels with 1 µm TTA-P2 reduced both the spontaneous and the evoked firing in response to current injections in SCI-nociceptors to a level similar to sham-nociceptors. Electrophysiology in vitro was then combined with the conditioned place preference (CPP) paradigm to determine the relationship between the increased activity of T-type channels in SCI-nociceptors and chronic neuropathic pain following SCI. The size of the interspike T-type calcium current recorded from nociceptors isolated from SCI rats showing TTA-P2-induced CPP (responders) was â¼6 fold greater than the interspike T-type calcium current recorded from nociceptors isolated from SCI rats without TTA-P2-induced CPP (non-responders). Taken together, our data suggest that the increased activity of T-type calcium channels induced by the injury plays a primary role in driving SCI-nociceptors to a hyperexcitable state and contributes to chronic neuropathic pain following SCI.SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major comorbidity of spinal cord injury (SCI), affecting up to 70-80% of patients. Anticonvulsant and tricyclic antidepressant drugs are first line analgesics used to treat SCI-induced neuropathic pain, but their efficacy is very limited. A hyperexcitable state and spontaneous activity of SCI-nociceptors have been proposed as a possible underlying cause for the development of chronic neuropathic pain following SCI. Here, we show that the increased activity of T-type calcium channels induced by the injury plays a major role in driving SCI-nociceptors to a hyperexcitable state and for promoting their spontaneous activity, suggesting that T-type calcium channels may represent a pharmacological target to treat SCI-induced neuropathic pain.
Assuntos
Potenciais de Ação , Canais de Cálcio Tipo T/metabolismo , Nociceptividade , Nociceptores/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Masculino , Nociceptores/fisiologia , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.
Assuntos
Aldosterona/farmacologia , Canais de Cálcio Tipo T/metabolismo , Proteína 2 Inibidora de Diferenciação/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Canais de Cálcio Tipo T/genética , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiologia , Proteína 2 Inibidora de Diferenciação/genética , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacosRESUMO
Expression patterns of voltage-gated ion channels determine the spatio-temporal dynamics of ion currents that supply excitable neurons in developing tissue with proper electrophysiological properties. The purpose of the study was to identify fast cationic inward currents in mouse retinal horizontal cells (HCs) and describe their biophysical properties at different developmental stages. We also aimed to reveal their physiological role in shaping light responses (LRs) in adult HCs. HCs were recorded in horizontal slices of wild-type mouse retina at postnatal stages ranging from p8 through p60. Voltage-dependent inward currents were isolated with appropriate voltage protocols and blockers specific for sodium and T-type calcium channels. LRs were evoked with full-field flashes (130 µW/cm2). Transient and steady inward currents were identified at all developmental stages. Transient currents were mediated by T-type calcium and TTX-sensitive sodium channels, whereas steady currents were blocked by cadmium, indicating the presence of high voltage-activated calcium channels. Activation and steady-state inactivation kinetics of T-type calcium channels revealed a contribution to the resting membrane potential during postnatal development. Additionally, both sodium and T-type calcium channels had an impact on HC LRs at light offset in adult animals. Our results showed that the voltage-dependent inward currents of postnatally developing mouse HCs consist of T-type calcium, TTX-sensitive sodium, and high voltage-activated calcium channels, and that transient ionic currents contributed to light-evoked responses of adult HCs, suggesting a role in HC information processing.
Assuntos
Canais de Cálcio/metabolismo , Potenciais da Membrana/fisiologia , Células Horizontais da Retina/metabolismo , Canais de Sódio/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Técnicas de Patch-Clamp , Células Horizontais da Retina/citologia , Células Horizontais da Retina/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Tetrodotoxina/farmacologiaRESUMO
T-type Ca2+ channel Cav3.1 promotes microvessel contraction ex vivo. It was hypothesized that in vivo, functional deletion of Cav3.1, but not Cav3.2, protects mice against angiotensin II (ANG II)-induced hypertension. Mean arterial blood pressure (MAP) and heart rate were measured continuously with chronically indwelling catheters during infusion of ANG II (30 ng·kg-1·min-1, 7 days) in wild-type (WT), Cav3.1-/-, and Cav3.2-/- mice. Plasma aldosterone and renin concentrations were measured by radioimmunoassays. In a separate series, WT mice were infused with ANG II (100 ng·kg-1·min-1) with and without the mineralocorticoid receptor blocker canrenoate. Cav3.1-/- and Cav3.2-/- mice exhibited no baseline difference in MAP compared with WT mice, but day-night variation was blunted in both Cav3.1 and Cav3.2-/- mice. ANG II increased significantly MAP in WT, Cav3.1-/-, and Cav3.2-/- mice with no differences between genotypes. Heart rate was significantly lower in Cav3.1-/- and Cav3.2-/- mice compared with control mice. After ANG II infusion, plasma aldosterone concentration was significantly lower in Cav3.1-/- compared with Cav3.2-/- mice. In response to ANG II, fibrosis was observed in heart sections from both WT and Cav3.1-/- mice and while cardiac atrial natriuretic peptide mRNA was similar, the brain natriuretic peptide mRNA increase was mitigated in Cav3.1-/- mice ANG II at 100 ng/kg yielded elevated pressure and an increased heart weight-to-body weight ratio in WT mice. Cardiac hypertrophy, but not hypertension, was prevented by the mineralocorticoid receptor blocker canrenoate. In conclusion, T-type channels Cav3.1and Cav3.2 do not contribute to baseline blood pressure levels and ANG II-induced hypertension. Cav3.1, but not Cav3.2, contributes to aldosterone secretion. Aldosterone promotes cardiac hypertrophy during hypertension.
Assuntos
Aldosterona/sangue , Angiotensina II , Pressão Arterial , Canais de Cálcio Tipo T/deficiência , Hipertensão/sangue , Glândulas Suprarrenais/enzimologia , Animais , Pressão Arterial/efeitos dos fármacos , Biomarcadores/sangue , Canais de Cálcio Tipo T/genética , Ácido Canrenoico/farmacologia , Cardiomegalia/sangue , Cardiomegalia/genética , Cardiomegalia/patologia , Citocromo P-450 CYP11B2/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertensão/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Miocárdio/metabolismo , Miocárdio/patologia , Receptores de Angiotensina/metabolismo , Renina/sangueRESUMO
The ventral subiculum (vSub), a representative output structure of the hippocampus, serves as a main limbic region in mediating the brain's response to stress. There are three subtypes of subicular pyramidal neurons based on their firing patterns: regular-spiking (RS), weak-bursting (WB) and strong-bursting (SB) neurons, located differently along proximal-distal axis. Here, we found that chronic social defeat stress (CSDS) in mice increased the population of SB neurons but decreased RS neurons in the proximal vSub. Specific blockers of T-type calcium channels inhibited the burst firings with a concomitant reduction of afterdepolarization, suggesting that T-type calcium channels underlie the burst-spiking activity. Consistently, CSDS increased both T-type calcium currents and expression of Cav3.1 proteins, a subtype of T-type calcium channels, in the proximal vSub. Therefore, we conclude that CSDS-induced enhancement of Cav3.1 expression increased bursting neuronal population in the vSub, which may contribute to stress-related behaviors.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo T/metabolismo , Hipocampo/fisiopatologia , Neurônios/fisiologia , Comportamento Social , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Potenciais de Ação/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Doença Crônica , Hipocampo/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacosRESUMO
BACKGROUND: Hypnotics and general anaesthetics impair memory by altering hippocampal synaptic plasticity. We recently reported on a neurosteroid analogue with potent hypnotic activity [(3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile; 3ß-OH], which does not cause developmental neurotoxicity in rat pups. Here, we investigated the effects of 3ß-OH on neuronal excitability in the subiculum, the major output structure of the hippocampal formation, and synaptic plasticity at two key hippocampal synapses in juvenile rats. METHODS: Biophysical properties of isolated T-type calcium currents (T-currents) in the rat subiculum were investigated using acute slice preparations. Subicular T-type calcium channel (T-channel) subtype mRNA expression was compared using qRT-PCR. Using electrophysiological recordings, we examined the effects of 3ß-OH and an endogenous neuroactive steroid, allopregnanolone (Allo), on T-currents and burst firing properties of subicular neurones, and on the long-term potentiation (LTP) in CA3-CA1 and CA1-subiculum pathways. RESULTS: Biophysical and molecular studies confirmed that CaV3.1 channels represent the dominant T-channel isoform in the subiculum of juvenile rats. 3ß-OH and Allo inhibited rebound burst firing by decreasing the amplitude of T-currents in a voltage-dependent manner with similar potency, with 30-80% inhibition. Both neurosteroids suppressed LTP at the CA1-subiculum, but not at the CA3-CA1 Schaffer collateral synapse. CONCLUSIONS: Neurosteroid effects on T-channels modulate hippocampal output and provide possible molecular mechanisms for the amnestic action of the novel hypnotic 3ß-OH. Effects on T-channels in the subiculum provide a novel target for amnestic effects of hypnotics.
Assuntos
Androstanóis/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Nitrilas/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/biossíntese , Canais de Cálcio Tipo T/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Masculino , RNA Mensageiro/genética , Ratos Sprague-DawleyRESUMO
While there is growing appreciation for diversity among ventral tegmental area dopamine neurons, much less is known regarding functional heterogeneity among the substantia nigra pars compacta (SNc) neurons. Here, we show that calbindin-positive dorsal tier and calbindin-negative ventral tier SNc dopaminergic neurons in mice comprise functionally distinct subpopulations distinguished by their dendritic calcium signaling, rebound excitation, and physiological responses to dopamine D2-receptor (D2) autoinhibition. While dopamine is known to inhibit action potential backpropagation, our experiments revealed an unexpected enhancement of excitatory responses and dendritic calcium signals in the presence of D2-receptor inhibition. Specifically, dopamine inhibition and direct hyperpolarization enabled the generation of low-threshold depolarizations that occurred in an all-or-none or graded manner, due to recruitment of T-type calcium channels. Interestingly, these effects occurred selectively in calbindin-negative dopaminergic neurons within the SNc. Thus, calbindin-positive and calbindin-negative SNc neurons differ substantially in their calcium channel composition and efficacy of excitatory inputs in the presence of dopamine inhibition.SIGNIFICANCE STATEMENT Substantia nigra dopaminergic neurons can be divided into two populations: the calbindin-negative ventral tier, which is vulnerable to neurodegeneration in Parkinson's disease, and the calbindin-positive dorsal tier, which is relatively resilient. Although tonic firing is similar in these subpopulations, we find that their responses to dopamine-mediated inhibition are strikingly different. During inhibition, calbindin-negative neurons exhibit increased sensitivity to excitatory inputs, which can then trigger large dendritic calcium transients due to strong expression of T-type calcium channels. Therefore, SNc neurons differ substantially in their calcium channel composition, which may contribute to their differential vulnerability. Furthermore, T-currents increase excitation efficacy onto calbindin-negative cells during dopamine inhibition, suggesting that shared inputs are differentially processed in subpopulations resulting in distinct downstream dopamine signals.
Assuntos
Calbindinas/metabolismo , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio/fisiologia , Neurônios Dopaminérgicos/classificação , Neurônios Dopaminérgicos/fisiologia , Substância Negra/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Dopamina/metabolismo , Antagonistas dos Receptores de Dopamina D2/administração & dosagem , Neurônios Dopaminérgicos/efeitos dos fármacos , Feminino , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Receptores de Dopamina D2/metabolismo , Substância Negra/efeitos dos fármacosRESUMO
KEY POINTS: In arterial smooth muscle, Ca2+ sparks are elementary Ca2+ -release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+ -sensitive K+ (BKCa ) channels. This study elucidated the contribution of T-type Cav 3.2 channels in caveolae and their functional interaction with L-type Cav 1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L-type Cav 1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. T-type Cav 3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T-type Cav 3.2 channels and RyRs to ignite Ca2+ sparks. ABSTRACT: Recent data suggest that T-type Cav 3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav 3.2 channels and their functional interaction with Cav 1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen-inducible smooth muscle-specific Cav 1.2-/- (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+ , Cd2+ and methyl-ß-cyclodextrin were used to inhibit Cav 3.2 channels, Cav 1.2 channels and caveolae, respectively. Ni2+ (50 µmol L-1 ) and methyl-ß-cyclodextrin (10 mmol L-1 ) decreased Ca2+ spark frequency by â¼20-30% in mesenteric VSMCs in a non-additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200 µmol L-1 ) suppressed Ca2+ sparks in mesenteric arteries by â¼70-80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl-ß-cyclodextrin. Our data demonstrate that Ca2+ influx through CaV 1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV 3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Artérias Mesentéricas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cavéolas/metabolismo , Células Cultivadas , Masculino , Artérias Mesentéricas/citologia , Camundongos , Miócitos de Músculo Liso/metabolismoRESUMO
Impairment of endothelial function with aging is accompanied by reduced nitric oxide (NO) production. T-type Cav3.1 channels augment nitric oxide and co-localize with eNOS. Therefore, the hypothesis was that T-type channels contribute to the endothelial dysfunction of aging. Endothelial function was determined in mesenteric arteries (perfusion) and aortae (isometric contraction) of young and old wild-type (WT), Cav3.1, and Cav3.2 knockout mice. NO production was measured by fluorescence imaging in mesenteric arteries. With age, endothelium-dependent subsequent dilatation (following depolarization with KCl) of mesenteric arteries was diminished in the arteries of WT mice, unchanged in Cav3.2-/- preparations but increased in those of Cav3.1-/- mice. NO synthase inhibition abolished the subsequent dilatation in mesenteric arteries and acetylcholine-induced relaxations in aortae. NO levels were significantly reduced in mesenteric arteries of old compared to young WT mice. In Cav3.1-/- and Cav3.2-/- preparations, NO levels increased significantly with age. Relaxations to acetylcholine were significantly smaller in the aortae of old compared to young WT mice, while such responses were comparable in preparations of young and old Cav3.1-/- and Cav3.2-/- mice. The expression of Cav3.1 was significantly reduced in aortae from aged compared to young WT mice. The level of phosphorylated eNOS was significantly increased in aortae from aged Cav3.1-/- mice. In conclusion, T-type calcium channel-deficient mice develop less age-dependent endothelial dysfunction. Changes in NO levels are involved in this phenomenon in WT and Cav3.1-/- mice. These findings suggest that T-type channels play an important role in age-induced endothelial dysfunction.
Assuntos
Envelhecimento/metabolismo , Canais de Cálcio Tipo T/metabolismo , Endotélio Vascular/fisiologia , Envelhecimento/fisiologia , Animais , Aorta/crescimento & desenvolvimento , Aorta/metabolismo , Aorta/fisiologia , Canais de Cálcio Tipo T/genética , Endotélio Vascular/metabolismo , Feminino , Deleção de Genes , Masculino , Artérias Mesentéricas/crescimento & desenvolvimento , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Vasoconstrição , VasodilataçãoRESUMO
Ion channels are very important in the peripheral sensitization in neuropathic pain. Our present study aims to investigate the possible contribution of CaV3.2 T-type calcium channels in damaged dorsal root ganglion neurons in neuropathic pain. We established a neuropathic pain model of rats with spared nerve injury. In these model rats, it was easy to distinguish damaged dorsal root ganglion neurons (of tibial nerve and common peroneal nerve) from intact dorsal root ganglion neurons (of sural nerves). Our results showed that CaV3.2 protein expression increased in medium-sized neurons from the damaged dorsal root ganglions but not in the intact ones. With whole cell patch clamp recording technique, it was found that after-depolarizing amplitudes of the damaged medium-sized dorsal root ganglion neurons increased significantly at membrane potentials of -85 mV and -95 mV. These results indicate a functional up-regulation of CaV3.2 T-type calcium channels in the damaged medium-sized neurons after spared nerve injury. Behaviorally, blockade of CaV3.2 with antisense oligodeoxynucleotides could significantly reverse mechanical allodynia. These results suggest that CaV3.2 T-type calcium channels in damaged medium-sized dorsal root ganglion neurons might contribute to neuropathic pain after peripheral nerve injury.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Tecido Nervoso/lesões , Neuralgia/metabolismo , Neuralgia/patologia , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Tamanho Celular , Inativação Gênica , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Masculino , Tecido Nervoso/patologia , Neurônios/metabolismo , Nociceptividade , RNA Antissenso/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/genéticaRESUMO
Adrenal catecholamine (CAT) secretion is a general physiological response of animals to environmental stressors such as hypoxia. This represents an important adaptive mechanism to maintain homeostasis and protect vital organs such as the brain. In adult mammals, CAT secretory responses are triggered by activation of the sympathetic nervous system that supplies cholinergic innervation of adrenomedullary chromaffin cells (AMC) via the splanchnic nerve. In the neonate, the splanchnic innervation of AMC is immature or absent, yet hypoxia stimulates a non-neurogenic CAT secretion that is critical for adaptation to extra-uterine life. This non-neurogenic, hypoxia-sensing mechanism in AMC is gradually lost or suppressed postnatally along a time course that parallels the development of splanchnic innervation. Moreover, denervation of adult AMC results in a gradual return of the direct hypoxia-sensing mechanism. The signaling pathways by which neonatal AMC sense acute hypoxia leading to non-neurogenic CAT secretion and the mechanisms that underlie the re-acquisition of hypoxia-sensing properties by denervated adult AMC, are beginning to be understood. This review will focus on current views concerning the mechanisms responsible for direct acute hypoxia sensing and CAT secretion in perinatal AMC and how they are regulated by innervation during postnatal development. It will also briefly discuss plasticity mechanisms likely to contribute to CAT secretion during exposures to chronic and intermittent hypoxia.
Assuntos
Catecolaminas/metabolismo , Células Cromafins/metabolismo , Hipóxia/metabolismo , Animais , Plasticidade Celular , Humanos , Canais Iônicos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Chronic hypoxia (CH)-induced pulmonary hypertension is associated with diminished production of endothelium-derived Ca2+-dependent vasodilators such as nitric oxide. Interestingly, ATP-induced endothelial Ca2+ entry as well as membrane cholesterol (Chol) are decreased in pulmonary arteries from CH rats (4 wk, barometric pressure = 380 Torr) compared with normoxic controls. Store-operated Ca2+ entry (SOCE) and depolarization-induced Ca2+ entry are major components of the response to ATP and are similarly decreased after CH. We hypothesized that membrane Chol facilitates both SOCE and depolarization-induced pulmonary endothelial Ca2+ entry and that CH attenuates these responses by decreasing membrane Chol. To test these hypotheses, we administered Chol or epicholesterol (Epichol) to acutely isolated pulmonary arterial endothelial cells (PAECs) from control and CH rats to either supplement or replace native Chol, respectively. The efficacy of membrane Chol manipulation was confirmed by filipin staining. Epichol greatly reduced ATP-induced Ca2+ influx in PAECs from control rats. Whereas Epichol similarly blunted endothelial SOCE in PAECs from both groups, Chol supplementation restored diminished SOCE in PAECs from CH rats while having no effect in controls. Similar effects of Chol manipulation on PAEC Ca2+ influx were observed in response to a depolarizing stimulus of KCl. Furthermore, KCl-induced Ca2+ entry was inhibited by the T-type Ca2+ channel antagonist mibefradil but not the L-type Ca2+ channel inhibitor diltiazem. We conclude that PAEC membrane Chol is required for ATP-induced Ca2+ entry and its two components, SOCE and depolarization-induced Ca2+ entry, and that reduced Ca2+ entry after CH may be due to loss of this key regulator.NEW & NOTEWORTHY This research is the first to examine the direct role of membrane cholesterol in regulating pulmonary endothelial agonist-induced Ca2+ entry and its components. The results provide a potential mechanism by which chronic hypoxia impairs pulmonary endothelial Ca2+ influx, which may contribute to pulmonary hypertension.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Células Endoteliais/metabolismo , Hipóxia/metabolismo , Artéria Pulmonar/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cavéolas/metabolismo , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/farmacologia , Doença Crônica , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Masculino , Potenciais da Membrana , Artéria Pulmonar/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
T-type calcium channels are prominently expressed in primary nociceptive fibers and well characterized in pain processes. Although itch and pain share many similarities including primary sensory fibers, the function of T-type calcium channels on acute itch has not been explored. We investigated whether T-type calcium channels expressed within primary sensory fibers of mouse skin, especially Cav3.2 subtype, involve in chloroquine-, endothelin-1- and histamine-evoked acute itch using pharmacological, neuronal imaging and behavioral analyses. We found that pre-locally blocking three subtypes of T-type calcium channels in the peripheral afferents of skins, yielded an inhibition in acute itch or pain behaviors, while selectively blocking the Cav3.2 channel in the skin peripheral afferents only inhibited acute pain but not acute itch. These results suggest that T-type Cav3.1 or Cav3.3, but not Cav3.2 channel, have an important role in acute itch processing, and their distinctive roles in modulating acute itch are worthy of further investigation.