RESUMO
1. PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in preclinical development for psoriasis. This study was undertaken to identify the major phase I metabolites of PAP-1 in Sprague-Dawley (SD) rats. 2. Five phase I metabolites, that is 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)butoxy]psoralen (M3), 5-[4-(3-hydroxyphenoxy)butoxy]psoralen (M4), and 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated from the bile of rats and identified by mass spectrometry and NMR spectroscopy. The last four metabolites are new compounds. 3. Incubation of PAP-1 with SD rat liver microsomes rendered the same five major metabolites in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6 (quinidine), CYP2E (diethyldithiocarbamate), or CYP2C9 (sulphaphenazole) blocked the metabolism of PAP-1 in rat microsomes. 4. Of the five metabolites M3, M4, and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism. 5. We further conducted a 6-month repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female Lewis rats and did not observe any toxic effects.
Assuntos
Ficusina/toxicidade , Canal de Potássio Kv1.3/metabolismo , Desintoxicação Metabólica Fase I , Bloqueadores dos Canais de Potássio/toxicidade , Animais , Bile/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Fezes/química , Ficusina/química , Ficusina/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Proteínas Associadas a Pancreatite , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade CrônicaRESUMO
Ziconotide (PRIALT) is a neuroactive peptide in the final stages of clinical development as a novel non-opioid treatment for severe chronic pain. It is the synthetic equivalent of omega-MVIIA, a component of the venom of the marine snail, Conus magus. The mechanism of action underlying ziconotide's therapeutic profile derives from its potent and selective blockade of neuronal N-type voltage-sensitive calcium channels (N-VSCCs). Direct blockade of N-VSCCs inhibits the activity of a subset of neurons, including pain-sensing primary nociceptors. This mechanism of action distinguishes ziconotide from all other analgesics, including opioid analgesics. In fact, ziconotide is potently anti-nociceptive in animal models of pain in which morphine exhibits poor anti-nociceptive activity. Moreover, in contrast to opiates, tolerance to ziconotide is not observed. Clinical studies of ziconotide in more than 2,000 patients reveal important correlations to ziconotide's non-clinical pharmacology. For example, ziconotide provides significant pain relief to severe chronic pain sufferers who have failed to obtain relief from opiate therapy and no evidence of tolerance to ziconotide is seen in these patients. Contingent on regulatory approval, ziconotide will be the first in a new class of neurological drugs: the N-type calcium channel blockers, or NCCBs. Its novel mechanism of action as a non-opioid analgesic suggests ziconotide has the potential to play a valuable role in treatment regimens for severe chronic pain. If approved for clinical use, ziconotide will further validate the neuroactive venom peptides as a source of new and useful medicines.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Dor/tratamento farmacológico , ômega-Conotoxinas/uso terapêutico , Sequência de Aminoácidos/genética , Animais , Bloqueadores dos Canais de Cálcio/química , Doença Crônica , Humanos , Dados de Sequência Molecular , ômega-Conotoxinas/química , ômega-Conotoxinas/genéticaRESUMO
Several novel N-type voltage sensitive calcium channel blockers showed high affinity in the IMR32 assay and efficacy in the anti-writhing model. Herein, we describe the design, synthesis, SAR studies, biological data, physicochemical properties and pharmacokinetics of this 4-piperidinylaniline series.
Assuntos
Analgésicos não Narcóticos/farmacologia , Compostos de Anilina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/síntese química , Camundongos , Relação Estrutura-AtividadeRESUMO
Our drug discovery efforts for N-type calcium channel blockers in the 4-piperidinylaniline series led to the discovery of an orally active analgesic agent 26.1-[4-Dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethyl-but yl)-phenyl]-(3-methyl-but-2-enyl)amine (26) showed high affinity to functionally block N-type calcium channels (IC50=0.7 microM in the IMR32 assay) and exhibited high efficacy in the anti-writhing analgesia test with mice (ED50=12 mg/kg by po and 4 mg/kg by iv). In this report, the rationale for the design, synthesis, biological evaluation, and pharmacokinetics of this series of blockers is described.
Assuntos
Analgésicos/química , Analgésicos/farmacologia , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/efeitos dos fármacos , Piperidinas/química , Piperidinas/farmacologia , Administração Oral , Analgésicos/síntese química , Compostos de Anilina/síntese química , Animais , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/síntese química , Linhagem Celular , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Camundongos , Estrutura Molecular , Piperidinas/síntese química , Ratos , Ratos WistarRESUMO
Exploration of the SAR around the leucine side chain in a series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC is presented. A novel analog is disclosed which possesses improved aqueous solubility, in vivo activity in an audiogenic seizure model, and reversible blockade in electrophysiological assays.
Assuntos
Alcanos/química , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio Tipo N/metabolismo , Leucina/química , Oligopeptídeos/química , Alcanos/síntese química , Alcanos/farmacologia , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/efeitos dos fármacos , Dipeptídeos/síntese química , Dipeptídeos/química , Dipeptídeos/farmacologia , Eletrofisiologia , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Solubilidade , Relação Estrutura-Atividade , ômega-Conotoxinas/síntese química , ômega-Conotoxinas/química , ômega-Conotoxinas/farmacologiaRESUMO
In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.
Assuntos
Analgésicos não Narcóticos/síntese química , Anticonvulsivantes/síntese química , Bloqueadores dos Canais de Cálcio/síntese química , Neurônios/metabolismo , Piperidinas/síntese química , Estimulação Acústica , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacocinética , Analgésicos não Narcóticos/farmacologia , Animais , Anticonvulsivantes/química , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacocinética , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Frequência Cardíaca/efeitos dos fármacos , Humanos , Técnicas In Vitro , Ativação do Canal Iônico , Masculino , Camundongos , Camundongos Endogâmicos DBA , Microssomos Hepáticos/metabolismo , Medição da Dor , Técnicas de Patch-Clamp , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Convulsões/tratamento farmacológicoRESUMO
Selective N-Type Voltage Sensitive Calcium Channel (VSCC) antagonists have shown utility in several models of pain and ischemia. We report the structure-activity relationship at the proximal phenyl group in a series of non-peptidyl VSCC blockers.
Assuntos
Compostos de Anilina/química , Compostos de Anilina/farmacologia , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Leucina/análogos & derivados , Estimulação Acústica , Sequência de Aminoácidos , Compostos de Anilina/uso terapêutico , Animais , Bloqueadores dos Canais de Cálcio/uso terapêutico , Leucina/química , Leucina/farmacologia , Leucina/uso terapêutico , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Convulsões/tratamento farmacológico , Convulsões/fisiopatologia , Relação Estrutura-AtividadeRESUMO
Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Dipeptídeos/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/efeitos dos fármacos , Dipeptídeos/química , Modelos Animais de Doenças , Humanos , Camundongos , Convulsões/tratamento farmacológico , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown utility in several models of stroke and pain. We are especially interested in small molecule N-type calcium channel blockers for therapeutic use. Herein, we report a series of N,N-dialkyl-dipeptidylamines with potent functional activity at N-type VSCCs and in vivo efficacy. The synthesis, SAR, and pharmacological evaluation of this series are discussed.
Assuntos
Bloqueadores dos Canais de Cálcio/síntese química , Diaminas/síntese química , Diaminas/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos DBA , Modelos Químicos , Convulsões/tratamento farmacológicoRESUMO
The interaction between the proteins syntaxin 1A and SNAP-25 is a key step in synaptic vesicle docking and fusion. To define the SNAP-25 binding domain on syntaxin, we have prepared peptides that span the syntaxin H3 domain (residues 191-266), the region previously shown to be important for binding to SNAP-25, and then determined the affinities of these peptides for binding to SNAP-25. A minimal binding domain was identified within a region of 32 amino acids (residues 189-220). Its affinity for SNAP-25 is substantially enhanced by C-terminal extension (residues 221-266). Circular dichroism revealed the presence of substantial alpha-helicity in the H3 domain and in the 32-mer minimal binding domain, but not in H3 peptides that do not bind to SNAP-25. At temperatures that denature the alpha-helix of the minimal binding domain peptide, SNAP-25 binding is lost. Selected mutations in evolutionarily conserved residues of the amphiphilic alpha-helix within the minimal binding domain (e.g., residues 205 and 209) greatly reduce the affinity for SNAP-25 but have no major effect on secondary structure, suggesting that these residues may interact directly with SNAP-25. The H3 domain peptide and the minimal binding domain peptide inhibit norepinephrine release from PC12 cells. These results suggest that specific amino acid residues in the H3 domain, positioned by the underlying alpha-helical structure, are important for its binding to SNAP-25 and support the notion that this interaction is important for presynaptic vesicular exocytosis.
Assuntos
Antígenos de Superfície/química , Proteínas de Membrana , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Superfície/metabolismo , Sítios de Ligação , Dicroísmo Circular , Exocitose/fisiologia , Dados de Sequência Molecular , Mutação , Norepinefrina/antagonistas & inibidores , Norepinefrina/metabolismo , Células PC12/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , TemperaturaRESUMO
Biochemical properties of the alpha 1 subunits of class A brain calcium channels (alpha 1A) were examined in adult rat brain membrane fractions using a site-directed anti-peptide antibody (anti-CNA3) specific for alpha 1A. Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM), but not receptor sites for the dihydropyridine isradipine or for omega-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive alpha 1A polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220- and 190-kDa alpha 1A polypeptides were also specifically recognized by both anti-BI-Nt and anti-BI-1-Ct antibodies, which are directed against the NH2- and COOH-terminal ends of alpha 1A predicted from cDNA sequence, respectively. These data indicate that the predicted NH2 and COOH termini are present in both size forms and therefore that these isoforms of alpha 1A are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct alpha 1A subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Canais de Cálcio/metabolismo , ômega-Conotoxinas , Sequência de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Di-Hidropiridinas/metabolismo , Ativação Enzimática , Imunoquímica , Dados de Sequência Molecular , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Sistemas do Segundo Mensageiro , ômega-Conotoxina GVIARESUMO
The solution structure of omega-conotoxin MVIIA (SNX-111), a peptide toxin from the fish hunting cone snail Conus magus and a high-affinity blocker of N-type calcium channels, was determined by 2D NMR spectroscopy. The backbones of the best 44 structures match with an average pairwise RMSD of 0.59 angstroms. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 20-21, and 24-25. The structure of this toxin is very similar to that of omega-conotoxin GVIA with which is has only 40% sequence homology, but very similar calcium channel binding affinity and selectivity.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Peptídeos/química , ômega-Conotoxinas , Sequência de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/metabolismo , Fenômenos Químicos , Físico-Química , Hidrogênio/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Caramujos/química , Software , ômega-Conotoxina GVIARESUMO
BACKGROUND: Voltage-gated calcium channels in small-cell lung carcinomas may initiate autoimmunity in the paraneoplastic neuromuscular disorder Lambert-Eaton syndrome. The calcium-channel subtype that is responsible is not known. METHODS: We compared the effects of antagonists of L-type, N-type, and P/Q-type neuronal calcium channels on the depolarization-dependent influx of calcium-45 in cultured carcinoma cells. Serum samples from patients with various disorders were tested for reactivity with P/Q-type channels solubilized from carcinoma and cerebellar membranes and N-type channels from cerebral cortex. RESULTS: P/Q-type calcium-channel antagonists were the most potent inhibitors of depolarization-induced 45Ca influx in cultured small-cell carcinoma cell lines. Anti-P/Q-type calcium-channel antibodies were found in serum from all 32 patients with Lambert-Eaton syndrome and a diagnosis of cancer and in 91 percent of the 33 patients with Lambert-Eaton syndrome without cancer. Anti-N-type calcium-channel antibodies were found in 49 percent of the 65 patients with the Lambert-Eaton Syndrome. Lower titers of anti-P/Q-type and anti-N-type calcium-channel antibodies were found in 54 percent of 70 patients with a paraneoplastic encephalomyeloneuropathic complication of lung, ovarian, or breast carcinoma, 24 percent of 90 patients with cancer but no evident neurologic complications, 23 percent of 78 patients with sporadic amyotrophic lateral sclerosis, and less than 3 percent of 69 patients with myasthenia gravis, epilepsy, or scleroderma. CONCLUSIONS: The high frequency of P/Q-type calcium-channel antibodies found in patients with Lambert-Eaton syndrome implies that antibodies of this specificity have a role in the presynaptic pathophysiology of this disorder.
Assuntos
Autoanticorpos/análise , Canais de Cálcio/imunologia , Síndrome Miastênica de Lambert-Eaton/imunologia , Neurônios/imunologia , Síndromes Paraneoplásicas/imunologia , Canais de Cálcio/efeitos dos fármacos , Carcinoma de Células Pequenas/imunologia , Carcinoma de Células Pequenas/patologia , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Células Tumorais CultivadasRESUMO
We have determined the solution structure of the omega-conotoxin MVIIC from Conus magus by 1H NMR. This conopeptide preferentially blocks P and Q type Ca2+ currents by binding with high affinity to voltage-sensitive Ca2+ channels in neurons. This 26 residue peptide with three disulfide bonds was chemically synthesized and refolded for NMR structural studies. The 1H NMR NOESY spectrum of this peptide was completely assigned, with stereospecific assignments made for 12 of the beta prochiral centers. Complete relaxation matrix analysis using MARDIGRAS was used to obtain initial interproton distances from peak intensities. The correlation time necessary for these calculations was determined by measuring 13C relaxation times using inversely detected natural abundance spectra. Distances were input to DG, which provided 15 starting structures which were then subjected to restrained molecular dynamics calculations using SANDER with the AMBER 91 force field in vacuo. 1H-1H vicinal coupling constants were obtained using a combination of line fitting of both E. COSY and NOESY spectra and used to generate angle restraints that were included explicitly in the restrained molecular dynamics calculations. The final set of the 15 best structures had a backbone rmsd of 0.84 A. The ensemble R1/6 factor calculated by CORMA for the final 15 structures was 11%. The final structure consists of an anti-parallel, triple-stranded beta-sheet, with four turns. In spite of significant differences in amino acid sequence and affinities for calcium channel subtypes, the backbone structure of omega-conotoxin MVIIC is very similar to the previously reported structure of omega-conotoxin GVIA.
Assuntos
Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio/metabolismo , Peptídeos/química , Conformação Proteica , ômega-Conotoxinas , Sequência de Aminoácidos , Animais , Hidrogênio , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Moluscos , Venenos de Moluscos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Homologia de Sequência de Aminoácidos , Soluções , ômega-Conotoxina GVIARESUMO
This investigation assessed the ability of a variety of calcium channel blocking peptides to block synaptic transmission in the isolated mouse phrenic nerve-hemidiaphragm. The synthetic version of the naturally occurring N-type voltage-sensitive calcium channel (VSCC) blocker omega-conopeptide MVIIA (SNX-111) had no effect on nerve-evoked muscle contractions. The non-N-, non-L-type VSCC blocker, omega-conopeptide MVIIC (SNX-230), blocked neuromuscular transmission completely, as did the selective P-type VSCC blocker, omega-Aga-IVA. Subsequent evaluation of other synthetic omega-conopeptides and analogs disclosed a significant positive correlation between the test compounds' affinities for high-affinity SNX-230 brain binding sites and their neuromuscular blocking potencies. Quantal analysis of transmitter release showed that SNX-230 abolished evoked endplate potentials completely, but had little effect on the amplitude and frequency of spontaneous miniature endplate potentials. Perineural focal recordings of presynaptic currents showed that SNX-230 did not block the neuronal action potential. These and other findings indicated that SNX-230 prevents transmitter release at the mouse neuromuscular junction by blocking calcium channels at presynaptic nerve endings. These calcium channels correspond pharmacologically to VSCCs associated with high-affinity binding sites in rat brain and are most probably either of the P- or Q-type.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , ômega-Conotoxinas , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Camundongos , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , ômega-Agatoxina IVARESUMO
This article reviews the structural and functional diversity of neuronal calcium channels and the therapeutic potential of antagonizing such channels. Through spatial and temporal control of intracellular calcium concentration, voltage-sensitive calcium channels regulate a host of neuronal processes, including neurotransmitter secretion, electrical activity, cytoskeletal function, cell metabolism and proliferation, and gene expression. Several genes elaborate a number of calcium channel isoforms or subtypes--each tailored to specific roles in neuronal function and possessing distinct biophysical properties, distribution, modulation, and pharmacological sensitivity. This diversity has raised the possibility that subtype-specific antagonists could provide novel treatments for some neuropathologies. In fact, neuroprotective and analgesic actions of N-type channel blockers in animals appear to confirm this supposition. These properties prompted human clinical studies evaluating these agents for prevention of neuronal degeneration following ischemic brain trauma and for relief of pain. Future medical applications for these blockers and antagonists of other channels subtypes are discussed.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Doenças do Sistema Nervoso/tratamento farmacológico , Neurônios/efeitos dos fármacos , Sequência de Aminoácidos , Analgesia , Animais , Doenças Autoimunes/diagnóstico , Isquemia Encefálica/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/química , Humanos , Hipertensão/tratamento farmacológico , Dados de Sequência Molecular , Neurônios/fisiologiaRESUMO
Voltage-sensitive Ca2+ channels are essential to transmitter release at the chemical synapse. To demonstrate the localization of voltage-sensitive Ca2+ channels in relation to the site of transmitter release, mouse neuromuscular junctions were double labelled with alpha-bungarotoxin and a novel voltage-sensitive Ca2+ channel probe, SNX-260, a synthetic analog of omega-conopeptide MVIIC. Similar to omega-conopeptide MVIIC, biotinylated SNX-260 blocked nerve-stimulated transmitter release at the mouse neuromuscular junction. Fluorescently-tagged biotinylated SNX-260 labelled the nerve terminal which appeared thinner than and was outlined by acetylcholine receptor clusters as seen in en face view. This SNX-260 labelling was inhibited by preincubation with unconjugated SNX-260. Side-views of the neuromuscular junction indicated that the SNX-260 labelling was on the synaptic side facing the acetylcholine receptor rather than on the nonsynaptic side of the nerve terminal. This presynaptic binding was confirmed by the absence of SNX-260 labelling in denervated muscles following a nerve cut or disjunction after collagenase treatment. Confocal microscopy revealed spots of SNX-260 labelling that may correlate with active zones. The SNX-260 labelling pattern was not affected by preincubation with unconjugated SNX-111 (omega-conopeptide MVIIA), an N-type voltage-sensitive Ca2+ channel blocker. These findings suggest that SNX-260 is a novel probe for localizing non-N type voltage-sensitive Ca2+ channels and that these voltage-sensitive Ca2+ channels are localized near the transmitter release sites at the mammalian motor nerve terminal membrane. The results are consistent with the suggestion that non-N, probably P/Q type voltage-sensitive Ca2+ channels mediate evoked transmitter release at the mammalian neuromuscular junction.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Peptídeos/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Denervação , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Sensibilidade e Especificidade , Coloração e RotulagemRESUMO
The use of subtype-selective voltage-sensitive calcium channel (VSCC) antagonists has established that neurotransmitter release in mammalian brain is mediated by N-like and P-like VSCCs, and that other subtypes also contribute significantly. To determine the roles presynaptic VSCCs play in nervous system function and to evaluate the therapeutic potential of their selective inhibition, it is necessary to define further the contributions of VSCC subtypes to neurotransmitter release. The novel conopeptide, SNX-230 (omega-conopeptide MVIIC), has revealed a new VSCC subtype, the Q-type, in cerebellar granule cells. We have compared the effects of SNX-230 on release of tritiated D-aspartate ([3H]D-Asp; a non-metabolizable analog of glutamate), gamma-aminobutyric acid ([3H]GABA), and norepinephrine ([3H]NE) from rat hippocampal slices to those of the N-type VSCC blocker, SNX-111 (omega-conopeptide MVIIA), and the P-type blocker, omega-agatoxin-IVA (AgaIVA). SNX-230 blocks both [3H]D-Asp and [3H]GABA release completely, whereas AgaIVA blocks them potently but partially and SNX-111 has no effect. These results suggest that glutamate and GABA release are mediated by two VSCC subtypes, a P-type and another, perhaps Q-like. SNX-111 blocks [3H]NE release potently but partially, while SNX-230 blockade is complete, consisting of one very potent phase and one less potent phase. AgaIVA also blocks [3H]NE release potently but partially. These results suggest that at least two VSCC subtypes, an N-type and a novel non-N-type, mediate NE release. Pair-wise combinations of the three ligands indicate that at least three pharmacologically distinct components comprise [3H]NE release in the hippocampus.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Hipocampo/metabolismo , Neurotransmissores/metabolismo , ômega-Conotoxinas , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Norepinefrina/metabolismo , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismoRESUMO
High-threshold voltage-sensitive calcium channels of the N-type, L-type, and P-type have been distinguished in the mammalian CNS predominantly on the basis of their sensitivity to selective antagonists. Matching them with genes identified by molecular cloning is an ongoing undertaking. Whereas L-type channels are characterized by their sensitivity to dihydropyridines and P-type channels by sensitivity to the funnel-web spider toxin AgaIVA, the N-type channel has been shown to be recognized by the omega-conopeptides GVIA and MVIIA. Recently, two new members of the family of omega-conopeptides--MVIIC from the marine snail Conus magus and SVIB from Conus striatus--have been described. Binding and electrophysiological data suggest that these two peptides, in addition to interacting with N-type calcium channels, interact with a widely distributed receptor in neuronal membranes that is distinct from N-type channels. In this report we demonstrate through biochemical and pharmacological differentiation at individual receptor polypeptide resolution, by affinity cross-linking, SDS-PAGE, and autoradiography, that SNX-230 (synthetic MVIIC) binds with high affinity to a calcium channel alpha 1 subunit distinct from the high-affinity alpha 1 target of SNX-111 (synthetic MVIIA). SNX-183 (synthetic SVIB) interacts with both alpha 1 subunits with lower affinity. Whereas the alpha 1 subunit recognized with high affinity by MVIIA corresponds to the N-type channel, the other represents a novel calcium channel distinct from N-, L-, and perhaps P-type channels.
Assuntos
Canais de Cálcio/classificação , Proteínas do Tecido Nervoso/classificação , Peptídeos/metabolismo , ômega-Conotoxinas , Animais , Ligação Competitiva , Química Encefálica , Canais de Cálcio/metabolismo , Masculino , Peso Molecular , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Sinaptossomos/metabolismoRESUMO
Neuronal voltage-sensitive calcium channels (VSCCs) are a diverse family of proteins that regulate entry of Ca2+ into neurons. Selective antagonists of VSCCs have proven to be powerful pharmacological tools for identifying and characterizing these channels. A new VSCC antagonist, SNX-230 (also known as omega-conopeptide MVIIC), binds with high affinity to receptors in rat brain and blocks one or more high-threshold VSCCs that are neither L- nor N-type. We have defined the neuroanatomical distribution of the high-affinity non-L, non-N VSCC receptors for SNX-230 using [125I]SNX-230 bound to rat brain sections and compared it with that of [125I]SNX-111, a reversible blocker of N-type VSCCs. Highest densities of binding for both ligands were seen in areas rich in synaptic connections, such as the oriens, radiatum and molecular layers of the hippocampus. In general, the density of [125I]SNX-230-binding was higher in cerebellum compared with that in forebrain. In contrast, this general distribution of density was reversed for [125I]SNX-111. In the glomeruli of the olfactory bulb, binding of [125I]SNX-230 was undetectable compared with the high density of [125I]SNX-111-binding. Differential localization of the two ligands was also seen in cervical spinal cord. The clearly different localization of [125I]SNX-230 compared with that of [125I]SNX-111 in the olfactory bulb and spinal cord suggested that the binding sites for [125I]SNX-230 in other brain regions, while co-localized macroscopically, are also distinct from those for [125I]SNX-111. This was confirmed when addition of saturating concentrations of SNX-111 did not affect the distribution pattern of [125I]SNX-230-binding.(ABSTRACT TRUNCATED AT 250 WORDS)