RESUMO
Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.
Assuntos
Dor do Câncer/tratamento farmacológico , Hiperalgesia/tratamento farmacológico , Peptídeos/administração & dosagem , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Analgésicos Opioides/efeitos adversos , Animais , Dor do Câncer/induzido quimicamente , Dor do Câncer/genética , Dor do Câncer/patologia , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/patologia , Ligantes , Camundongos , Camundongos Knockout , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Neuralgia/genética , Neuralgia/patologia , Antagonistas Nicotínicos/administração & dosagem , Compostos Organoplatínicos/efeitos adversos , Oxaliplatina , Receptores de GABA-B/genéticaRESUMO
The activity of persistent Ca²âº sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²âº entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²âº sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca²âº sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²âº sparklets? With the use of total internal reflection fluorescence microscopy, Ca²âº sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca²âº sparklets. Persistent Ca²âº sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³9F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³9F Cav1.2c abrogated the potentiating effect of c-Src on Ca²âº sparklet activity. We could not detect a significant change in persistent Ca²âº sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²âº sparklets. However, our results indicate that persistent Ca²âº sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Cav1.2c COOH terminus.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Neurônios/metabolismo , Proteína Quinase C-alfa/metabolismo , Quinases da Família src/metabolismo , Animais , Proteína Tirosina Quinase CSK , Canais de Cálcio Tipo L/genética , Linhagem Celular Transformada , Regulação da Expressão Gênica , Humanos , Microscopia de Fluorescência , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação , Proteína Quinase C-alfa/genética , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Pirimidinas/farmacologia , Ratos , Transdução de Sinais , Transfecção , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
Voltage-gated, dihydropyridine-sensitive L-type Ca(2+) channels are multimeric proteins composed of a pore-forming transmembrane α(1) subunit (Ca(v)1.2) and accessory ß, α(2)δ, and γ subunits. Ca(2+) entry via Ca(v)1.2 channels shapes the action potential (AP) of cardiac myocytes and is required for excitation-contraction coupling. Two de novo point mutations of Ca(v)1.2 glycine residues, G406R and G402S, cause a rare multisystem disorder called Timothy syndrome (TS). Here, we discuss recent work on the mechanisms by which Ca(v)1.2 channels bearing TS mutations display slowed inactivation that leads to increased Ca(2+) influx, prolonging the cardiac AP and promoting lethal arrhythmias. Based on these studies, we propose a model in which the scaffolding protein AKAP79/150 stabilizes the open conformation of Ca(v)1.2-TS channels and facilitates physical interactions among adjacent channels via their C-tails, increasing the activity of adjoining channels and amplifying Ca(2+) influx.
Assuntos
Canais de Cálcio Tipo L , Canalopatias/patologia , Síndrome do QT Longo/patologia , Sindactilia/patologia , Transtorno Autístico , Humanos , Síndrome do QT Longo/genética , Mutação , Miócitos Cardíacos , Sindactilia/genéticaRESUMO
Plasmid vectors used for mammalian expression or for in vitro cRNA translation can differ substantially and are rarely cross-compatible. To make comparisons between mammalian and Xenopus oocyte expression systems, it would be advantageous to use a single vector without the need for shuttle vectors or subcloning. We have designed such a vector, designated pUNIV for universal, with elements that will allow for in vitro or ex vivo expression in multiple cell types. We tested the expression of pUNIV-based cDNA cassettes using enhanced green fluorescent protein and two forms of the type A gamma-aminobutyric acid receptor (GABA(A)R) and compared pUNIV to vectors optimized for expression in either Xenopus oocytes or mammalian cells. In HEK293 cells, radioligand binding was robust, and patch clamp experiments showed that subtle macroscopic GABA(A)R kinetics were indistinguishable from our previous results. In Xenopus oocytes, agonist median effective concentration measurements matched previous work using a vector optimized for oocyte expression. Furthermore, we found that expression using pUNIV was significantly enhanced in oocytes and was remarkably long-lasting in both systems.
Assuntos
Vírus do Mosaico da Alfafa/genética , Vetores Genéticos , Canais Iônicos/genética , Rim/metabolismo , Oócitos/metabolismo , Animais , Linhagem Celular , Corantes Fluorescentes/metabolismo , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Rim/citologia , Cinética , Técnicas de Patch-Clamp , Plasmídeos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Ensaio Radioligante , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiologia , Fatores de Tempo , XenopusRESUMO
Estudio descriptivo retrospectivo realizado en el servicio de cirugía vascular periférica del Hospital de San José de Bogotá D.C. Colombia, en un período de seis años. Se encontraron 21 pacientes con diagnóstico de aneurisma de aorta abdominal roto, los cuales se dividieron en dos grupos, uno contenido y otro no contenido. La principal causa de mortalidad fue choque hipovolémico, seguida de estado de falla multisistémica. Los orígenes más comunes de morbilidad fueron infarto agudo del miocardio y síndrome de dificultad respiratoria del adulto. El índice de mortalidad global fue de 57.14%.
This is a descriptive retrospective study conducted in the Peripheral Vascular Surgery Service at the San José Hospital in Bogotá D.C., Colombia during a six-year period. Twenty-one patients with a diagnosis of a ruptured abdominal aortic aneurysm were found and distributed into two groups: contained and not contained ruptures. The main mortality cause was hypovolemic shock followed by a state of multisystem failure. The most common sources of morbility were related to acute myocardial infarction and adult respiratory distress syndrome. The global mortality rate was 57,14%.