RESUMO
Gabapentinoids have clinically been used for treating epilepsy, neuropathic pain, and several other neurologic disorders for >30 years; however, the definitive molecular mechanism responsible for their therapeutic actions remained uncertain. The conventional pharmacological observation regarding their efficacy in chronic pain modulation is the weakening of glutamate release at presynaptic terminals in the spinal cord. While the α2/δ-1 subunit of voltage-gated calcium channels (VGCCs) has been identified as the primary drug receptor for gabapentinoids, the lack of consistent effect of this drug class on VGCC function is indicative of a minor role in regulating this ion channel's activity. The current review targets the efficacy and mechanism of gabapentinoids in treating chronic pain. The discovery of interaction of α2/δ-1 with thrombospondins established this protein as a major synaptogenic neuronal receptor for thrombospondins. Other findings identified α2/δ-1 as a powerful regulator of N-methyl-D-aspartate receptor (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) by potentiating the synaptic expression, a putative pathophysiological mechanism of neuropathic pain. Further, the interdependent interactions between thrombospondin and α2/δ-1 contribute to chronic pain states, while gabapentinoid ligands efficaciously reverse such pain conditions. Gabapentin normalizes and even blocks NMDAR and AMPAR synaptic targeting and activity elicited by nerve injury. SIGNIFICANCE STATEMENT: Gabapentinoid drugs are used to treat various neurological conditions including chronic pain. In chronic pain states, gene expression of cacnα2/δ-1 and thrombospondins are upregulated and promote aberrant excitatory synaptogenesis. The complex trait of protein associations that involve interdependent interactions between α2/δ-1 and thrombospondins, further, association of N-methyl-D-aspartate receptor and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor with the C-tail of α2/δ-1, constitutes a macromolecular signaling complex that forms the crucial elements for the pharmacological mode of action of gabapentinoids.
Assuntos
Analgesia , Dor Crônica , Neuralgia , Humanos , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido Glutâmico , Dor Crônica/tratamento farmacológico , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Trombospondinas/metabolismo , IsoxazóisRESUMO
INTRODUCTION: Transdermal cannabinoids may provide better safety and bioavailability profiles compared with other routes of administration. This single-arm, open-label study investigated a novel topical transdermal delivery system on the pharmacokinetics of cannabidiol (CBD) and tetrahydrocannabinol (THC). METHODS: Participants were 39.5 ± 7.37 years old and healthy, based on a review by the Medical Director. Blood was collected pre-dose and 10, 20, 30, and 45 min, and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 12 h after topical application of 100 mg CBD:100 mg THC. Psychoactive effects were assessed prior to each timepoint. Area-under-the-curve (AUC0-12 h), maximum concentration (Cmax), time to maximum concentration (Tmax), area-under-the-curve to infinity (AUCI), terminal elimination rate constant (λ), terminal half-life (t½), and absorption rate constant (ka) were measured individually for CBD and THC. Safety was assessed by clinical chemistry, hematology, and adverse events. RESULTS: AUC0-12 h for CBD and THC was 3329.8 ± 3252.1 and 2093.4 ± 2090.6 pg/mL/h, with Cmax of 576.52 ± 1016.18 and 346.57 ± 776.85 pg/mL, respectively. Tmax for CBD and THC was 8 h, ranging from 2.5 h to 12 h and 10 min to 12 h, respectively. AUCI for CBD and THC was 6609.2 ± 7056.4 and 3721.0 ± 3251.7 pg/mL/h, with t1/2 of 5.68 ± 1.5 and 5.38 ± 1.25 h, respectively. CBD was absorbed at a faster rate compared with THC (123.36 ± 530.97 versus 71.5 ± 1142.19 h-1) but with similar λ (0.12 ± 0.029 versus 0.13 ± 0.03 h-1). No psychoactive effects were reported. Transdermal cannabinoid delivery was safe and well tolerated in the population studied. CONCLUSION: To our knowledge, this is the first pharmacokinetic study in humans that demonstrated CBD and THC entering systemic circulation via transdermal administration . This study represents an important contribution to understanding the pharmacokinetics of transdermal cannabinoids. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier-NCT05121506 (November 16, 2021).
Assuntos
Canabidiol , Dronabinol , Adulto , Humanos , Pessoa de Meia-Idade , Administração Cutânea , Disponibilidade Biológica , Canabidiol/administração & dosagem , Canabidiol/farmacocinética , Canabinoides/administração & dosagem , Canabinoides/efeitos adversos , Dronabinol/administração & dosagem , Dronabinol/farmacocinéticaRESUMO
OBJECTIVES: To identify predictors of the specific (difference between treatment and placebo) and overall (change from baseline in treatment arm) treatment effects of topical NSAIDs in OA. METHODS: Randomized controlled trials (RCTs) of topical NSAIDs in OA were identified through systematic literature searching and inquiry to pharmaceutical companies. The raw, de-identified data were analysed in one-stage individual patient data meta-analysis (IPD-MA). Negative values for treatment effects (0-100 scale) indicate pain reduction. RESULTS: Of 63 eligible RCTs, 15 provided IPD (n = 1951 on topical NSAID), including 11 placebo-controlled RCTs (n = 1587 on topical NSAIDs, 1553 on placebo). Seven potential predictors of response were examined. Topical NSAIDs were superior to placebo [-6 (95% CI -9, -4)], with a small, but statistically significant greater effect in women than men [difference -4 (95% CI -8, -1)]. The overall treatment effect was 4-fold larger than the specific effect [-25 (95% CI -31, -19)] and increased with greater baseline pain severity (P < 0.001). No differences in efficacy were observed for age, BMI, features of inflammation, duration of complaints or radiographic OA severity. CONCLUSION: Topical NSAIDs are effective for OA pain relief. Greater overall pain relief in individuals with more baseline pain might be due to contextual and non-specific effects, including regression to the mean. Additional factors that have been linked either mechanistically or through empirical evidence to outcomes should be selected for inclusion across future RCTs in order to facilitate the identification of response predictors through IPD-MA.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Osteoartrite/tratamento farmacológico , Manejo da Dor/estatística & dados numéricos , Administração Tópica , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Manejo da Dor/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do TratamentoRESUMO
Solutions of very low concentrations cannot be treated by the usual concept of concentration. Stochastic calculations are performed for the analysis of such solutions containing one or a few molecule(s). It is concluded that these systems escape the usual concentration parameters. Two "case histories" are also shown for demonstration of the practical consequences of the theoretical analysis.
RESUMO
AIMS: Accumulation of advanced glycation endpoints is a trigger to the development of diabetic peripheral neuropathy, which is a common complication of diabetes. Oral administration of benfotiamine (BFT) has shown some preclinical and clinical promise as a treatment for diabetic peripheral neuropathy. The purpose of this study was to evaluate the method of transdermal delivery of BFT as a possible, viable route of administration for the treatment of diabetic peripheral neuropathy. METHODS: A single application of 10 mg of BFT was given to guinea pigs topically. The levels of thiamine (T), thiamine monophosphate, thiamine diphosphate, S-benzoylthiamine and BFT were measured in the blood, skin and muscle at different time points within 24 h. RESULTS: At the 24-h time point, following the single BFT dose, the T level was increased 10× in the blood, more than 7× in the skin and almost 4× in the muscle compared to the untreated animals. The total T content (total) was increased 7× in the blood, 17× in the skin and 3× in the muscle compared to the untreated animals. CONCLUSIONS: This strong increase in the tissue levels of T and the associated metabolic derivatives levels found in the blood and local tissues following a single dose indicate that topically applied BFT may be a viable and advantageous delivery method for the treatment of diabetic peripheral neuropathy.
Assuntos
Tiamina/análogos & derivados , Administração Cutânea , Animais , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/metabolismo , Sistemas de Liberação de Medicamentos , Cobaias , Masculino , Músculo Esquelético/metabolismo , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/metabolismo , Pele/metabolismo , Tiamina/administração & dosagem , Tiamina/sangue , Tiamina/metabolismo , Tiamina/farmacocinéticaRESUMO
BACKGROUND: Osteoarthritis is a common condition, typically treated with orally administered analgesics and non-steroidal anti-inflammatory drugs (NSAIDs). Chronic administration of NSAIDs, serotonin-norepinephrine reuptake inhibitors (SNRIs, i.e., duloxetine), and opioid medications (i.e., tramadol) is regularly associated with multiple, serious side effects, in part due to the route of administration. Transdermal delivery of NSAIDs, such as ibuprofen, represents a potentially alternative treatment for this inflammatory pain condition with a better therapeutic profile. OBJECTIVE: Investigate the safety and efficacy of a novel transdermal ibuprofen formulation (VALE®-ibuprofen) containing 10% ibuprofen, compared to a placebo in a randomized, double-blinded clinical trial, for clinical improvement in patients with moderate to severe painful osteoarthritis of the knee. STUDY DESIGN: A randomized, placebo-controlled, double blind, multi-center Phase 2 clinical trial. SETTING: An academic medical center, and private rheumatology and interventional pain management practices. METHODS: The Phase 2 clinical study included patients with primary osteoarthritis in a single knee joint with a progression level of moderate to severe based in part on a grade II or III designation according to the Kellgren and Lawrence classification system. Patients received the corresponding, randomly assigned study formulation (VALE-ibuprofen or placebo) for application to the target knee at a dose of 2.0 grams of drug product (200 mg ibuprofen) twice daily for 14 days. The evaluation of the efficacy of the treatments utilized the widely accepted methods of the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index and the Visual Analog Scale (VAS) scores for the patients. RESULTS: The results indicate that the transdermal VALE-ibuprofen formulation was very well tolerated from a safety perspective during the 2-week trial and also produced significant, positive clinical improvements superior to the placebo in all clinical endpoints tested. In particular, the WOMACTotal and WOMACPhysical Functioning, for the VALE-ibuprofen, were superior compared to the placebo (P = 0.0283 and P = 0.0201, respectively). Other clinical endpoints including the WOMACPain, WOMACStiffness, and VASResting scores were superior to those obtained from the placebo group, trending towards statistical significance compared to placebo (P = 0.0811, 0.1103, and 0.0785, respectively). Based on the Patient and Physician Global Impression of Change survey, patient satisfaction slightly improved across both groups; however, no statistical significance was detectable as compared to the baseline. LIMITATIONS: The sample size of 64 subjects in the final data analysis and the lack of including an orally administered drug group are limitations of this study. CONCLUSIONS: The use of transdermal VALE-ibuprofen has beneficial clinical effects on the pain levels experienced in some patients with moderate to severe osteoarthritis of the knee as measured by the WOMAC Osteoarthritis Indices for stiffness, pain, physical function, and total. Visual Analog Scales (VAS) tests, VASMotion and VASWeight-bearing, again while appeared superior to placebo, were not statistically different from placebo. CLINICAL TRIAL REGISTRATION: NCT01496326.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Ibuprofeno/administração & dosagem , Osteoartrite do Joelho/tratamento farmacológico , Administração Cutânea , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da DorRESUMO
The purpose of the study was to evaluate the effect of adding peripheral vasodilators, tolazoline, or papaverine, to transdermal drug delivery vehicles with the goal of improving the tissue bioavailability of transdermally delivered ibuprofen. Ibuprofen (150 mg) formulations with several concentrations of two different vasodilators and/or a penetration enhancer (PE) complex were topically applied to rabbits. Plasma levels of ibuprofen were determined by a validated high-performance liquid chromatography method and evaluated at 0, 0.5, 1, 2, and 3 h. The PE complex enhanced the plasma ibuprofen level approximately sevenfold versus control, and tolazoline (0.005%) added to the PE complex increased the plasma levels of ibuprofen approximately another twofold compared with the PE. Higher concentrations of tolazoline paradoxically did not exhibit vasodilator enhancement to ibuprofen delivery. Papaverine was tested in the same manner. In this set of experiments, PE increased the plasma ibuprofen 3.7-fold versus control, and addition of papaverine (0.0005%) increased plasma ibuprofen an additional 3.3-fold compared with the PE formulation. Transdermal formulations of ibuprofen containing low concentrations of tolazoline or papaverine increased plasma ibuprofen levels in the presence of passive PE components.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Ibuprofeno/administração & dosagem , Papaverina/farmacologia , Absorção Cutânea/efeitos dos fármacos , Tolazolina/farmacologia , Vasodilatadores/farmacologia , Administração Cutânea , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/farmacocinética , Ibuprofeno/sangue , Ibuprofeno/farmacocinética , Permeabilidade/efeitos dos fármacos , Coelhos , Pele/efeitos dos fármacos , Pele/metabolismo , Tolazolina/administração & dosagem , Vasodilatadores/administração & dosagemRESUMO
Naturally selected atrazine-resistant (AR) weeds possessing a Ser(264) --> Gly D1 protein encoded by a mutant psbA allele in the chloroplast-DNA have increased photosensitivity and lower fitness. The D1 mutant lines of S. nigrum revealed impaired regulation of photosystem II (PSII) activity as compared with the wild-type plants resulting in a less effective photochemical light utilization and in addition, a lower capacity of non-photochemical thermal dissipation (NPQ), one of the main photoprotective mechanisms in oxygenic photosynthetic organisms. In this work, comparative chlorophyll fluorescence analysis in attached leaves of wild-type and AR Solanum nigrum L. and in their reciprocal crosses has been used to establish how the lower NPQ is inherited. Both a 50% reduction in steady-state NPQ and a 60-70% reduction in the rapidly reversible, energy-dependent (qE) component of NPQ were common phenomena in the parent and hybrid lines of D1 mutant S. nigrum. The nuclear hybrid status of the F2 plant material was confirmed by morphological observations on fully developed leaves. No alteration was found in the nucleotide sequence and the deduced amino acid sequences of the nuclear psbS gene isolated from different biotypes of S. nigrum, and there were no differences in the expressions of both the PsbS and the D1 proteins. All things considered, co-inheritance of the lower photoprotective NPQ capacity and the Ser(264) --> Gly D1 protein mutation was clearly observed, suggesting that the evolutionarily conserved D1 structure must be indispensable for the efficient NPQ process in higher plants.
Assuntos
Sequência Conservada , DNA de Cloroplastos/genética , Luz , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Solanum nigrum/metabolismo , Temperatura , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Cruzamentos Genéticos , Fluorescência , Genes de Plantas/genética , Hibridização Genética , Immunoblotting , Dados de Sequência Molecular , Fotossíntese/efeitos da radiação , Folhas de Planta/anatomia & histologia , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Reprodutibilidade dos Testes , Alinhamento de Sequência , Solanum nigrum/efeitos da radiação , Relação Estrutura-Atividade , Xantofilas/metabolismoRESUMO
Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio/fisiologia , Aminas/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Western Blotting , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Ácidos Cicloexanocarboxílicos/metabolismo , Eletrofisiologia , Éxons/genética , Gabapentina , Genótipo , Coração/efeitos dos fármacos , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido gama-Aminobutírico/metabolismoRESUMO
Heat sensitivity of grapevine (Vitis vinifera L. cv. Kékfrankos) photosynthesis was studied in two vineyards (Eger-Kolyukteto, flat; and Eger-Nagyeged hill, steep slope) with different mesoclimates and water supply conditions in two climatically different years. 2007 was drier and warmer, with higher vapour pressure deficit (VPD) than 2005. Pre-dawn water potential measurements indicated mild water deficit at the steep-sloped vineyard. In July 2005 mild water deficit enhanced the thermostability of grapevine photosynthesis, as reflected in the temperature dependence of optimal quantum yield (Fv/Fm) and in the critical temperature of initial fluorescence (F0Tc). Decreased Fv/Fm and actual quantum yield (ΔF/Fm') was recorded at most temperatures in September at the water-stressed (steep slope) site. This time, F0Tcs were also lower due to early leaf senescence. In September 2007, heat sensitivity of Fv/Fm was similar to 2005, and ΔF/Fm' indicated higher thermostability at both sites, but keeping the consistent difference between the two vineyards. The critical points of steady-state fluorescence (FsTc) were higher by 3-6°C at both vineyards in 2007 than in 2005. Although, in September thermolabile F0 signals were measured at the water-stressed vineyard, the heat sensitivity was not decreased in light adapted state, assumingly as a result of enhanced xanthophyll cycle pigment pool size. The higher xanthophyll pigments pool size (V + A + Z) in 2007 (compared to 2005) at the unstressed (flat) vineyard suggests that high temperature and VPD play a role in changing (V + A + Z)/(chl a + b), and, thus, results in higher thermostability under high light conditions.
RESUMO
Polychromatic flow cytometry enables detailed identification of cell phenotype using multiple fluorescent parameters. The photomultiplier tubes (PMTs) used to detect fluorescence in current instruments limit the sensitivity in the long wavelength spectral range. We demonstrate the flow cytometric applications of silicon avalanche photodiodes (APDs), which have improved red sensitivity and a working fluorescence detection range beyond 1,000 nm. A comparison of the wavelength-dependent performance of the APD and PMT was carried out using pulsed light-emitting diode sources, calibrated test beads, and biological samples. A breadboard flow cytometer test bench was constructed to compare the performance of PMTs and APD detectors. The APD used an additional amplifier stage to match the internal gain of the PMT. The resolution of the APD and PMT was compared for flow cytometry applications using a pulsed light-emitting diode source over the 500-1060 nm spectral range. These measurements showed the relative changes in the signal-to-noise performance of the APD and PMT over a broad spectral range. Both the APD and PMTs were used to measure the signal-to-noise response for a set of six peak calibration beads over the 530-800 nm wavelength range. CD4-positive cells labeled with antibody-conjugated phycoerythrin or 800 nm quantum dots were identified by simultaneous detection using the APD and the PMT. The ratios of the intensities of the CD4- and CD4+ populations were found to be similar for both detectors in the visible wavelengths, but only the APD was able to separate these populations at wavelengths above 800 nm. These measurements illustrate the differences in APD and PMT performance at different wavelengths and signal intensity levels. While the APD and PMT show similar signal-to-noise performance in the visible spectral range, the dark noise of the APD detector reduces the sensitivity at low signal levels. At wavelengths longer than 650 nm, the high quantum efficiency of the APD contributes to better signal-to-noise performance. The APD detector provides enhanced performance in the long wavelength region and may be used to extend the working range of the flow cytometer beyond 1,000 nm.
Assuntos
Citometria de Fluxo/instrumentação , Raios Infravermelhos , Luz , Anticorpos/farmacologia , Antígenos CD4/metabolismo , Calibragem , Fluorescência , Humanos , Ficoeritrina/metabolismoRESUMO
Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Monóxido de Carbono/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/metabolismo , Mitocôndrias Cardíacas/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/genética , Humanos , Mitocôndrias Cardíacas/genética , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Miócitos Cardíacos/enzimologia , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Compostos Organofosforados/farmacologia , Mutação Puntual , Estrutura Terciária de Proteína/genética , Ratos , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Xantina Oxidase/genética , Xantina Oxidase/metabolismoRESUMO
In order to investigate the role of potential genotypic differences in three economically important barley cultivars, experiments were carried out to determine the influence of supplemental ultraviolet-B (UV-B, 280-320 nm) radiation on reactive oxygen species (ROS), antioxidant activity and photosynthesis. Greenhouse-grown barley (Hordeum vulgare L.) cultivars 'Cork', 'Prestige' and 'Golden Promise' showed different responses to supplemental 280-320 nm (UV-B) representing 100, 138 and 238% levels of ambient biologically active UV-B radiation, respectively. Among the three cultivars studied, cv. Golden Promise was the most tolerant to UV-B, cv. Prestige was slightly more sensitive than cv. Cork. A comparison with the other two cultivars showed that under supplemental UV-B, Golden Promise leaves (i) retained a higher quantum yield of photosynthesis under photosynthetically active radiation (PAR, 400-700 nm) corresponding to growth conditions; (ii) had the smallest decrease in both electron transport rate and non-photochemical quenching under high PAR; (iii) contained less oxidized ascorbate [measured as dehydroascorbate or electron paramagnetic resonance (EPR) detectable monodehydroascorbate radicals] than either Cork or Prestige. Under the highest UV-B level applied, Golden Promise leaves maintained the same activity of both monodehydroascorbate-reductase (MDAR) and ascorbate-peroxidase (APX) enzymes, as untreated controls, while MDAR markedly decreased in the other two cultivars and APX slightly increased in cv. Prestige. These features, together with the observation of directly EPR-trappable free radicals and the light-independent accumulation of monodehydroascorbate radicals in Cork and Prestige but not in Golden Promise leaves under high UV-B suggest that Golden Promise plants suffered less oxidative stress than the two other cultivars.
RESUMO
We examined the effects of chronic hypoxia on recombinant human L-type Ca2+ channel alpha1C subunits stably expressed in HEK 293 cells, using whole-cell patch-clamp recordings. Current density was dramatically increased following 24 h exposure to chronic hypoxia (CH), and membrane channel protein levels were enhanced. CH also increased the levels of Alzheimer's amyloid beta peptides (AbetaPs), determined immunocytochemically. Pharmacological prevention of AbetaP production (via exposure to inhibitors of secretase enzymes that are required to cleave AbetaP from its precursor protein) prevented hypoxic augmentation of currents, as did inhibition of vesicular trafficking with bafilomycin A1. The enhancing effect of AbetaPs or CH were abolished following incubation with the monoclonal 3D6 antibody, raised against the extracellular N' terminus of AbetaP. Immunolocalization and immunoprecipitation studies provided compelling evidence that AbetaPs physically associated with the alpha1C subunit, and this association was promoted by hypoxia. These data suggest an important role for AbetaPs in mediating the increase in Ca2+ channel activity following CH and show that AbetaPs act post-transcriptionally to promote alpha1C subunit insertion into (and/or retention within) the plasma membrane. Such an action will likely contribute to the Ca2+ dyshomeostasis of Alzheimer's disease and may contribute to the mechanisms underlying the known increased incidence of this neurodegenerative disease following hypoxic episodes.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Canais de Cálcio Tipo L/genética , Regulação da Expressão Gênica/fisiologia , Hipóxia/genética , Peptídeos/metabolismo , Regulação para Cima/fisiologia , Linhagem Celular , Humanos , Rim/química , Rim/citologia , Rim/embriologia , Rim/metabolismoAssuntos
Canais de Cálcio Tipo L/metabolismo , Oxigênio/metabolismo , Canais de Cálcio Tipo L/genética , DNA/isolamento & purificação , Humanos , Mutação , Técnicas de Patch-Clamp/métodos , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas/metabolismo , Deleção de Sequência , Transfecção/métodosRESUMO
OBJECTIVE: Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca(2+)-free experimental conditions in our previous study. METHODS: Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba(2+), or a mixture of 30 mM Ca(2+) and 60 mM Ba(2+) to facilitate Ca(2+) permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 microM) was used to reveal a putative role for calcineurin in regulation of LTCC. RESULTS: A mixture of Ca(2+) and Ba(2+) as charge carriers allowed for Ca(2+) permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba(2+) as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca(2+) permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity. CONCLUSIONS: We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba(2+) permeation but can be compensated or overcome by a specific Ca(2+)-dependent stimulation with Ca(2+) permeation. More complete restoration of physiological Ca(2+) movements (e.g., Ca(2+) release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC.
Assuntos
Inibidores de Calcineurina , Canais de Cálcio Tipo L/fisiologia , Cálcio/farmacologia , Ciclosporina/farmacologia , Coração/fisiologia , Bário/farmacologia , Cálcio/fisiologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Linhagem Celular , Coração/efeitos dos fármacos , Átrios do Coração , Insuficiência Cardíaca/fisiopatologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Células Musculares/efeitos dos fármacos , Células Musculares/fisiologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Função VentricularRESUMO
The carboxyl terminal of the L-type calcium channel alpha1C subunit comprises approximately one third of the primary structure of the alpha1 subunit (> 700 amino acids residues). This region is sensitive to limited posttranslational processing. In heart and brain the alpha1C subunits are found to be truncated but the C-terminal domain remains functionally present. Based on our previous data we hypothesized that the distal C-terminus (approximately residues 1650-1950) harbors an important, predominantly inhibitory domain. We generated C-terminal-truncated alpha1C mutants, and after expressing them in combination with a beta3 subunit in HEK-293 cells, electrophysiological experiments were carried out. In order to dissect the important inhibitory part of the C-terminus, trypsin was dialyzed into the cells. The data provide evidence that there are multiple residues within the inhibitory domain that are crucial to the inhibitory process as well as to the enhancement of expressed current by intracellular application of proteases. In addition, the expression of the chimeric mutant alpha(1C)delta1673-DRK1 demonstrated that the C-terminal is specific for the heart channel.
Assuntos
Regulação da Expressão Gênica , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/química , Processamento Alternativo , Northern Blotting , Encéfalo/metabolismo , Canais de Cálcio Tipo L , Linhagem Celular , Células Cultivadas , DNA Complementar/metabolismo , Eletrofisiologia , Deleção de Genes , Humanos , Modelos Biológicos , Mutação , Proteínas do Tecido Nervoso/biossíntese , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Tripsina/farmacologiaRESUMO
OBJECTIVES: This study was designed to identify possible electrical remodeling (ER) in transgenic (Tg) mice with over-expressed L-type Ca(2+) channels. Transient outward K(+) current (I(to)) and action potential duration (APD) were studied in 2-, 4-, 8-, and 9- to 12-month-old mice to determine linkage to ventricular remodeling (VR), ER, and heart failure (HF). BACKGROUND: Prolongation of APD and reduction in current density of I(to) are thought to be hallmarks of VR and HF. Mechanisms are not understood. METHODS: Patch-clamp, perfused hearts, echocardiography, and Western blots were employed using 2-, 4-, 8-, and 9- to 12-month-old Tg mice. RESULTS: Transgenic mice developed slow VR statistically manifesting at four months and continuing through death at 12 to 14 months, despite a slight up-regulation of I(to). A slight decrease or no change in APD was observed up to eight months; however, at 9 to 12 months, a small increase in APD was detected. Early afterdepolarizations were observed after application of 4-aminopyridine in Tg mice. No change was detected in protein of Kv4.3 and Kv4.2 up to eight months. At 9 to 12 months, Tg mice showed a slight decrease (41.4 +/- 6.9%, p < 0.05) in Kv4.2, consistent with a decrease in I(to). Surprisingly, Kv1.4 (the "fetal" K(+)-channel form) was up-regulated, and restitution of I(to) was slowed. Echocardiography revealed cardiac enlargement with impaired chamber function in hearts that were taken from the older animals. CONCLUSIONS: Contrary to accepted dogma, APD and I(to) in a mouse model of hypertrophy and HF are not hallmarks of pathophysiology. We suggest that [Ca(2+)](i) (i.e., [Ca(2+)] concentration) is the primary factor in triggering cardiac enlargement and arrhythmogenesis.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Cardiomegalia/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Remodelação Ventricular/fisiologia , Animais , Cardiomegalia/complicações , Cardiomegalia/diagnóstico por imagem , Modelos Animais de Doenças , Ecocardiografia , Eletrofisiologia , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/etiologia , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Fatores de Risco , Fatores de TempoRESUMO
Atrazine-resistant (AR) weeds have a modified D1 protein structure, with a Ser264-->Gly mutation on the D1 protein, near the plastoquinone binding niche. The photosynthetic performance, the light response of the xanthophyll cycle and chlorophyll fluorescence quenching-related parameters were compared in attached leaves of susceptible (S) and AR biotypes of the C3 dicot Chenopodium album L., Epilobium adenocaulon Hausskn., Erigeron canadensis L., Senecio vulgaris L. and Solanum nigrum L. and the C4 dicot Amaranthus retroflexus L. grown under natural high-light conditions. No significant difference in CO2 assimilation rate per leaf area unit was found between the S and AR biotypes of the investigated C3 plants, whereas the AR biotype of A. retroflexus exhibited a relatively poor photosynthetic performance. The D1 protein mutant plants expressed a reduced activity of light-stimulated zeaxanthin formation. Neither the lower violaxanthin de-epoxidase activity nor the depletion of ascorbate seems to be the cause of the lower in vivo zeaxanthin formation in the AR plants. All the D1 mutant weeds had limited light-induced non-photochemical (NPQ) and photochemical (qP) quenching capacities, and displayed a higher photosensitivity, as characterized by the ratio (1-qP)/NPQ and a higher susceptibility to photoinhibition. Analysis of the chlorophyll fluorescence parameters showed that a lower proportion of excitation energy was allocated to PSII photochemistry, while a higher excess of excitation remained in the AR weeds relative to the S plants.
RESUMO
l-Type calcium channels are multiprotein complexes composed of pore-forming (CaV1.2) and modulatory auxiliary alpha2delta- and beta-subunits. We demonstrate expression of two different isoforms for the beta2-subunit (beta2a, beta2b) and the beta3-subunit (beta3a, beta3trunc) in human non-failing and failing ischemic myocardium. Quantitatively, in the left ventricle expression of beta2b transcripts prevails in the order of > beta3 >> beta2a. The expressed cardiac full-length beta3-subunit is identical to the beta3a-isoform, and beta3trunc results from deletion of exon 6 (20 nn) entailing a reading frameshift and translation stop at nucleotide position 495. In failing ischemic myocardium beta3trunc expression increases whereas overall beta3 expression remains unchanged. Heterologous coexpression studies demonstrated that beta2 induced larger currents through rabbit and human cardiac CaV1.2 pore subunits than beta3 isoforms. All beta-subunits increased channel availability at single channel level, but beta2 exerted an additional, marked stimulation of rapid gating (open and closed times, first latency), leading to higher peak current values. We conclude that cardiac beta-subunit isoforms differentially modulate calcium inward currents because of regulatory effects within the channel protein complex. Moreover, differences in the various beta-subunit gene products present in human heart might account for altered single channel behavior found in human heart failure.