RESUMO
Trimeric intracellular cation channels (TRIC-A and TRIC-B) are thought to provide counter-ion currents to enable charge equilibration across the sarco/endoplasmic reticulum (SR) and nuclear membranes. However, there is also evidence that TRIC-A may interact directly with ryanodine receptor type 1 (RyR1) and 2 (RyR2) to alter RyR channel gating. It is therefore possible that the reverse is also true, where the presence of RyR channels is necessary for fully functional TRIC channels. We therefore coexpressed mouse TRIC-A or TRIC-B with mouse RyR2 in HEK293 cells to examine if after incorporating membrane vesicles from these cells into bilayers, the presence of TRIC affects RyR2 function, and to characterize the permeability and gating properties of the TRIC channels. Importantly, we used no purification techniques or detergents to minimize damage to TRIC and RyR2 proteins. We found that both TRIC-A and TRIC-B altered the gating behavior of RyR2 and its response to cytosolic Ca2+ but that TRIC-A exhibited a greater ability to stimulate the opening of RyR2. Fusing membrane vesicles containing TRIC-A or TRIC-B into bilayers caused the appearance of rapidly gating current fluctuations of multiple amplitudes. The reversal potentials of bilayers fused with high numbers of vesicles containing TRIC-A or TRIC-B revealed both Cl- and K+ fluxes, suggesting that TRIC channels are relatively non-selective ion channels. Our results indicate that the physiological roles of TRIC-A and TRIC-B may include direct, complementary regulation of RyR2 gating in addition to the provision of counter-ion currents of both cations and anions.
Assuntos
Retículo Endoplasmático , Canal de Liberação de Cálcio do Receptor de Rianodina , Humanos , Animais , Camundongos , Células HEK293 , Biofísica , Citosol , Canais IônicosRESUMO
BACKGROUND/AIM: We have previously reported the protection of doxorubicin-induced keratinocyte toxicity by alkaline extract of the leaves of Sasa senanensis Rehder (SE). In order to extend the generality of the cell protective effect of SE, we investigated whether it also protects rat PC12 and human SH-SY5Y neuron model cells from amyloid ß-peptide (Aß)-induced injury. MATERIALS AND METHODS: Viability of cells was determined by the MTT method. Cytotoxicity was evaluated by the concentration that reduces the cell viability by 50% (CC50). Protection from Aß-induced cytotoxicity was evaluated by the concentration that reversed the Aß-induced reduction of viability by 50% (EC50). The selectivity index (SI) of neuroprotective activity was defined as the ratio of EC50 to CC50 Aß1-42 aggregation was assayed using Aß1-42 ammonium hydroxide. RESULTS: SE showed hormetic growth stimulation at lower concentrations in both neuron precursors and differentiated cells. SE reproducibly inhibited Aß-induced cytotoxicity against both undifferentiated and differentiated neuron cells. Both the extent of differentiation induction and viability depended on the cell density, suggesting the release of growth and differentiation stimulation substances into culture supernatant. Higher concentrations of SE partially reduced the Aß1-42 aggregation. CONCLUSION: Hormetic growth stimulation and inhibition of aggregation may be involved in the neuroprotective activity of SE.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Sasa/química , Peptídeos beta-Amiloides/farmacologia , Animais , Antioxidantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neurônios/patologia , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/metabolismo , RatosRESUMO
Myricetin (3,3',4',5,5',7-hexahydroxyflavone), a major flavonoid in berries and red wine, has been recently used as a health food supplement based on its antioxidant and antitumor properties. We report here that myricetin preferentially exerts inhibitory effects on gastric H+, K+-ATPase. Myricetin inhibited H+, K+-ATPase with a sub-micromolar IC50 value in an enzyme assay using freeze-dried tubulovesicles prepared from hog stomach. Na+, K+-ATPase and Ca2+-ATPase were also inhibited by myricetin in a dose-dependent manner, but the IC50 values for these enzymes were approximately an order of magnitude higher compared to the H+, K+-ATPase. In structure-inhibitory functional analysis of sixteen myricetin derivatives, several phenolic hydroxy groups attached to the flavonoid backbone were highlighted as essential modifications for the inhibition of P2-type ATPases. Furthermore, oral administration of myricetin significantly attenuated histamine-induced gastric acid secretion in an in vivo mouse assessment. Therefore, myricetin derivatives seem to be useful seed compounds for developing new drugs and supplements to alleviate gastric acid secretion.
Assuntos
Produtos Biológicos/farmacologia , Flavonoides/farmacologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Inibidores da Bomba de Prótons/farmacologia , Estômago/enzimologia , Animais , Produtos Biológicos/química , Cálcio/metabolismo , Flavonoides/química , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Inibidores da Bomba de Prótons/química , Bombas de Próton/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
BACKGROUND/AIM: Most of the previous investigators have used various types of media for the culture of nerve cells. In order to optimize the culture conditions, we compared the growth rate and amino acid consumption by two popular neuron models, rat PC12 and human SH-SY5Y, grown in DMEM or DMEM: Ham's F-12 (1:1): non-essential amino acids, supplemented with 10% fetal bovine serum (referred to DMEM and Mix, respectively). MATERIALS AND METHODS: Cell growth was monitored by the MTT method. Amino acids in the culture medium were quantitated by amino acid analysis after deproteinization. RESULTS: Efficient cell attachment could be achieved even if PC12 cells were inoculated at extreme lower cell density in a non-coated plain dish, without addition of its condition medium. Both PC12 and SH-SY5Y cells proliferated up to slightly higher cell density in DMEM than in Mix. Approximately 2-fold higher utilization rate of glutamine and essential amino acids was observed in DMEM. Amyloid peptides such as Aß1-42 and Aß25-35 suppressed their growth nearly by 50%. CONCLUSION: The present study suggests the usefulness of DMEM for the study of searching neuroprotective substances, based on its favorable effects on cell attachment, cell growth and amino acid utilization as well as amyloid peptide sensitivity.
Assuntos
Aminoácidos/isolamento & purificação , Proliferação de Células/genética , Células PC12/química , Aminoácidos/genética , Animais , Sobrevivência Celular/genética , Células Cultivadas , Meios de Cultura/química , Humanos , Neurônios/química , Neurônios/metabolismo , Células PC12/metabolismo , RatosRESUMO
Trimeric intracellular cation channels (TRIC) represents a novel class of trimeric intracellular cation channels. Two TRIC isoforms have been identified in both the human and the mouse genomes: TRIC-A, a subtype predominantly expressed in the sarcoplasmic reticulum (SR) of muscle cells, and TRIC-B, a ubiquitous subtype expressed in the endoplasmic reticulum (ER) of all tissues. Genetic ablation of either TRIC-A or TRIC-B leads to compromised K(+) permeation and Ca(2+) release across the SR/ER membrane, supporting the hypothesis that TRIC channels provide a counter balancing K(+) flux that reduces SR/ER membrane depolarization for maintenance of the electrochemical gradient that drives SR/ER Ca(2+) release. TRIC-A and TRIC-B seem to have differential functions in Ca(2+) signaling in excitable and nonexcitable cells. Tric-a(-/-) mice display defective Ca(2+) sparks and spontaneous transient outward currents in arterial smooth muscle and develop hypertension, in addition to skeletal muscle dysfunction. Knockout of TRIC-B results in abnormal IP3 receptor-mediated Ca(2+) release in airway epithelial cells, respiratory defects, and neonatal lethality. Double knockout mice lacking both TRIC-A and TRIC-B show embryonic lethality as a result of cardiac arrest. Such an aggravated lethality indicates that TRIC-A and TRIC-B share complementary physiological functions in Ca(2+) signaling in embryonic cardiomyocytes. Tric-a(-/-) and Tric-b(+/-) mice are viable and susceptible to stress-induced heart failure. Recent evidence suggests that TRIC-A directly modulates the function of the cardiac ryanodine receptor 2 Ca(2+) release channel, which in turn controls store-overload-induced Ca(2+) release from the SR. Thus, the TRIC channels, in addition to providing a countercurrent for SR/ER Ca(2+) release, may also function as accessory proteins that directly modulate the ryanodine receptor/IP3 receptor channel functions.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/fisiologia , Canais Iônicos/metabolismo , Retículo Sarcoplasmático/fisiologia , Animais , Homeostase/fisiologia , Humanos , Canais Iônicos/genética , CamundongosRESUMO
Sarcoplasmic/endoplasmic reticulum (SR) and nuclear membranes contain two related cation channels named TRIC-A and TRIC-B. In many tissues, both subtypes are co-expressed, making it impossible to distinguish the distinct single-channel properties of each subtype. We therefore incorporated skeletal muscle SR vesicles derived from Tric-a-knockout mice into bilayers in order to characterise the biophysical properties of native TRIC-B without possible misclassification of the channels as TRIC-A, and without potential distortion of functional properties by detergent purification protocols. The native TRIC-B channels were ideally selective for cations. In symmetrical 210 mM K(+), the maximum (full) open channel level (199 pS) was equivalent to that observed when wild-type SR vesicles were incorporated into bilayers. Analysis of TRIC-B gating revealed complex and variable behaviour. Four main sub-conductance levels were observed at approximately 80 % (161 pS), 60 % (123 pS), 46 % (93 pS), and 30 % (60 pS) of the full open state. Seventy-five percent of the channels were voltage sensitive with Po being markedly reduced at negative holding potentials. The frequent, rapid transitions between TRIC-B sub-conductance states prevented development of reliable gating models using conventional single-channel analysis. Instead, we used mean-variance plots to highlight key features of TRIC-B gating in a more accurate and visually useful manner. Our study provides the first biophysical characterisation of native TRIC-B channels and indicates that this channel would be suited to provide counter current in response to Ca(2+) release from the SR. Further experiments are required to distinguish the distinct functional properties of TRIC-A and TRIC-B and understand their individual but complementary physiological roles.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Animais , Cálcio/metabolismo , Ativação do Canal Iônico/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Potássio/metabolismo , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/fisiologiaRESUMO
Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca2+ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca2+, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5-10 mM. With Ca2+ as permeant ion, 5-10 mM ATP consistently inactivated recombinant channels (15/16 experiments). Such inactivation was rarely observed with native RyR2 isolated from cardiac muscle (1 in 16 experiments). However, if the channels were purified, inactivation by ATP was then revealed in all experiments. This action of ATP may be relevant for inactivation of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling or may represent unnatural behavior that is revealed when RyR2 is purified or expressed in noncardiac systems.
Assuntos
Trifosfato de Adenosina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , DNA Complementar/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Coelhos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Suramina/farmacologiaRESUMO
The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL/6J and 129/SV mice, Tcore was continuously recorded under 12:12 h light:dark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity and/or cortisol regulation.