RESUMEN
In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25(OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Vitamina D/análogos & derivados , Vitamina K 2/farmacología , Factor de Transcripción Activador 4 , Fosfatasa Alcalina/metabolismo , Animales , Calcitriol , Calcio/metabolismo , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Sinergismo Farmacológico , Proteínas de Homeodominio , Masculino , Ratones , Osteocalcina/biosíntesis , Factor de Transcripción Sp7 , Factores de Transcripción/biosíntesis , Vitamina D/farmacologíaRESUMEN
Ergothioneine is a thiourea derivative of histidine found in food, especially mushrooms. Experiments in cell-free systems and chemical assays identified this compound as a powerful antioxidant. Experiments were designed to test the ability of endothelial cells to take up ergothioneine and hence benefit from protection against oxidative stress. Reverse-transcription polymerase chain reaction and Western blotting demonstrated transcription and translation of an ergothioneine transporter in human brain microvascular endothelial cells (HBMECs). Uptake of [(3)H]ergothioneine occurred by the organic cation transporter novel type-1 (OCTN-1), was sodium-dependent, and was reduced when expression of OCTN-1 was silenced by small interfering RNA (siRNA). The effect of ergothioneine on the production of reactive oxygen species (ROS) in HBMECs was measured using dichlorodihydrofluorescein and lucigenin, and the effect on cell viability was studied using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. ROS production and cell death induced by pyrogallol, xanthine oxidase plus xanthine, and high glucose were suppressed by ergothioneine. The antioxidant and cytoprotective effects of ergothioneine were abolished when OCTN-1 was silenced using siRNA. The expression of NADPH oxidase 1 was decreased, and those of glutathione reductase, catalase, and superoxide dismutase enhanced by the compound. In isolated rat basilar arteries, ergothioneine attenuated the reduction in acetylcholine-induced relaxation caused by pyrogallol, xanthine oxidase plus xanthine, or incubation in high glucose. Chronic treatment with the compound improved the response to acetylcholine in arteries of rats with streptozotocin-induced diabetes. In summary, ergothioneine is taken up by endothelial cells via OCTN-1, where the compound then protects against oxidative stress, curtailing endothelial dysfunction.
Asunto(s)
Citoprotección/fisiología , Células Endoteliales/metabolismo , Ergotioneína/metabolismo , Ergotioneína/farmacología , Estrés Oxidativo/fisiología , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Citoprotección/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Humanos , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Rapid non-genomic effects of 17ß-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17ß-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17ß-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17ß-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17ß-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Sitios de Unión , Línea Celular , Membrana Celular , Estradiol/metabolismo , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Expresión Génica , Humanos , Ligandos , Lipoilación , Fitoestrógenos/metabolismo , Unión Proteica , Isoformas de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
The serotonin (5-HT) uptake system is supposed to play a crucial part in vascular functions by "fine-tuning" the local concentration of 5-HT in the vicinity of 5-HT(2) receptors in vascular smooth muscle cells. In this study, the mechanism of 5-HT uptake in human brain vascular smooth muscle cells (HBVSMCs) was investigated. [(3)H]5-HT uptake in HBVSMCs was Na(+)-independent. Kinetic analyses of [(3)H]5-HT uptake in HBVSMCs revealed a K(m) of 50.36 ± 10.2 mM and a V(max) of 1033.61 ± 98.86 pmol/mg protein/min. The specific serotonin re-uptake transporter (SERT) inhibitor citalopram, the specific norepinephrine transporter (NET) inhibitor desipramine, and the dopamine transporter (DAT) inhibitor GBR12935 inhibited 5-HT uptake in HBVSMCs with IC(50) values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 µM, respectively. These IC(50) values were 100-fold higher than data reported by other authors, suggesting that those inhibitors were not blocking their corresponding transporters. Reverse transcription-polymerase chain reaction results demonstrated the presence of mRNA for organic cation transporter (OCT)-3 and plasma membrane monoamine transporter (PMAT), but the absence of OCT-1, OCT-2, SERT, NET, and DAT. siRNA knockdown of OCT-3 and PMAT specifically attenuated 5-HT uptake in HBVSMCs. It is concluded that 5-HT uptake in HBVSMCs was mediated predominantly by a low-affinity and Na(+)-independent mechanism. The most probable candidates are OCT-3 and PMAT, but not the SERT.
RESUMEN
Adenosine modulates various vascular functions such as vasodilatation and anti-inflammation. The local concentration of adenosine in the vicinity of adenosine receptors is fine tuned by 2 classes of nucleoside transporters: equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs). In vascular smooth muscle cells, 95% of adenosine transport is mediated by ENT-1 and the rest by ENT-2. In endothelial cells, 60%, 10%, and 30% of adenosine transport are mediated by ENT-1, ENT-2, and CNT-2, respectively. In vitro studies show that glucose per se increases the expression level of ENT-1 via mitogen-activating protein kinase-dependent pathways. Similar results have been demonstrated in diabetic animal models. Hypertension is associated with the increased expression of CNT-2. It has been speculated that the increase in the activities of ENT-1 and CNT-2 may reduce the availability of adenosine to adenosine receptors, thereby weakening the vascular functions of adenosine. This may explain why patients with diabetes and hypertension suffer greater morbidity from ischemia and atherosclerosis. No oral hypoglycemic agents can inhibit ENTs, but an exception is troglitazone (a thiazolidinedione that has been withdrawn from the market). ENTs are also sensitive to dihydropyridine-type calcium-channel blockers, particularly nimodipine, which can inhibit ENT-1 in the nanomolar range. Those calcium-channel blockers are noncompetitive inhibitors of ENTs, probably working through the reversible interactions with allosteric sites. The nonsteroidal anti-inflammatory drug sulindac sulfide is a competitive inhibitor of ENT-1. In addition to their original pharmacological actions, it is believed that the drugs mentioned above may regulate vascular functions through potentiation of the effects of adenosine.
Asunto(s)
Adenosina/metabolismo , Proteínas de Transporte de Nucleósido Equilibrativas/fisiología , Proteínas de Transporte de Membrana/fisiología , Enfermedades Vasculares , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Proteínas de Transporte de Nucleósido Equilibrativas/genética , Proteínas de Transporte de Nucleósido Equilibrativas/metabolismo , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Enfermedades Vasculares/tratamiento farmacológico , Enfermedades Vasculares/metabolismoRESUMEN
Dihydropyridine-type calcium channel antagonists, in addition to having a vasodilatory effect, are known to inhibit cellular uptake of nucleosides such as adenosine. However, the nucleoside transporter subtypes involved and the mechanism by which this occurs are not known. Therefore, we have studied the inhibitory effects of dihydropyridines on both human equilibrative nucleoside transporters, hENT-1 and hENT-2, which are the major transporters mediating nucleoside transport in most tissues. Among the dihydropyridines tested, nimodipine proved to be the most potent inhibitor of hENT-1, with an IC(50) value of 60+/-31 muM, whereas nifedipine, nicardipine, nitrendipine, and felodipine exhibited 100-fold less effective inhibitory activity. Nifedipine, nitrendipine, and nimodipine inhibited hENT-2 with IC(50) values in the micromolar range; however, nicardipine and felodipine had no significant effect on hENT-2. Removal of the 4-aryl ring or changing the nitro group at the 4-aryl ring proved not to be detrimental to the inhibitory effects of dihydropyridines on hENT-1, but resulted in a drastic decrease in their inhibitory effects on hENT-2. Kinetic studies revealed that nimodipine and nifedipine reduced V(max) of [(3)H]uridine transport without affecting K(m). The inhibitory effects of nimodipine and nifedipine could be washed out. In addition, nimodipine and nifedipine inhibited the rate of NBTGR-induced dissociation of [(3)H]NBMPR from hENT-1 cell membrane. We conclude that dihydropyridines are non-competitive inhibitors of hENT-1 and hENT-2, probably working through reversible interactions with the allosteric sites. The inhibitory potencies of dihydropyridines may be associated with the structure of the 4-aryl ring, as well as the ester groups at the C-3 and C-5 positions.