No abstract

No abstract

The CYP2C19 gene plays a detrimental role in the metabolism of clopidogrel. This study aimed to investigate the association between CYP2C19 polymorphisms and the clinical efficacy of clopidogrel therapy in patients who have undergone carotid artery stenting (CAS). CYP2C19 genotype screening was performed on 959 ischemic stroke patients. Of these patients, 241 who had undergone CAS were enrolled in the study. They were all followed up for 1 year after stent surgery, and the primary clinical end-points were ischemic events. The frequencies of the CYP2C19*2 and *3 alleles among the 959 patients were 31.80% and 5.06%, respectively. Regarding the 241 participants who had undergone CAS, multivariate Cox regression analysis showed that the CYP2C19 loss-of-function (LOF) alleles (*2 and *3) were risk factors for post-CAS prognosis. Within 1 year of follow-up, the patients carrying the CYP2C19 LOF alleles were more likely to experience ischemic events than those carrying none. The occurrence of ischemic events did not significantly differ between the *2 and *3 allele carriers. Our results suggest that CYP2C19 LOF alleles (*2 and *3) significantly impact the prognosis of patients on clopidogrel therapy after CAS and that the CYP2C19*2 and CYP2C19*3 alleles have the same effects on prognosis.

No abstract

BackgroundSecreted phospholipase A2-IIA (sPLA2-IIA) is an inducible enzyme released under several inflammatory conditions. It has been shown that sPLA2-IIA is released from rat brain astrocytes after inflammatory stimulus, and lipopolysaccharide (LPS) and nitric oxide (NO) have been implicated in regulation of this release. Here, brain microvascular endothelial cells (BMVECs) were treated with LPS to uncover whether sPLA2-IIA was released, whether nitric oxide regulated this release, and any related signal mechanisms.MethodsSupernatants were collected from primary cultures of BMVECs. The release of sPLA2-IIA, and the expression of inducible nitric oxide synthase (iNOS), phospho-JAK3, phospho-STAT1, total JAK3 and STAT1, β-actin, and bovine serum albumin (BSA) were analyzed by Western blot or ELISA. NO production was calculated by the Griess reaction. sPLA2 enzyme activity was measured with a fluorometric assay. Specific inhibitors of NO (L-NAME and aminoguanidine, AG), JAK3 (WHI-P154,WHI), STAT1 (fludarabine, Flu), and STAT1 siRNA were used to determine the involvement of these molecules in the LPS-induced release of sPLA2-IIA from BMVECs. Nuclear STAT1 activation was tested with the EMSA method. The monolayer permeability of BMVECs was measured with a diffusion assay using biotinylated BSA.ResultsTreatment of BMVECs with LPS increased the release of sPLA2-IIA and nitrite into the cell culture medium up to 24 h. Pretreatment with an NO donor, sodium nitroprusside, decreased LPS-induced sPLA2-IIA release and sPLA2 enzyme activity, and enhanced the expression of iNOS and nitrite generation after LPS treatment. Pretreatment with L-NAME, AG, WHI-P154, or Flu notably reduced the expression of iNOS and nitrite, but increased sPLA2-IIA protein levels and sPLA2 enzyme activity. In addition, pretreatment of the cells with STAT1 siRNA inhibited the phosphorylation of STAT1, iNOS expression, and nitrite production, and enhanced the release of sPLA2-IIA. Pretreatment with the specific inhibitors of NOS, JAK2, and STAT3 decreased the permeability of BMVECs. In contrast, inhibition of sPLA2-IIA release increased cell permeability. These results suggest that sPLA2-IIA expression is regulated by the NO-JAK3-STAT1 pathway. Importantly, sPLA2-IIA augmentation could protect the LPS-induced permeability of BMVECs.ConclusionOur results demonstrate the important action of sPLA2-IIA in the permeability of microvascular endothelial cells during brain inflammatory events. The sPLA2 and NO pathways can be potential targets for the management of brain MVEC injuries and related inflammation.

No abstract

We have investigated the effect of orexin A on the intracellular free calcium concentration ([Ca2+]i) in primary cultured cortical neurons and explored the exact mechanisms of orexin A-evoked changes of [Ca2+]i. In the present study, changes of [Ca2+]i induced by orexin A in primary cultured cortical neurons were first detected by confocal laser scanning microscopy using Ca2+-sensitive dye fluo-4 as a novel calcium fluorescent probe. Our results showed that 1-0.1 microM orexin A induced the increase in [Ca2+]i in cortical neurons. The increase in [Ca2+]i by acute application of orexin A occurred in a dose-dependent manner. Orexin A-induced increase in [Ca2+]i was not observed under the condition of Ca2+-free Dulbecco's modified Eagle's medium. Pretreatment on the cells with 1 microM thapsigargin did not block orexin A-evoked response. These findings first illuminated the fact that orexin A-induced increase in [Ca2+]i may be mainly from extracellular calcium influx in cortical neurons.

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X(1 -7)). P2X(3) homomultimer and P2X(2/3) heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X(5) receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X(5) receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X(5) receptors in cat. In mouse DRG, the P2X(5) receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X(5) receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X(5) receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X(5)-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X(5)-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X(5) receptors were coexpressed with NF-200 or with NOS. Only a few P2X(5)-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X(5) receptors.