To further determine the cell death
To further determine the cell death, the Piericidin A in flasks were harvested after OGD or LTD4 treatment, washed twice with PBS, and evaluated with Annexin V (AV)-FITC apoptosis detection kit I (BD Biosciences Pharmingen, USA) on a flow cytometer (FACSCalibur, Becton–Dickinson, USA). Briefly, 106 cells per ml were resuspended in a binding buffer, stained with AV-FITC and PI working solutions in the kit for 15min at 25°C in dark, and then analyzed with flow cytometer (totally 10,000 cells). “AV−PI−” represents the normal cells, “AV+PI−” the early phase apoptotic cells, “AV+PI+” the late phase apoptotic cells, and “AV−PI+” the necrotic cells.
Statistical analysis. Values are reported as means±SEM. Statistical comparisons were made by one-way ANOVA to detect significant difference using SPSS 10.0 for windows. P<0.05 was considered to be statistically significant.
Discussion The most important finding in the present study is that over-expression of CysLT1 receptor reduced but over-expression of CysLT2 receptor increased OGD-induced PC12 cell death, indicating the distinct roles of CysLT1 and CysLT2 receptors in ischemic neuronal injury. The distinct roles of the two receptors have also been reported in mouse studies in which bleomycin-induced pulmonary fibrosis is enhanced in CysLT1 receptor-deficient mice but reduced in CysLT2 receptor-deficient mice , , . This distinction may be caused by a difference in the activation of MAPKs that is one of the important signal transduction pathways for CysLT receptors , , , . In human mast cells, two CysLT receptors initiate different MAPK signaling cascades, CysLT1 receptor activates ERK phosphorylation whereas CysLT2 activates Gαi/o and p38 MAPK . In PC12 cells, OGD activates MAPK isoforms (such as ERK, JNK, and p38) that play an important role in OGD-induced apoptosis . Thus, the different MAPK signaling cascades might be also responsible for the distinct roles of two receptor types in OGD-induced PC12 cell death. Moreover, we found that exogenous LTD4, a CysLT receptor agonist, induced apoptosis, especially in PC12/mCysLT2 cells. The concentration of LTD4 (10−7M) used here is about 2- to 10-fold higher than the EC50 for mouse CysLT1 or CysLT2 receptor , . Otherwise, a line of evidence has supported the effects of endogenous CysLTs on ischemic cerebral/neuronal injury. For example, the production of CysLTs is increased in rat or gerbil brain after cerebral ischemia , , during cerebral ischemia in human , and also in the cultured neurons after OGD . In addition, we found that 5-lipoxygenase, a key enzyme for production of CysLTs, is activated after OGD in PC12 cells . In the present study, a longer duration (48h) was needed for LTD4 to induce a larger effect and its effect seemed to be less potent than that of OGD. This difference might result from OGD-induced more complex responses that may potentiate the action of CysLTs, such as inducible NOS , apoptotic protease activating factor-1 , mitochondrial dysfunction, and oxidative stress ; while CysLTs may mediate OGD-induced cell death only as one of the modulating systems. On the other hand, the effects of antagonists further confirmed the distinct roles of the two receptors. The selective CysLT1 receptor antagonist montelukast (10−6M) did not affect OGD-induced apoptosis. In previous studies, we have found the protective effect of CysLT1 receptor antagonists (pranlukast and montelukast) on in vivo cerebral ischemia , , , , but did not find their protective effects on OGD-induced injury in cultured neurons (unpublished observations). This discrepancy in the effects of CysLT1 receptor antagonists in vivo and in vitro might imply that the in vivo protective effects of pranlukast and montelukast might be due to their anti-inflammatory activities, such as reducing brain–blood barrier permeability and brain edema, inhibiting inflammatory and glial cells. Since no selective CysLT2 receptor antagonist is available, Bay u9773, a non-selective antagonist with partial agonist property , was used to determine the role of CysLT2 receptor. A relatively lower concentration (10−7M) of Bay u9773 was used in the experiments because it had toxic effect on PC12 cells at concentrations over 10−6M (data not shown); but this concentration (10−7M) was higher than that used in the aequorin assay (0.1–0.5×10−7M) for functional activation of the human CysLT2 receptor-expressing HG57 cells by CysLTs . We found that Bay u9773 attenuated OGD- or LTD4-induced apoptosis in PC12/mCysLT2 cells, and OGD-induced apoptosis in PC12/WT and PC12/mCysLT1 cells (Hoechst 33258 and PI staining). This finding confirmed the roles of CysLT2 receptor in the ischemic-like injury in PC12 cells.