Distinct roles of CysLT1 and CysLT2 receptors in oxygen glucose deprivation-induced PC12 cell death

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Abstract

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT1 and CysLT2) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT1 and CysLT2 receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT1 receptor decreased and over-expression of CysLT2 receptor increased OGD-induced cell death. An agonist LTD4 (10−7 M) also induced apoptosis, especially in CysLT2 receptor over-expressing cells. A selective CysLT1 receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT2 receptor over-expressing cells. Thus, CysLT1 and CysLT2 receptors play distinct roles in OGD-induced PC12 cell death; CysLT1 attenuates while CysLT2 facilitates the cell death.

Section snippets

Materials and methods

Cell culture and receptor gene transfection. PC12 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. The cDNA for mouse CysLT1 or CysLT2 receptor (mCysLT1 and mCysLT2, subcloned into pcDNA3.0) was kindly gifted by Professor C.D. Funk (University of Pennsylvania, USA). The pcDNA3.0 null vector was purchased from Invitrogen (Carlsbad, California, USA). The receptor cDNA expressing vectors and the null vector were lineared by PvuI and transfected

Expressions of CysLT1 and CysLT2 receptor mRNAs

The endogenous mRNA expression of rat CysLT2 receptor (rCysLT2) was higher than that of rCysLT1 in normal PC12 cells. Permanent transfection with mCysLT1 and mCysLT2 receptors increased their mRNA expressions (Fig. 1A). The over-expression of CysLT1 receptor did not alter whereas over-expression of CysLT2 receptor significantly increased the cell size (Fig. 1B).

Hoechst 33258 and PI double staining

OGD mainly induced PC12 cell apoptosis. After 6-h OGD, apoptotic cells were less in PC12/mCysLT1 cells (29.0%, P < 0.01) and more in

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 [17], [18],

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 30500613) and the Scientific Foundation of Education Ministry of China (20050335105).

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    These authors contributed equally to this work.

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