Roles Of 5-lipoxygenase And Cysteinyl Leukotriene Receptors In Oxygen-glucose Deprivation-induced Injury In Rat Cortical Neurons

5-Lipoxygenase (5-LOX, EC 1.13.11.34) is a key enzyme metabolizing arachidonic acid (AA) to form inflammatory mediators, leukotriene B₄ (LTB₄) and cysteinyl leukotrienes (CysLTs, including LTC₄, LTD₄ and LTE₄). CysLTs are potent inflammatory mediators and involved in various diseases. The actions of CysLTs, such as smooth muscle contraction, microvascular leakage and other pathophysiological changes, are mediated via activating their receptors (CysLT₁ and CysLT₂ receptors). In early studies, 5-LOX, CysLT₁ and CysLT₂ receptors are initially found in peripheral organs and cells. Therefore, most studies focus on their roles in peripheral organ diseases, such as the disease of respiratory system. In recent studies, 5-LOX. CysLT₁ and CysLT₂ are reported to be also expressed in the brain, and they may be involved in the pathogenesis of various CNS diseases, such as cerebral ischemia, trauma, tumor, epilepsy and aging. After cerebral ischemia, both the expression and activity of 5-LOX in the brain are increased, and the contents of CysLTs are elevated in the brain tissue. Furthermore, the expression of CysLT₁ and CysLT₂ receptors are induced after cerebral ischemia. Inhibitors of 5-LOX, such as AA861, MK-886 and nordihydroguaiaretic acid, exert protective effects on cerebral ischemia. Our previous studies have also shown the protective effect of CysLT₁receptor antagonist, pranlukast (ONO-1078) and montelukast, on experimental cerebral ischemia.However, there are lots of questions in the involvement of 5-LOX, CysLT) and CysLT2 receptors in ischemic injury in the neurons. For example, are 5-LOX, CysLTi and CysLT2 receptors expressed in neurons? Are they involved in oxygen-glucose deprivation (OGD)-induced ischemic injury in neurons? How do they play their roles in the ischemic injury? Up to date, there are still no clear answers for these questions.Therefore, in the present study, we want to answer the above questions using the in vitro model of OGD-induced ischemic injury in the primary rat cortical neurons.Parti5-LOX pathway is involved in OGD-induced injury in rat corticalneuronsMembrane phospholipids are catalyzed to form arachidonic acid (AA) through activation of phospholipases A? (PLA2). Free AA is metabolized by two major enzyme systems, cyclooxygenase (COX) and 5-LOX. To determine whether the COX pathway and 5-LOX pathway are involved in OGD-induced injury in rat cortical neurons, the effects of PLA2 inhibitor (quinacrine), COX inhibitor (indomethacin), 5-LOX inhibitor (caffeic acid), 5-LOX activating protein (FLAP) inhibitor (MK-886) and excitory acid NMDA receptor antagonist (MK-801) on OGD-induced injury in rat cortical neurons were observed. After 1.5-h OGD following 24-h recovery, neuronal cell bodies and neuritis were destroyed, and neuron lysis appeared;MTT reduction assay indicated that neuron viability was significantly decreased. An NMDA receptor antagonist MK-801 (5 and 10 uM) almost completely reversed the OGD-induced injury. A PLA2 inhibitor quinacrine (1 and 5 uM), an FLAP inhibitor MK-886 (2 and 10 uM) and a 5-LOX inhibitor caffeic acid (5 and 25 uM) concentration-dependently ameliorated OGD-reduced neuron viability, while a non-specific COX inhibitor indomethacin (0.4-10 u.M) did not show this effect.MK-801 almost completely, but quinacrine, MK-886 and caffeic acid only partially inhibited the reduction in neuron viability. The results demonstrate that 5-LOX, not COX, is primarily involved in OGD-induced neuron injury.Part 2OGD-induced 5-LOX activation may be partly mediated via NMDAreceptor in rat cortical neuronsIn Part 1, we have confirmed that 5-LOX is involved in the OGD-induced injury in rat cortical neurons. In this part, we determined whether 5-LOX is activated (translocation and production of leukotrienes) after OGD in the primary rat cortical neurons;if so, whether this activation is mediated by NMDA receptor. By immunocytochemistry, Western blotting and green fluorescent protein-5-LOX transfection, we found that 5-LOX was evenly distributed in the cytosols, nuclei and neurites in the normal neurons. After OGD, 5-LOX was translocated to the nuclear envelope (one of the phenomenons of 5-LOX activation). 5-LOX metabolites, cysteinyl leukotrienes (CysLTs), but not leukotriene B4, were increased in the culture media 0.5-1.5 h after recovery. To determine whether excitory acids modulate 5-LOX activation, we measured the release of endogenous glutamate and observed the effects of glutamate and NMDA on 5-LOX activation. The results showed that NMDA (100 |.iM for 0.5-1 h) and glutamate (100 uM for 1 h) also induced 5-LOX translocation, and NMDA increased the production of CysLTs during 0.5-1 h exposure. NMDA, glutamate and OGD reduced neuron viability. The results of antagonist/inhibitors showed that NMDA receptor antagonist MK-801 inhibited 5-LOX translocation, production of CysLTs and the reduction of neuron viability after treatments of OGD and NMDA (or glutamate). Otherwise, 5-LOX activating protein inhibitor MIC-886 and 5-LOX inhibitor caffeic acid inhibited the reduction of neuron viability and the production of CysLTs induced by OGD and NMDA (or glutamate), but did not affect 5-LOX translocation. From these findings, we conclude that OGD activates 5-LOX inthe primary rat cortical neurons;the mechanisms of 5-LOX activation may be mediated through releasing endogenous exicitoiy acid (glutamate) by OGD, then activating NMDA receptor, and finally activating 5-LOX.Part 3Expression and distribution of CysLTi and CysLTi receptors in ratcortical neuronsIn Part 1 and Part 2, it has been clarified that 5-LOX is expressed and activated to produce CysLTs after OGD in rat cortical neurons. Furthermore, in previous studies, we have demonstrated the neuroprotective effect of CysLTi receptor antagonists, pranlukast (ONO-1078) and montelukast, on cerebral ischemia. These findings strongly suggest that CysLT] and CysLT2 receptors may be expressed in rat cortical neurons and may play a role in OGD-induced injury. Therefore, in this part, we determined whether CysLTi and CysLT2 receptors are expressed in rat cortical neurons, if so, where they are localized in the neurons. The results of RT-PCR indicated that CysLTi and CysLT2 receptor mRNAs were expressed in rat cortical neurons;the mRNA expression of CysLT? receptor was higher than that of CysLTi receptor. The immunostaining results indicated that CysLTi receptor was distributed in the cytoplasm membrane and CysLT2 receptor in thr nuclei. Taken together, these results indicated that CysLTi and CysLT2 receptors are actually expressed in rat cortical neurons. CysLTi as well as CysLTi receptors, especially CysLT2 receptor, may be involved in OGD-induced injury.Part 4Effects of the agonist and antagonists of CysLT! and CysLTjreceptors on the responses in rat cortical neuronsBoth CysLTi and CysLT2 receptors are classic G protein-coupled receptors with 7 transmembrane domains, which can evoke intracellular calcium elevation after activation. In Part 3, we have indicated that two receptors are expressed in rat cortical neurons. In this part, we determined whether an agonist for CysLTi and CysLT2 receptors, LTD4, affects the intracellular calcium and the neuron viability, and CysLTi receptor antagonists (montelukast and pranlukast) and a CysLT ]/CysLT2 receptor non-selective antagonist (BAY u9773) affect OGD-induced injury in rat cortical neurons. LTD4 (0.01-1 uM) did not induce the elevation of intracellular calcium;while NMDA (100 uM) did elevate in rat cortical neurons. LTD4 (0.01-1 uM) did not alter neuron viability and death as detected by MTT, propidium iodide (PI) and Hoechst 33258 assays. In addition, the reduction of neuron viability was significantly ameliorated by NMDA receptor antagonist MK-801 (5 uM) as detected by MTT reduction assay after 1.5-h OGD following 24-h recovery. However, pranlukast (0.4-10 uM), montelukast (0.4-10 uM) and BAY u9773 (0.04-1 uM) did not show this effect. From these findings, we conclude that both agonist and antagonists of CysLTi and CysLT2 receptors do not affect the neuron responses. These findings are not consistent with our experctation and the results from previous studies of cerebral ischemia. This inconsistency is important and necessary to further investigate. However, the up-regulation of CysLTi and CysLTi receptors in the brain after cerebral ischemia in vivo may result from complex intercellular interactions or interactions with other modulating molecules;the protection by CysLTi receptor antagonists against cerebral ischemic injury may secondarily result from inhibition of blood-brain barrier disruption, brain edema and inflammation.Conclusions1. 5-LOX pathway is involved in OGD-induced injury in rat cortical neurons, and inhibiting the activity of each key enzyme in the pathway is beneficial for neuroprotection.2. 5-LOX is normally expressed in the cytosols, nuclei and neurites of rat cortical neurons. OGD or excitory acids (NMDA and glutamate) activates 5-LOX, leading to translocation to the nuclear envelope and production of cysteinyl leukotrienes. The mechnanism of 5-LOX activation may be mediated through releasing endogenous exicitory acid (glutamate) by OGD, then activating NMDA receptor, and finally activating 5-LOX.3. CysLTi and CysLT2 receptors are expressed in rat cortical neurons. CysLTi receptor is weakly expressed and localized in the neuron membranes;while CysLTi receptor is higher expressed and localized in the nuclei.4. CysLTi and CysLT? receptor agonist, LTD4, does not affect neuron viability and death. CysLTi receptor antagonists, montelukast and pranlukast, and CysLTi/CysLTi receptor non-selective antagonist, BAY u9773, do not affect OGD-induced neuron injury as well.