HAMI 3379, a CysLT₂ Receptor Antagonist, Attenuates Ischemia-Like Neuronal Injury by Inhibiting Microglial Activation

Abstract

The cysteinyl leukotrienes (CysLTs) are inflammatory mediators closely associated with neuronal injury after brain ischemia through the activation of their receptors, CysLT1R and CysLT2R. Here we investigated the involvement of both receptors in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemic neuronal injury and the effect of the novel CysLT2R antagonistin comparison with the CysLT1R antagonist montelukast. In primary neurons, neither the nonselective agonist leukotriene D4 (LTD4) nor the CysLT2R agonist N-methyl-leukotriene C4 (NMLTC4) induced neuronal injury, and HAMI 3379 did not affect OGD/R-induced neuronal injury. However, in addition to OGD/R, LTD4 and NMLTC4 induced cell injury and neuronal loss in mixed cultures of cortical cells, and neuronal loss and necrosis in neuron-microglial cocultures. Moreover, they induced phagocytosis and cytokine release (interleukin-1b and tumor necrosis factor-a) from primary microglia, and conditioned medium from the treated microglia induced neuronal necrosis. HAMI 3379 inhibited all of these responses, and its effects were the same as those of CysLT2R interference by CysLT2R short hairpin RNA, indicating CysLT2R dependence. In comparison, montelukast moderately inhibited OGD/R-induced primary neuronal injury and most OGD/R-and LTD4-induced (but not NMLTC4-induced) responses in mixed cultures, cocultures, and microglia. The effects of montelukast were both dependent and independent of CysLT1Rs because interference by CysLT1R small interfering RNA had limited effects on neuronal injury in neuron-microglial cocultures and on cytokine release from microglia. Our findings indicated that HAMI 3379 effectively blocked CysLT2R-mediated microglial activation, thereby indirectly attenuating ischemic neuronal injury. Therefore, CysLT2R antagonists may represent a new type of therapeutic agent in the treatment of ischemic stroke.

Cysteinyl Leukotriene Receptor 1/2 Antagonists Nonselectively Modulate Organic Anion Transport by Multidrug Resistance Proteins (MRP1-4)

“…The CysLT 2 R-selective LTMs have only recently become available, but their usefulness in elucidating the role of CysLT 2 R in multiple cellular processes including vascular permeability and ischemic injury is already well established (Ni et al, 2011;Zhang et al, 2013). HAMI 3379 is reported to be 10,000-fold more selective and BayCysLT 2 500-fold more selective for CysLT 2 R than CysLT 1 R (Wunder et al, 2010;Ni et al, 2011).…”

Cysteinyl Leukotrienes as Potential Pharmacological Targets for Cerebral Diseases

“…Data indicate that in microglia, both CysLTs and CysLTRs participate in the inflammatory response [45, 46]; nevertheless, the impact of CysLTR-1 and CysLTR-2 in the process is controversial. A number of in vitro evidence indicate a relevant role of CysLTR-1 in microglial activation.…”

“…On the other hand, another study showed that, in primarily cultured microglia, the CysLTR-2 resulted the main regulator of microglia activation. Indeed, the CysLTR-2 antagonist HAMI 3379 inhibited phagocytosis and cytokine release induced by oxygen-glucose deprivation/reperfusion (OGD/R) and by LTD4, whereas montelukast was effective only against OGD/R [46]. …”

“…Indeed, in vitro culture of neuron-like PC12 cells transfected with CysLTR-1 and CysLTR-2 showed distinct sensitivities to ischemic injury, which resulted prominent in CysLTR-2-transfected cells [62], but neither CysLTR-1 nor CysLTR-2 were able to directly induce neuronal injury [46, 63]. Moreover, OGD/R-induced ischemic injury was not attenuated by the selective CysLTR-2 antagonist HAMI 3379 and by CysLTRs RNA interference in primary neurons [46].…”

Cysteinyl leukotriene receptor 1 mediates LTD4-induced activation of mouse microglial cells in vitro

“…Moreover, the responses mediated by CysLT 2 R and CysLT 1 R may vary in different experiments. We recently reported that CysLT 2 R plays a major regulatory role in the activation of rat primary microglia (phagocytosis and cytokine release), whereas CysLT 1 R only regulates cytokine release from microglia [31] . These findings reflect differences between species (rat and mouse) and cell types (primary microglia and BV2 cells [18] ); therefore, the roles of CysLT 2 R in BV2 cell activation should be further clarified.…”