Preferential inhibition by antidiarrheic 2-methoxy-4-methylphenol of Ca²⁺ influx across acquired N-methyl-D-aspartate receptor channels composed of NR1/NR2B subunit assembly

In our previous studies, particular phenolic ingredients, such as 2-methoxy-4-methylphenol (2M4MP), of the antidiarrheic drug wood creosote significantly prevented cell death by both hydrogen peroxide and glutamate in cultured rat hippocampal neurons. In this study, we further evaluated the pharmacological properties of 2M4MP on Ca²⁺ influx across native and acquired N-methyl-D-aspartate (NMDA) receptor (NMDAR) channels. The addition of 2M4MP significantly prevented the loss of cellular viability and the increase in intracellular free Ca²⁺ levels as determined by Fluo-3 in cultured rat hippocampal neurons briefly exposed to NMDA. Brief exposure to NMDA also led to a marked increase in mitochondrial free Ca²⁺ levels determined by Rhod-2, in addition to intracellular free Ca²⁺ levels, in HEK293 cells expressing either NR1/NR2A or NR1/NR2B subunit channels. The further addition of the general NMDAR channel blocker dizocilpine similarly inhibited the increase of intracellular Ca²⁺ levels by NMDA in both types of acquired NMDAR channels, whereas the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in HEK293 cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similarly, 2M4MP significantly and selectively inhibited the NMDA-induced influx of Ca²⁺ across acquired NR1/NR2B channels in a concentration-dependent manner. Moreover, prior daily oral administration of 2M4MP significantly reduced the infarct volume in the ipsilateral cerebral hemisphere in rats with middle cerebral artery occlusion 1 day after reperfusion. These results suggest that 2M4MP may protect neurons from excitotoxicity through preferential inhibition of Ca²⁺ influx across NMDAR channels composed of NR1/NR2B subunits.