Figure 5.

IP₃ and PMA induced NR2B tyr-P in vitro. A, d-IP₃ (100 μm; n = 4), an IP₃ receptor activator, induced a significant increase in NR2B tyr-P compared with untreated (naive) slices. The increased NR2B tyr-P was blocked by pretreatment with CGP 77675 (10 μm; n = 4). l-IP₃ (100 μm; n = 4), an inactive analog of d-IP₃, did not produce a significant effect. The slices were permeabilized by exposure to a brief (10 sec) application of saponin (0.001%) to allow penetration of IP₃ through cell membrane. Saponin itself [saponin plus vehicle (Veh)] did not affect the level of NR2B tyr-P. B, Direct application of PMA to spinal slices to activate PKC, a downstream event to IP₃-mediated intracellular release, also mimicked CFA-induced NR2B tyr-P. PMA (1 μm; n = 5) produced a significant increase in NR2B tyr-P in the spinal slice. The inactive analog 4αPDD (10 μm; n = 5) did not produce a significant effect. The PMA-induced NR2B tyr-P was blocked by inhibitors of Src (CGP 77675, 10 μm) and PKC [chelerythrine (Che)]. ^*p < 0.05 versus naive untreated slices.