Search

We investigated whether chronic sciatic ligation modifies the glutamate release in spinal cord nerve endings (synaptosomes) as well as the expression and the function of presynaptic release-regulating mGlu2/3 autoreceptors and 5-HT 2A heteroreceptors in these particles. Synaptosomes were from the spinal cord of animals suffering from the sciatic ligation that developed on day 6 post-surgery a significant decrease of the force inducing paw-withdrawal in the lesioned paw. The exocytosis of glutamate (quantified as release of preloaded [ 3 H]D-aspartate, [ 3 H]D-Asp) elicited by a mild depolarizing stimulus (15 mM KCl) was significantly increased in synaptosomes from injured rats when compared to controls (uninjured rats). The mGlu2/3 agonist LY379268 (1000 pM) significantly inhibited the 15 mM KCl-evoked [ 3 H]D-Asp overflow from control synaptosomes, but not in terminals isolated from injured animals. Differently, a low concentration (10 nM) of (±) DOI, unable to modify the 15 mM KCl-evoked [ 3 H]D-Asp overflow in control spinal cord synaptosomes, significantly reduced the glutamate exocytosis in nerve endings isolated from the injured rats. Acute oral trazodone (TZD, 0.3 mg/kg on day 7 post-surgery) efficiently recovered glutamate exocytosis as well as the efficiency of LY379268 in inhibiting this event in spinal cord synaptosomes from injured animals. The sciatic ligation significantly reduced the expression of mGlu2/3, but not of 5-HT 2A , receptor proteins in spinal cord synaptosomal lysates. Acute TZD recovered this parameter. Our results support the use of 5-HT 2A antagonists for restoring altered spinal cord glutamate plasticity in rats suffering from sciatic ligation., (Copyright © 2020 Cisani, Roggeri, Olivero, Garrone, Tongiani, Di Giorgio and Pittaluga.)

Rationale: The development of inhibitors of microsomal prostaglandin (PG)E 2 synthase-1 (mPGES-1) was driven by the promise of attaining antiinflammatory agents with a safe cardiovascular profile because of the possible diversion of the accumulated substrate, PGH 2 , towards prostacyclin (PGI 2 )., Objectives: We studied the effect of the human mPGES-1 inhibitor, AF3485 (a benzamide derivative) on prostanoid biosynthesis in human whole blood in vitro . To characterize possible off-target effects of the compound, we evaluated: i)the impact of its administration on the systemic biosynthesis of prostanoids in a model of complete Freund's adjuvant (CFA)-induced monoarthritis in rats; ii) the effects on cyclooxygenase (COX)-2 expression and the biosynthesis of prostanoids in human monocytes and human umbilical vein endothelial cells (HUVECs) in vitro ., Methods: Prostanoids were assessed in different cellular models by immunoassays. The effect of the administration of AF3485 (30 and 100 mg/kg,i.p.) or celecoxib (20mg/kg, i.p.), for 3 days, on the urinary levels of enzymatic metabolites of prostanoids, PGE-M, PGI-M, and TX-M were assessed by LC-MS., Results: In LPS-stimulated whole blood, AF3485 inhibited PGE 2 biosynthesis, in a concentration-dependent fashion. At 100μM, PGE 2 levels were reduced by 66.06 ± 3.30%, associated with a lower extent of TXB 2 inhibition (40.56 ± 5.77%). AF3485 administration to CFA-treated rats significantly reduced PGE-M (P < 0.01) and TX-M (P < 0.05) similar to the selective COX-2 inhibitor, celecoxib. In contrast, AF3485 induced a significant (P < 0.05) increase of urinary PGI-M while it was reduced by celecoxib. In LPS-stimulated human monocytes, AF3485 inhibited PGE 2 biosynthesis with an IC 50 value of 3.03 µM (95% CI:0.5-8.75). At 1μM, AF3485 enhanced TXB 2 while at higher concentrations, the drug caused a concentration-dependent inhibition of TXB 2 . At 100 μM, maximal inhibition of the two prostanoids was associated with the downregulation of COX-2 protein by 86%. These effects did not involve AMPK pathway activation, IkB stabilization, or PPARγ activation. In HUVEC, AF3485 at 100 μM caused a significant (P < 0.05) induction of COX-2 protein associated with enhanced PGI 2 production. These effects were reversed by the PPARγ antagonist GW9662., Conclusions: The inhibitor of human mPGES-1 AF3485 is a novel antiinflammatory compound which can also modulate COX-2 induction by inflammatory stimuli. The compound also induces endothelial COX-2-dependent PGI 2 production via PPARγ activation, both in vitro and in vivo , which might translate into a protective effect for the cardiovascular system., (Copyright © 2020 Di Francesco, Bruno, Ricciotti, Tacconelli, Dovizio, Guillem-Llobat, Alisi, Garrone, Coletta, Mangano, Milanese, FitzGerald and Patrignani.)