Effects Of HS014 On Excitatory Amino Acids Release Of Spinal Astrocytes

Objective To investigate the influence of HS014, a selective MC4 antagonist, on excitatory amino acids (EAA) release of the astrocytes in the spinal cord of rat s activated by tumor necrosis factor-a(TNF-a) and spinal astrocytes chronically treated with morphine.Methods All the Astrocytes of spinal cords purified cultured in vitro were cultured in serum-free Neurobasal/B27 medium containing no serum. The experiment contains two parts:the first part, Astrocytes of spinal cords purified cultured in vitro were divided into 5 groups(n=6 each):(1) Control group (group C), (2) containing 10ug/L TNF-a(group T), (3) containing 10ug/L TNF-aand 0.1μmol/L HS014(group TH0.1), (4) containing 10ug/L TNF-a and 1μmol/L HS014(group TH1), (5) containing and 10μmol/L HS014(group TH10). The contents of glutamic acid (Glu) and aspatic acid (Asp) in the supernatants of astrocytes were measured by the means of HPLC-MS/MS;the second part, Astrocytes of spinal cords purified cultured in vitro were divided into 5 groups:control group and group A, B, C, D. The cells A, B, C, D were cultured for 72h in the presence of 10μmol·L-1 morphine. The group A was added nothing, 10μmol·L-1 naloxone was added in group B,0.1μmol·L HS014 was added in group C, 10μmnol·L-1 naloxone and 0.1μmol·L HS014 were added in group D. The contents of glutamic acid (Glu) in the supernatants of astrocytes were measured by the means of HPLC-MS/MS at 0,30,60 and 120 min.Results1 The contents of Glu and Asp in group T were significantly increased in compared with that in group C(P<0.01), The contents of Glu and Asp in group TH0.1,TH1,TH10 were evidently were lower than those in group T, and than those in group T(P<0.01).2 The contents of Glu in group A were significantly increased in compared with that in control group (P<0.05), Glutamate release was significantly increased in group B compared with group A, the glutamate release in gourp C was lower than it in group A.Conclusion HS014 can suppress the release of excitatory amino acids when the astrocytes stimulated by TNF-a; The glutamate release increase when cultured in the serum with morphine, naloxone can increase the glutamate release, but HS014 can decrease the the glutamate release.