Studies On The Effect Of Morphine On The Glutamate Uptake, [Ca～(2+)]i And Proliferation Of Astrocytes In Hippocampus Of Rats

Morphine is widely used for therapeutic purposes because of its potent analgesic effect. However, it is known that chronic use of morphine leads to tolerance and addiction. The mechanisms of tolerance and addiction have been investigated in the present years. The opiate and its receptors in locus ceruleus and accummbens nucleus were deeply involved. However, It still remains unclear whether astrocytes in hippocampus take part in morphine tolerance. We cultured astrocytes of hippocampus and observed the alteration of glutamate uptake, [Ca2+]i, proliferation after morphine administration with methods of glutamate uptake , flow cytometry (FCM) , confocal laser scanning microscopy (CLSM), reverse transcription PCR (RT-PCR). The main results as follows:1. The astrocytes in hippocampus of rats were cultured and purified to more than 95% yield (confirmed with GFAP immunohistochemistry).2. The glutamate uptake of astrocytes was significantly increased 3 days after morphine or naloxone administration (0.5, 1, 2, 10(M). The glutamate uptake of astrocytes was significantly decreased 10 days after 2(M morphine administration . The effects of morphine could be blocked by pretreatment of the opioid receptor antagonist naloxone .3. The expression of GLT1 and GS mRNA in astrocytes was significantly increased 3 days after 2(M morphine treatment, while treated with 2(M morphine for 10 days, the expression of GLT1 mRNA was decreased and the level of GS mRNA has no significant changes compared to control group. Naloxone had no effects on the expression of GLT1 and GS mRNA.4. The intracellular Ca2+ of astrocyte has no significant changes 3 days after 0.5,1,2,10(M morphine treatment. And acute morphine administration induced astrocyte [Ca2+]i responses of spike ;To astrocytes of morphine pretreatment, the response of [Ca2+]i was evoked by higher concentration of morphine pretreated. The [Ca2+]i response of astrocyte to morphine could be blocked by naloxone and by not MK-801(NMDA receptor antagonist);5. Morphine(2(M) induced up-regulation of the expression of Cx43 mRNA in astrocytes 3 days after morphine administration and down-regulation 10 days after morphine treatment and transfer of astrocyte culture.The effects of morphine could be blocked by pretreatment of the naloxone .6. The cell proliferation and volume was changed markedly after morphine administration. However, death rates, apoptosis and proliferation index(PI) has no significant changes compared with the control group. These results suggested that: (1) The expression of GLT1 and GS in the astrocytes was up-regulated and glutamate uptake was enhanced 3 days after morphine administration, and decreased 10 days after morphine administration. It demonstrated that morphine regulated the expression of GLT1 and GS mRNA and affected glutamate uptake; and the alteration of glutamate uptake was involved in morphine tolerance. (2) Chronic morphine administration did not lead to increaseing of [Ca2+]i which suggested that morphine did not result in Ca2+ overload of astrocyte . Acute morphine treatment could evoke the spike response of [Ca2+]i in astrocytes. It suggested that the morphine-induced response of [Ca2+]i in astrocytes may play an important role in morphine tolerance. While NMDA receptor was not involved in the effect of morphine .Morphine could enhance transmission among astrocytes in short period(3days) after treatment, but the effect would be reversed in long(3) period(10 days ) after treatment, because morphine-induced expression of Cx43 mRNA in astrocytes changed in different time phase.(4) Morphine inhibited astrocyte proliferation but did not affect its living, apoptosis and PI, which suggested that morphine prolonged the cell cycle.(5) The effects of naloxone on astrocytes should be investigated deeply.