Influence of morphine on levels of type II inhibitory guanine nucleotide binding protein in primary hippocampal neurons

BACKGROUND: The pharmacological action of opioid drugs is related to signal transduction of inhibitory guanine nucleotide binding protein. OBJECTIVE: To quantitatively and qualitatively analyze the influence of morphine on levels of type 11 inhibitory guanine nucleotide binding protein (G(i2) protein) in primary cultured hippocampal neurons at different time points. DESIGN, TIME AND SETTING: A randomized controlled study, which was performed at the Department of Neurobiology, Changzheng Hospital, Second Military Medical University of Chinese PLA between September 2002 and March 2004. MATERIALS: Cerebral hippocampal neurons were obtained from newborn SD rats at 1-2 days of age. Biotin-antibody II-avidin fluorescein isothiocyanate (Avidin-FITC) was purchased from Sigma Company (USA) and the G(i2) protein polyclonal antibody from Santa Cruz Biochemistry Company (USA). METHODS: Seven days after culture, mature hippocampal neurons were randomly divided into six groups: 4-, 8-, 16-, 24-, and 48-hour morphine groups, and a blank control group. Neurons in the morphine groups received morphine (10 mu mol/L), which could cause alterations of G-protein mRNA and cAMP expression in the prefrontal cortex. Neurons in the blank control group were given the same volume of saline. MAIN OUTCOME MEASURES: G(i2) protein levels were detected by an immunofluorescence technique, and were analyzed by the image analytic system with the use of green fluorescence intensity. RESULTS: G(i2) protein levels in hippocampal neurons gradually decreased in the 4-, 8-, 16-, 24-, and 48-hour morphine groups. In particular, G(i2) protein levels in the 16-, 24-, and 48-hour morphine groups were significantly lower than that in the blank control group (P < 0.05-0.01). CONCLUSION: Morphine may decrease G(i2) protein level in primary hippocampal neurons, and the decreasing trend is positively related to morphine-induced time.