| Co-Investigator(Kenkyū-buntansha) |
NAKAGAWA Takayuki Kyoto University, Faculty of Pharmaceutical Sciences, Instructor, 薬学研究科, 助手 (30303845)
MINAMI Masabumi Kyoto University, Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学研究科, 助教授 (20243040)
SAJI Hideo Kyoto University, Faculty of Pharmaceutical Sciences, Professor, 薬学研究科, 教授 (40115853)
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| Research Abstract |
We investigated the molecular mechanism responsible for supersensitization of the AC system induced by sustained opioid treatment using CHO cells expressing one of the cloned opioid receptors. In cells treated for 4 h with opioid agonists, challenge with opioid antagonists induced supersensitization of the AC system over the naive level, but had no effect on GTPase activity. This phenomenon was not affected by cycloheximide. To examine the involvement of the interaction between G-protein and AC in the development of supersensitization, we used CHO cells co-expressing the opioid receptor and some chimeric G-protein alpha_<i2>/alpha_q subunits. Our results revealed that a specific region of which is responsible for the interaction with AC, is closely related. Furthermore, we observed that supersensitization is also induced by activation of PTX-insensitive G_z.
On the other hand, we examined the regulation of RGS (regulators of G-protein signaling) 4 mRNA, which has been reported to stimulate the GTPase activity of G-protein alpha subunits, in PC12 cells stably expressing cloned opioid receptors by northern blot analysis. Treatment with opioid agonists increased the expression of RGS4 mRNA with a peak at 1-4 h. The inductions of RGS4 mRNA were blocked by antagonists and pertussis toxin. Furthermore, expression of recombinant RGS4 protein in HEK293 cells suppressed the opioid receptor-mediated inhibtory effect on forskolin-stimulated cAMP accumulation.
In transverse slices with the dorsal root attached, which were prepared from the spinal cord of adult rats, the repetitive stimulation to excite C-fibers but not Adelta-fibers evoked slow depolarization (SD) of the substantia gelatinosa neuron in spinal cord. SD was not inhibited by activation of neither glutamate- nor tachykinin NK1-receptor. On the other hand, SD was inhibited by activation of presynaptic mu-opioid, GABA_A, GABA_B, and 5-HT_<1A> receptors.
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