|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1998 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1997 : ¥2,600,000 (Direct Cost : ¥2,600,000)
The machinery similar to native dopaminergic nerve terminals were expressed in Xenopus oocytes by injecting mRNA extracted from the rat brain. Furthermore, GABAB receptor is known to function as an inhibition of the neurotransmitter release, therefore we attempted to develop the expression of GABA_B receptor in Xenopus oocytes, and further to analyze its structure.
1) Stimulation with KC1 (10 mM-50 mM) of mRNA-injected oocytes preloaded with dopamine evoked external Ca^2^+-dependent release of dopamine. The high KC1 (50 mM) stimulated-release of dopamine reduced to 40 % in the oocytes injected with rat brain rmPNA together with antibody to synaptophysin. Immunoblot analysis demonstrated that synaptophysin was expressed in the brain mRNA-injected oocytes, but not in the non-injected and water-injected oocytes. Thus, synaptophysin may play a role in the exocytotic release of dopamine.
2) In over 65 % of the Xenopus oocytes coinjected with poly(A)^+ RNA derived from the rat cerebellum and eRNA for the cloned G-protein-gated inwardly rectifying K^+ channel (GIRK), the GABA_3B agonist baclofen elicited inwardly rectifying K^+ currents. The inward K^+ currents elicited by baclofen were inhibited by the selective GABA_B antagonists (2-OH saclofen and CGP 35348). Thus, the functional GABA_B receptor was expressed in Ken opus oocytes by coinjecting rat cerebellar mRNA and GIRK cRNA.
3) We have identified two novel splice variants of the GABA_B receptor (GABA_BRi), designated GABAaR1c and GABA_BRId, when screening a rat cerebellum cDNA library. GABA_BRic-mIRNA was ubiquitously expressed (forebrain, cerebellum, eye, heart, lung, stomach, small intestine, colon, liver, spleen, kidney, urinary bladder and skeletal muscle) and GABABR1d-mRNA Lu forebrain, cerebellum, eye, kidney, and urinary bladder.
Thus, GABA_BRi isoforms may function not only in the central nervous system but also in various peripheral tissues.