흰쥐 해마(hippocampus)에서 acetylcholine (ACh) 유리에 미치는 A1-adenosine 수용체의 post-receptor 기전에 관한 지견을 얻고자 하여 3H-choline으로 평형시킨 해마 slice를 사용하여 3H-ACh 유리에 미치는 여러가지 약물들의 영향을 관찰하였다. Adenosine (0.3 ~ 300μM)은 전기자극(3Hz, 2 ms, 5 VCm-1, 구형파)에 의한 ACh 유리를 용량 의존적으로 감소 시켰으며, 이러한 효과는 A1-adenosine 수용체의 선택적 차단제인 8-cyclopentyl-1, 3-dipropylxanthine (2μM)에 의해 차단됨을 볼 수 있었다. G-단백 억제제인 N-ethylmaleimide (NEM, 10과 30μM)는 그 자체에 의하여 자극유발성 ACh 유리를 증가시켰으며, adenosine의 효과는 NEM 전처리에 의하여 완전히 소실되었다. Protein kinase C 활성화제인 4β-phorbol 12, 13-dibutyrate (PDB, 1 ~ 10μM)는 ACh 유리 증가를 일으켰으며 억제제인 polymyxin B (PMB, 0.03 ~ 1 mg)는 감소를 일으켰으나, adenosine에 의한 ACh 유리 감소효과는 PDB 및 PMB에 의해 영향을 받지 않았다. Ca++-통로 차단제인 nifedipine (1μM)은 adenosine의 효과를 길항함을 볼 수 있었으나 K+-통로 차단제인 glibenclamide는 adenosine의 효과에 영향을 미치지 못하였다. 8-Bromo-cAMP (100과 300μM) 그 자체로는 ACh 유리에 별다른 영향을 미치치 못하였으나 300μM 8-bromo-cAMP 전처리에 의하여 30μM adenosine의 효과가 억제됨을 볼 수 있었다. 이상의 실험결과로 흰쥐 해마에서 A1-adenosine 수용체를 통한 adenosine의 ACh유리 감소는 G-단백에 의존적이며, 이러한 효과에 nifedipine에 예민한 Ca++-통로와 adenylate cyclase계가 일부 관여함은 확실하나 proteinkinase C 및 glibenclamide에 예민한 K+통로는 관여하지 않는 것으로 사료된다.
Since it was been reported that the depolarization-induced ACh release is inhibited by activation of presynaptic A1-adenosine heteroreceptor in hippocampus, a large body of experimental data on the post-receptor mechanism of this process has been accumulated. But, the post-receptor mechanism of presynaptic A1-adenosine receptor on the ACh release has not been clearly elucidated yet. Therefore, it was attempted to clarify the post-receptor mechanisms of the A1-adenosine receptor-mediated control of ACh release in this study. Slices from rat hippocampus were equilibrated with 3H-choline and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 VCm-1, 2ms, rectangular pulses), and the influence of various agents on the evoked tritium-outflow was investigated. Adenosine, in concentrations ranging from 0.3 ~ 300μM, decreased the ACh release in a dose-dependent manner, without affecting the basal rate of release. The adenosine effects were significantly inhibited by DPCPX (2μM), a selective A1-receptor antagonist. The responses to N-ethylmaleimide (10&30μM), a SH-alkylating agent of G-protein, were characterized by increments of the evoked ACh-release and the basal release, and the adenosine effects were completely abolished by NEM pretreatment. PDB (1 ~ 10μM), a specific protein kinase C (PKC) activator, increased, whereas PMB (0.03 ~ 1 mg), a PKC inhibitor, decreased the evoked ACh-release, and the adenosine effects were not affected by these agents. Nifedipine (1μM), a Ca2+ -channel blocker of dihydropyridine analogue, significantly inhibited the adenosine effect, but glibenclamide, a K+-channel blocker, did not. Finally, 8-bromo cyclic AMP (100 & 300μM), a membrane-permeable analogue of cAMP, did not alter the ACh release, but adenosine effects were inhibited by pretreatment with large dose of 8-br-cAMP (300μM). These results indicate that the decrement of the evoked ACh-release by A1-adenosine receptor is mediated by the G-protein, and nifedipine-sensitive Ca2+-channel and adenylate cyclase system are coupled partly to this effect, and that protein kinase C and glibenclamide-sensitive K+-channel are not involved in this process.
