The effects of barbiturates on substrate inhibition of Na, K-ATPase activity

バルビツール酸系薬物の作用機序は，GABAA受容体への作用を除き，不明な点が多い．Na, K-ATPaseは神経細胞の興奮性の維持を担う酵素であり，バルビツール酸系薬物の作用に関連する可能性もあると推測される．しかし，バルビツール酸系薬物のNa, K-ATPase活性に対する報告は少ないので，ラット及びウサギ脳ミクロソームのNa, K-ATPaseを使用し，本研究を行った．バルビツール酸系薬物として，pentobarbital， phenobarbital及びthiamylalを使用した．Na, K-ATPase活性のATP濃度依存性を測定すると，2.5 mMで最大活性を示し，5 mMATPでは基質阻害により活性は低下した．5 mM ATP存在下では，pentobarbitalとphenobarbitalはNa, K-ATPase活性を濃度依存的に促進したが， 2.5 mM ATP存在下ではその作用は認められなかった．すなわち，pentobarbitalとphenobarbitalには基質阻害を抑える作用が認められた． Thiamylalは5 mM あるいは2.5 mM ATP存在下のいずれの場合もNa, K-ATPase活性を抑制した．5 mM ATP存在下では，pentobarbitalとphenobarbitalはNa, K-ATPaseのNa+に対する親和性を増加させ，K+に対する親和性を減少させた．これらの結果は，基質阻害下では，pentobarbitalとphenobarbitalはNa, K-ATPaseの構造をE1型に変化し，ATPに対する親和性を増加させることにより活性を促進することを示唆した．Reaction mechanism of barbiturates has widely been explored but not as much for GABAA receptors. Na, K-ATPase is the enzyme responsible for the maintenance of neuronal excitability and may be related to the reaction of barbiturates. As there are few reports about the effect of barbiturates on Na, K-ATPase activity, we studied the effects of thiamylal, pentobarbital, and phenobarbital on Na, K-ATPase activity using extracts of rat and rabbit brains. Na, K-ATPase activity increased depending on the ATP concentration and showed the maximum activity at 2.5 mM, but it decreased at 5 mM ATP by substrate inhibition. Pentobarbital and phenobarbital reduced this decrease of activity by substrate inhibition depending on their concentrations at 5 mM ATP, but not at 2.5 mM. This suggests that pentobarbital and phenobarbital suppresses the substrate inhibition of Na, K-ATPase activity by ATP. Thiamylal simply decreased Na, K-ATPase activity depending on its concentration at both 5 and 2.5 mM ATP without any suppression of substrate inhibition. Pentobarbital and phenobarbital increased the affinity of Na, K-ATPase for Na+ and decreased for K+ at 5 mM ATP. These results suggest that under the condition of substrate inhibition by ATP, pentobarbital and phenobarbital increases Na, K-ATPase activity by changing the conformational state to E1 conformation and increased in the affinity for ATP