Nobe K, Ohata H, Momose K
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Biochem Pharmacol. 1994 Nov 29;48(11):2005-14. doi: 10.1016/0006-2952(94)90499-5.
Changes in diacylglycerol kinase (DG kinase) activity in carbachol (CCh)-stimulated guinea pig taenia coli were investigated. In a mixed micellar assay system, added 1,2-dioctanoyl-sn-glycerol (diC8) and endogenous DG were competitively bound to common DG kinase isozymes from guinea pig taenia coli and phosphorylated, suggesting that diC8 is useful as a probe of agonist effects on DG kinase activity. In phosphorus-32 ([32P]Pi)- and diC8-prelabeled guinea pig taenia coli, diC8 was phosphorylated by DG kinase to [32P]dioctanoyl-phosphatidic acid ([32P]diC8-PA). CCh increased the accumulation of both [32P]diC8-PA and endogenous [32P]phosphatidic acid ([32P]PA) in a time- and dose-dependent manner (0.1-100 microM CCh). CCh-induced increases in [32P]diC8-PA and [32P]PA were inhibited by 1 microM atropine and 3 microM DG kinase inhibitor (R59022). These findings indicated the activation of DG kinase by muscarinic receptor stimulation in guinea pig taenia coli. Therefore, DG kinase activation may play an important role in CCh-induced PA formation. CCh-induced [32P]diC8-PA and [32P]PA accumulation was dependent on intracellular calcium concentrations. However, a KCl-induced increase in intracellular calcium, without receptor stimulation, was ineffective. Moreover, treatment with phorbol ester also increased accumulation of both PA species in KCl-treated tissues. These findings suggest that muscarinic receptor mediated activation of DG kinase may require both an increase in intracellular calcium and PKC activation in guinea pig taenia coli.
研究了卡巴胆碱（CCh）刺激的豚鼠结肠带中甘油二酯激酶（DG激酶）活性的变化。在混合胶束测定系统中，添加的1,2 - 二辛酰 - sn - 甘油（diC8）和内源性二酰甘油（DG）竞争性结合豚鼠结肠带中的常见DG激酶同工酶并被磷酸化，这表明diC8可用作激动剂对DG激酶活性影响的探针。在磷 - 32（[32P]Pi）和diC8预标记的豚鼠结肠带中，DG激酶将diC8磷酸化为[32P]二辛酰磷脂酸（[32P]diC8 - PA）。CCh以时间和剂量依赖性方式（0.1 - 100μM CCh）增加了[32P]diC8 - PA和内源性[32P]磷脂酸（[32P]PA）的积累。1μM阿托品和3μM DG激酶抑制剂（R59022）抑制了CCh诱导的[32P]diC8 - PA和[32P]PA的增加。这些发现表明豚鼠结肠带中通过毒蕈碱受体刺激激活了DG激酶。因此，DG激酶激活可能在CCh诱导的PA形成中起重要作用。CCh诱导的[32P]diC8 - PA和[32P]PA积累依赖于细胞内钙浓度。然而，KCl诱导的细胞内钙增加，无受体刺激，是无效的。此外，佛波酯处理也增加了KCl处理组织中两种PA的积累。这些发现表明，豚鼠结肠带中毒蕈碱受体介导的DG激酶激活可能需要细胞内钙增加和蛋白激酶C（PKC）激活两者。
