Dickenson J M, Hill S J
Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham, UK. mqzjmd@mqn 1.phpharm.nottingham.ac.uk
Eur J Pharmacol. 1997 Feb 19;321(1):77-86. doi: 10.1016/s0014-2999(96)00917-x.
Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A1 receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P2U purinoceptors and CCKA receptors. In this study we have investigated whether adenosine A1 receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [3H]arachidonic acid release and total [3H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [3H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [3H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-¿1-[3-(2-isothioureido)propyl] indol-3-yl¿-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [3H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [3H]arachidonic acid release elicited by PMA. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) augmented the release of [3H]arachidonic acid produced by thrombin. Co-activation of the adenosine A1 receptor also potentiated thrombin-stimulated [3H]inositol phosphate accumulation. The synergistic interactions between the adenosine A1 receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [3H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1 H-indol-3-yl]-4-(1 H-indol-3-yl)- 1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [3H]inositol phosphates and the release of [3H]arachidonic acid through pertussis-toxin-insensitive G-proteins. Experiments using PMA suggest that protein kinase C differentially regulates thrombin receptor activation of phospholipase C and phospholipase A2. Co-activation of the transfected human adenosine A1 receptor augments thrombin-stimulated phospholipase C and phospholipase A2 activity. Finally, the augmentation of phospholipase A2 activity by the adenosine A1 receptor is inhibited by selective protein kinase C inhibitors, suggesting the involvement of protein kinase C.
中国仓鼠卵巢（CHO）细胞中的凝血酶受体激活可刺激肌醇磷脂的水解和花生四烯酸的释放。我们之前的研究表明，CHO细胞（CHO-A1）中人类转染的腺苷A1受体的激活可增强内源性P2U嘌呤受体和CCKA受体引发的肌醇磷酸的积累。在本研究中，我们调查了腺苷A1受体激活是否能调节CHO-A1细胞中凝血酶刺激的花生四烯酸释放和/或肌醇磷脂水解。凝血酶刺激了CHO-A1细胞中[3H]花生四烯酸的释放和总[3H]肌醇磷酸的积累。这两种对凝血酶的反应均对百日咳毒素不敏感。蛋白激酶C激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）增强了凝血酶刺激的[3H]花生四烯酸释放。与之形成鲜明对比的是，PMA抑制了凝血酶刺激的[3H]肌醇磷酸积累。选择性蛋白激酶C抑制剂Ro 31-8220（3-｛1-[3-(2-异硫脲基)丙基]吲哚-3-基｝-4-(1-甲基吲哚-3-基)-3-吡咯啉-2,5-二酮）对凝血酶刺激的[3H]花生四烯酸释放没有影响，但逆转了PMA引发的凝血酶刺激的[3H]花生四烯酸释放的增强作用。选择性腺苷A1受体激动剂N6-环戊基腺苷（CPA）增加了凝血酶产生的[3H]花生四烯酸的释放。腺苷A1受体的共激活也增强了凝血酶刺激的[3H]肌醇磷酸积累。在经百日咳毒素处理的细胞中，腺苷A1受体与凝血酶之间的协同相互作用被消除。CPA对[3H]花生四烯酸释放的增强作用被蛋白激酶C抑制剂Ro 31-8220和GF 109203X（3-[1-[3-(二甲氨基)丙基]-1H-吲哚-3-基]-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮）阻断。总之，CHO-A1细胞中的凝血酶受体激活通过百日咳毒素不敏感的G蛋白刺激[3H]肌醇磷酸的积累和[3H]花生四烯酸的释放。使用PMA的实验表明，蛋白激酶C对凝血酶受体激活磷脂酶C和磷脂酶A2具有不同的调节作用。转染的人类腺苷A1受体的共激活增强了凝血酶刺激的磷脂酶C和磷脂酶A2活性。最后，腺苷A1受体对磷脂酶A2活性的增强作用被选择性蛋白激酶C抑制剂抑制，提示蛋白激酶C参与其中。
