Stevens P A, Pyne S, Grady M, Pyne N J
Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, Scotland, U.K.
Biochem J. 1994 Jan 1;297 ( Pt 1)(Pt 1):233-9. doi: 10.1042/bj2970233.
Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5'-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.
用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）（100 nM）或缓激肽（100 nM）处理培养的气管平滑肌细胞（TSM），可使随后分离的细胞膜中基础的和鸟苷5'-[βγ - 亚氨基] - 三磷酸刺激的腺苷酸环化酶活性增强。对细胞的联合刺激无相加作用，表明两种试剂通过相似途径激活腺苷酸环化酶。PMA（100 nM）和缓激肽（100 nM）均可使Gs的α亚基在体外更有利于霍乱毒素催化的ADP - 核糖基化反应。PMA对对照细胞中的细胞内环磷酸腺苷无影响。然而，体内用霍乱毒素（0.5 ng/ml，18小时）处理使Gs组成性激活，使细胞对PMA刺激敏感，导致细胞内环磷酸腺苷积累呈浓度依赖性增加（EC50 = 7.3±2.5 nM，n = 5）。缓激肽也引起细胞内环磷酸腺苷浓度依赖性增加（EC50 = 63.3±14.5 nM，n = 3）。Gs的组成性激活导致对缓激肽的最大反应增加（10倍）和效力增加（EC50 = 6.17±1.6 nM，n = 3）。该反应不受B2受体拮抗剂NPC567 [其选择性阻断缓激肽刺激的磷脂酶C（PLC），对磷脂酶D（PLD）活性的活性较小]的影响。去 - 精氨酸9 - 缓激肽（一种B1受体激动剂）无活性。这些结果表明，能够激活PLD的受体亚型也可能刺激环磷酸腺苷积累。此外，用丁醇（0.3％，v/v）预处理细胞，丁醇可捕获PLD反应产生的磷脂酸，阻断缓激肽刺激的细胞内环磷酸腺苷增加。这些研究表明，PLD衍生的磷脂酸与腺苷酸环化酶活性的正向调节之间可能存在因果关系。支持这一点的是，缓激肽刺激的腺苷酸环化酶活性的浓度依赖性与缓激肽刺激的磷脂酶D活性的浓度依赖性相同（EC50 = 5 nM）。缓激肽而非PMA还能够通过未知机制在TSM细胞中抑制环磷酸腺苷磷酸二酯酶活性（EC50> 100 nM）。这些研究表明，TSM细胞中环磷酸腺苷途径与磷脂衍生的第二信使之间的交叉调节不是PLC催化的磷脂酰肌醇（4,5）二磷酸水解的结果，而是可能部分涉及PLD催化的磷脂酰胆碱水解。
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