BRADYKININ-DEPENDENT ACTIVATION OF ADENYLATE-CYCLASE ACTIVITY AND CYCLIC-AMP ACCUMULATION IN TRACHEAL SMOOTH-MUSCLE OCCURS VIA PROTEIN-KINASE C-DEPENDENT AND C-INDEPENDENT PATHWAYS

被引:29
作者
STEVENS, PA [1 ]
PYNE, S [1 ]
GRADY, M [1 ]
PYNE, NJ [1 ]
机构
[1] UNIV STRATHCLYDE,DEPT PHYSIOL & PHARMACOL,GLASGOW G1 1XW,SCOTLAND
基金
英国惠康基金;
关键词
D O I
10.1042/bj2970233
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 G(s) 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 G(s) 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 (EC(50)=7.3+/-2.5 nM, n=5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC(50)=63.3+/-14.5 nM, n=3). Constitutive activation of C-s resulted in an increased maximal response (10-fold) and potency (EC(50)=6.17+/-1.6 nM, n=3) to bradykinin. This response was not affected by the B-2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg(9)-bradykinin (a B-1-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-o1 (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 (EC(50)=5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC(50) > 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)P-2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.
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页码:233 / 239
页数:7
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