title: Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways.
abstract: 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.
authors.affiliation: Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, Scotland, U.K.
authors.forename: P A
authors.initials: PA
authors.lastname: Stevens
authors.forename: S
authors.initials: S
authors.lastname: Pyne
authors.forename: M
authors.initials: M
authors.lastname: Grady
authors.forename: N J
authors.initials: NJ
authors.lastname: Pyne
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D000440:Butanols / Q000494:pharmacology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D010739:Phospholipase D / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014132:Trachea / Q000187:drug effects / Q000201:enzymology*
chemical_list: D000440:Butanols; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; D002772:Cholera Toxin; D000242:Cyclic AMP; D011493:Protein Kinase C; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin