Differential effects of B2 receptor antagonists upon bradykinin-stimulated phospholipase C and D in guinea-pig cultured tracheal smooth muscle.
1. Guinea-pig tracheal smooth muscle cells were isolated and maintained in culture for 14-21 days prior to the study of the effect of a selective bradykinin B1 agonist and B2 antagonists upon bradykinin-stimulated phospholipase C and D activities. 2. Bradykinin-stimulated phospholipase C activity was determined by mass measurement of inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) in unlabelled cells, whereas phospholipase D activity was assayed by the accumulation of [3H]-phosphatidylbutanol ([3H]-PtdBut) in [3H]-palmitate-labelled cells, which were stimulated in the presence of butan-1-o1 (0.3%, v/v). 3. Bradykinin elicited the rapid and transient formation of Ins(1,4,5)P3, in a concentration-dependent manner (log EC50 = -7.55 +/- 0.1 M, N = 3). Bradykinin also rapidly activated the concentration-dependent (log EC50 = -8.3 +/- 0.4 M, n = 3) phospholipase D-catalysed accumulation of [3H]-PtdBut; the accumulation of [3H]-PtdBut was sustained. These effects were not inhibited by pretreatment of the cells with indomethacin (1 microM). 4. The bradykinin B1 agonist, desArg9-bradykinin (1 microM) was without effect upon phospholipase C or phospholipase D activity. Bradykinin-stimulated (10 nM, EC40) Ins(1,4,5)P3 formation was inhibited by B2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-bradykinin (NPC 567) and D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (NPC 349), with log IC50 values of -6.3 +/- 0.5 M and -6.3 +/- 0.4 M, respectively. However, bradykinin-stimulated (10 nM, EC100) [3H]-PtdBut accumulation was poorly inhibited and with low potency by each B2 receptor antagonist and bradykinin-stimulated phospholipase D activity persisted at concentrations of antagonist that completely blocked bradykinin-stimulated Ins(1,4,5)P3 formation (30 microM). 5. These observations suggest that the activation of phospholipase C by bradykinin may be mediated through a bradykinin B2 receptor population, whereas bradykinin-stimulated phospholipase D may be activated via a distinct population of bradykinin receptors that do not appear to be either B1 or B2 receptor types, based upon pharmacological specificity. The mechanism of the activation of phospholipase D by bradykinin and the role of the putative B3 bradykinin receptor are discussed.
Pyne
S
S
Department of Physiology and Pharmacology, University of Strathclyde, Royal College, Glasgow.
Pyne
N J
NJ
D000818:Animals; D001920:Bradykinin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D065168:Bradykinin Receptor Antagonists; D046508:Culture Techniques; D020404:Glycerophospholipids; D006168:Guinea Pigs; D007213:Indomethacin / Q000494:pharmacology; D007293:Inosine Triphosphate / Q000378:metabolism; D008297:Male; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018002:Receptors, Bradykinin / Q000502:physiology; D014132:Trachea / Q000187:drug effects / Q000201:enzymology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D065168:Bradykinin Receptor Antagonists; D020404:Glycerophospholipids; D010169:Palmitic Acids; D010712:Phosphatidic Acids; D018002:Receptors, Bradykinin; C068564:phosphatidylbutanol; D007293:Inosine Triphosphate; C024626:bradykinin, des-Arg(9)-; D019308:Palmitic Acid; C053010:NPC 567; D010738:Type C Phospholipases; D010739:Phospholipase D; D001920:Bradykinin; D007213:Indomethacin