Differential effects of B(2) receptor antagonists upon bradykinin-stimulated phospholipase C and phospholipase D in guinea-pig cultured tracheal smooth muscle

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Abstract

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. 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). 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). 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). 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.
Original languageEnglish
Pages (from-to)477-481
Number of pages5
JournalBritish Journal of Pharmacology
Volume110
Issue number1
Publication statusPublished - Sep 1993

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Phospholipase D
Type C Phospholipases
Bradykinin
Smooth Muscle
Guinea Pigs
Bradykinin Receptors
Bradykinin Receptor Antagonists
Bradykinin B2 Receptors
Inositol 1,4,5-Trisphosphate
Palmitates
Indomethacin
Population
Inhibitory Concentration 50
Smooth Muscle Myocytes

Keywords

  • animals
  • bradykinin
  • culture techniques
  • glycerophospholipids
  • guinea pigs
  • indomethacin
  • inosine triphosphate
  • male
  • muscle, smooth
  • palmitic acid
  • palmitic acids
  • phosphatidic acids
  • phospholipase D
  • receptors, bradykinin
  • trachea
  • type C phospholipases

Cite this

@article{84fe90a9757048829002ba82e50baf3f,
title = "Differential effects of B(2) receptor antagonists upon bradykinin-stimulated phospholipase C and phospholipase D in guinea-pig cultured tracheal smooth muscle",
abstract = "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. 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). 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). 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). 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.",
keywords = "animals, bradykinin, culture techniques, glycerophospholipids, guinea pigs, indomethacin, inosine triphosphate, male, muscle, smooth, palmitic acid, palmitic acids, phosphatidic acids, phospholipase D, receptors, bradykinin, trachea, type C phospholipases",
author = "S Pyne and Pyne, {N J}",
year = "1993",
month = "9",
language = "English",
volume = "110",
pages = "477--481",
journal = "British Journal of Pharmacology",
issn = "0007-1188",
number = "1",

}

TY - JOUR

T1 - Differential effects of B(2) receptor antagonists upon bradykinin-stimulated phospholipase C and phospholipase D in guinea-pig cultured tracheal smooth muscle

AU - Pyne, S

AU - Pyne, N J

PY - 1993/9

Y1 - 1993/9

N2 - 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. 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). 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). 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). 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.

AB - 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. 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). 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). 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). 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.

KW - animals

KW - bradykinin

KW - culture techniques

KW - glycerophospholipids

KW - guinea pigs

KW - indomethacin

KW - inosine triphosphate

KW - male

KW - muscle, smooth

KW - palmitic acid

KW - palmitic acids

KW - phosphatidic acids

KW - phospholipase D

KW - receptors, bradykinin

KW - trachea

KW - type C phospholipases

M3 - Article

VL - 110

SP - 477

EP - 481

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 0007-1188

IS - 1

ER -