Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle

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Abstract

Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11% (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11% (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2% inhibited by staurosporine, 87 +/- 6% inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10%, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6%, N = 3 experiments). [AIF4](-)-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.
LanguageEnglish
Pages593-603
Number of pages11
JournalBiochemical Pharmacology
Volume45
Issue number3
DOIs
Publication statusPublished - 1993

Fingerprint

Phospholipase D
Bradykinin
Smooth Muscle
Muscle
Guinea Pigs
Protein Kinase C
Acetates
Chemical activation
Staurosporine
Down-Regulation
Western Blotting
Experiments
Cells
Inositol 1,4,5-Trisphosphate
Protein C Inhibitor
Palmitates
Diglycerides
Pertussis Toxin
Type C Phospholipases
Second Messenger Systems

Keywords

  • bradykinin
  • calcium
  • cells
  • GTP-binding proteins
  • glycerophospholipids
  • indomethacin
  • palmitates
  • pertussis toxin
  • phosphatidic acids
  • phosphatidylcholines
  • phospholipase D
  • prostaglandin-endoperoxide synthases
  • protein kinase C
  • tetradecanoylphorbol acetate
  • time factors
  • trachea
  • tritium
  • virulence factors, bordetella

Cite this

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title = "Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle",
abstract = "Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11{\%} (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11{\%} (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2{\%} inhibited by staurosporine, 87 +/- 6{\%} inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10{\%}, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6{\%}, N = 3 experiments). [AIF4](-)-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.",
keywords = "bradykinin, calcium, cells, GTP-binding proteins, glycerophospholipids, indomethacin, palmitates, pertussis toxin, phosphatidic acids, phosphatidylcholines, phospholipase D, prostaglandin-endoperoxide synthases, protein kinase C, tetradecanoylphorbol acetate, time factors, trachea, tritium, virulence factors, bordetella",
author = "S Pyne and Pyne, {N J}",
year = "1993",
doi = "10.1016/0006-2952(93)90132-G",
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}

Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle. / Pyne, S; Pyne, N J.

In: Biochemical Pharmacology, Vol. 45, No. 3, 1993, p. 593-603.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle

AU - Pyne, S

AU - Pyne, N J

PY - 1993

Y1 - 1993

N2 - Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11% (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11% (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2% inhibited by staurosporine, 87 +/- 6% inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10%, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6%, N = 3 experiments). [AIF4](-)-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.

AB - Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11% (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11% (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2% inhibited by staurosporine, 87 +/- 6% inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10%, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6%, N = 3 experiments). [AIF4](-)-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.

KW - bradykinin

KW - calcium

KW - cells

KW - GTP-binding proteins

KW - glycerophospholipids

KW - indomethacin

KW - palmitates

KW - pertussis toxin

KW - phosphatidic acids

KW - phosphatidylcholines

KW - phospholipase D

KW - prostaglandin-endoperoxide synthases

KW - protein kinase C

KW - tetradecanoylphorbol acetate

KW - time factors

KW - trachea

KW - tritium

KW - virulence factors, bordetella

U2 - 10.1016/0006-2952(93)90132-G

DO - 10.1016/0006-2952(93)90132-G

M3 - Article

VL - 45

SP - 593

EP - 603

JO - Biochemical Pharmacology

T2 - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 3

ER -