Impaired activation of cytosolic phospolipase A2 in inflamed canine colonic circular muscle

Irshad Ali, William B. Campbell, Sushil K. Sarna

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background and Aims: Arachidonic acid (AA) is a critical second messenger in several cell types. We examined whether cholinergic AA acts as a second messenger in contraction of colonic circular muscle and if this role is altered by inflammation. Methods: The experiments were performed on single dispersed cells. AA release was measured by high-performance liquid chromatography. Escherichia coli membranes labeled with 3H-AA were used as a substrate for determining phospholipase A2 (PLA2) activity, and Western immunoblotting for protein expression. Results: Acetylcholine and the PLA2 activator melittin induced cell contractions and AA release. Both effects were inhibited by the PLA2 inhibitor ONO-RS-082. Cytosolic and membrane PLA2 activities increased in response to acetylcholine. These were blocked by ONO-RS-082 and cytosolic PLA2 100-kilodalton antibody, but not by dithiothreitol, a secretory PLA2 inhibitor. Acetylcholine- and melittin- stimulated release of AA and their contractile response were attenuated in inflamed cells. Immunoblotting indicated that the protein expression of cPLA2 was suppressed during inflammation. Conclusions: AA acts as a second messenger in muscarinic receptor-activated contractions of colonic circular muscle cells. cPLA2 is the primary enzyme that releases AA in these cells; its expression as well as activation are significantly attenuated by inflammation. The attenuated release of AA may partly account for the inhibition of colonic circular muscle tone and phasic contractions observed during inflammation.

Original languageEnglish (US)
Pages (from-to)62-70
Number of pages9
JournalGastroenterology
Volume119
Issue number1
StatePublished - Jul 2000
Externally publishedYes

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varespladib methyl
Arachidonic Acid
Canidae
Muscles
Second Messenger Systems
Cytosolic Phospholipases A2
Melitten
Acetylcholine
Inflammation
Phospholipases A2
Membranes
Dithiothreitol
Muscarinic Receptors
Immunoblotting
Muscle Cells
Cholinergic Agents
Proteins
Western Blotting
High Pressure Liquid Chromatography
Escherichia coli

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Impaired activation of cytosolic phospolipase A2 in inflamed canine colonic circular muscle. / Ali, Irshad; Campbell, William B.; Sarna, Sushil K.

In: Gastroenterology, Vol. 119, No. 1, 07.2000, p. 62-70.

Research output: Contribution to journalArticle

Ali, I, Campbell, WB & Sarna, SK 2000, 'Impaired activation of cytosolic phospolipase A2 in inflamed canine colonic circular muscle', Gastroenterology, vol. 119, no. 1, pp. 62-70.
Ali, Irshad ; Campbell, William B. ; Sarna, Sushil K. / Impaired activation of cytosolic phospolipase A2 in inflamed canine colonic circular muscle. In: Gastroenterology. 2000 ; Vol. 119, No. 1. pp. 62-70.
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abstract = "Background and Aims: Arachidonic acid (AA) is a critical second messenger in several cell types. We examined whether cholinergic AA acts as a second messenger in contraction of colonic circular muscle and if this role is altered by inflammation. Methods: The experiments were performed on single dispersed cells. AA release was measured by high-performance liquid chromatography. Escherichia coli membranes labeled with 3H-AA were used as a substrate for determining phospholipase A2 (PLA2) activity, and Western immunoblotting for protein expression. Results: Acetylcholine and the PLA2 activator melittin induced cell contractions and AA release. Both effects were inhibited by the PLA2 inhibitor ONO-RS-082. Cytosolic and membrane PLA2 activities increased in response to acetylcholine. These were blocked by ONO-RS-082 and cytosolic PLA2 100-kilodalton antibody, but not by dithiothreitol, a secretory PLA2 inhibitor. Acetylcholine- and melittin- stimulated release of AA and their contractile response were attenuated in inflamed cells. Immunoblotting indicated that the protein expression of cPLA2 was suppressed during inflammation. Conclusions: AA acts as a second messenger in muscarinic receptor-activated contractions of colonic circular muscle cells. cPLA2 is the primary enzyme that releases AA in these cells; its expression as well as activation are significantly attenuated by inflammation. The attenuated release of AA may partly account for the inhibition of colonic circular muscle tone and phasic contractions observed during inflammation.",
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N2 - Background and Aims: Arachidonic acid (AA) is a critical second messenger in several cell types. We examined whether cholinergic AA acts as a second messenger in contraction of colonic circular muscle and if this role is altered by inflammation. Methods: The experiments were performed on single dispersed cells. AA release was measured by high-performance liquid chromatography. Escherichia coli membranes labeled with 3H-AA were used as a substrate for determining phospholipase A2 (PLA2) activity, and Western immunoblotting for protein expression. Results: Acetylcholine and the PLA2 activator melittin induced cell contractions and AA release. Both effects were inhibited by the PLA2 inhibitor ONO-RS-082. Cytosolic and membrane PLA2 activities increased in response to acetylcholine. These were blocked by ONO-RS-082 and cytosolic PLA2 100-kilodalton antibody, but not by dithiothreitol, a secretory PLA2 inhibitor. Acetylcholine- and melittin- stimulated release of AA and their contractile response were attenuated in inflamed cells. Immunoblotting indicated that the protein expression of cPLA2 was suppressed during inflammation. Conclusions: AA acts as a second messenger in muscarinic receptor-activated contractions of colonic circular muscle cells. cPLA2 is the primary enzyme that releases AA in these cells; its expression as well as activation are significantly attenuated by inflammation. The attenuated release of AA may partly account for the inhibition of colonic circular muscle tone and phasic contractions observed during inflammation.

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