Phospholipase C Activity in Human Polymorphonuclear Leukocytes: Partial Characterization and Effect of Indomethacin
Abstract
Several hormones act at the cellular level to increase diacylglycerol via increased catabolism of phosphatidylinositol by phospholipase C (PLC). Diacyglycerol stimulates protein kinase C, leading to protein phosphorylation and hormone action. Since PLC activity has not been well studied in man, we have established an assay for PLC in human neutrophils. In this assay sonicates of neutrophils were incubated with L-3- phosphatidyl (U14C)-inositol and the incubation mixture extracted with chloroform:methanol. Following the additions of 2MKC1 and chloroform, PLC activity was determined by counting (U14C) in the aqueous phase. The PLC activity was linear with respect to time and the quantity of added enzyme. Optimum substrate concentration and pH were 2mM and 7.0 respectively. Optimal activity was dependent on Ca2+ (2mM) and deoxycholate (2MM). Naloxone, FMLP and PGD2, which affect various aspects of Leucocyte function, had no significant effects on neutrophil PLC activity. The effects of various compounds with phospholipase A2 inhibitory activity were also tested on this enzyme. Of these, mepacrine, lidocaine, and indomethacin inhibited the enzyme activity. From these data we conclude that indomethacin is capable of inhibiting phospholipase C activity in neutrophils at clinically significant levels and that this may be relevant in the therapeutic action of this drug. Keywords: Phosphatidylinositol; Naloxone, Indomethacin; Phospholipase; C activity; Neutrophils; Calcium ions.
Document Details
- Document Type
- Technical Report
- Publication Date
- Dec 01, 1988
- Accession Number
- ADA212702
Entities
People
- C. O. Simpkins
- D. O. Sobel
- K. M. Shakir
- S. L. Gartner
- T. J. Williams
Organizations
- Naval Medical Research Center