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Measurement of phosphoinositide-specific phospholipase C activity in mononuclear leucocytes from atopic and normal
R B Mallett1, S Myint, C A Holden
1Department of Dermatology, St Helier Hospital, Carshalton, Surrey, U.K.
Insights
Atopic dermatitis (AD) patients show increased phospholipase C (PLC) activity in mononuclear cells (MNL). This enzyme is more sensitive to stimulation, suggesting a key role in AD pathogenesis.
Area of Science:
- Biochemistry
- Immunology
- Dermatology
Background:
- The intracellular inositol pathway is crucial for cell activation, mediated by phosphoinositide-specific phospholipase C (PLC).
- Previous indirect studies indicated inositol metabolism abnormalities in mononuclear cells (MNL) of atopic dermatitis (AD) patients.
- A direct method for measuring PLC activity in MNL is needed to confirm these findings.
Purpose of the Study:
- To develop and utilize a direct method for measuring PLC activity in MNL from AD patients.
- To compare PLC activity in AD patients with healthy controls.
- To investigate the effects of substrate concentration and nucleotide stimulation on PLC activity in AD.
Main Methods:
- Developed a direct assay for measuring phospholipase C (PLC) activity.
- Isolated membrane and cytosol fractions from mononuclear cells (MNL).
- Assayed PLC activity in samples from atopic dermatitis (AD) patients and normal controls, varying substrate and nucleotide concentrations.
Main Results:
- Significantly increased membrane-bound PLC activity was observed in MNL from AD patients compared to controls.
- Nucleotide stimulation non-specifically increased PLC activity in AD samples.
- These findings suggest heightened sensitivity of PLC in atopics to substrate-driven activity.
Conclusions:
- Direct measurement confirms elevated membrane-bound PLC activity in AD MNL.
- The enzyme's increased sensitivity to stimulation may contribute to the pathophysiology of atopic dermatitis.
- This study provides a direct method for assessing PLC activity in AD research.
Abstract:
The intracellular inositol pathway is an important route for cell activation and relies on the stimulation of membrane-bound phosphoinositide-specific phospholipase C (PLC). Previously we have shown abnormalities of inositol metabolism in mononuclear cells (MNL) in atopic dermatitis (AD) using an indirect method. We now describe a direct method of measuring PLC activity in membrane and cytosol preparations of MNL in AD. We compare PLC activity in AD with that in normal controls and examine the effect of substrate concentration and nucleotide stimulation on the system. Our findings show increased membrane-bound PLC activity in AD compared with normal controls. Non-specific stimulation of AD PLC activity by nucleotides suggests that the enzyme of atopics is more sensitive to substrate-driven activity than that of non-atopics.