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Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Published on: August 18, 2012
Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: a
Madhu Dikshit1, Prashant Sharma
1Division of Pharmacology, Central Drug Research Institute, Lucknow-226001, India. madhudikshit@yahoo.com
Abstract:
Nitric oxide (NO) synthesis and free radical generation from polymorphonuclear leukocytes (PMNs) play an important role in several pathological conditions. It is therefore important to understand the regulatory mechanisms of free radical generation from PMNs. Flowcytometry can be used to assess generation of reactive oxygen and nitrogen species from PMNs by using fluorescent probes. In the present study regulation of NO synthesis in the control and lipopolysaccharide (LPS) treated rat PMNs has been investigated. Free radical generation was assessed by flow cytometry using a dye, 2'7'-dichlorodihydrofluorescein diacetate (DCFDA), dihydrorhodamine-123 (DHR) and 4,5-diaminofluorescein diacetate (DAF). Superoxide dismutase (SOD), and catalase significantly attenuated the arachidonic acid (AA, 1 x 10(-6) M) induced free radical generation, while 4-aminobenzoicacid hydrazide (ABH), myeloperoxidase (MPO) inhibitor had no significant effect. Intracellular and extracellular calcium levels also modulated FR generation. AA induced free radical generation from PMNs was also enhanced significantly after LPS treatment. NO synthase (NOS) inhibitors, aminoguanidine (AG) and 7-nitroindazole (NI) inhibited arachidonic acid induced free radical generation from LPS treated PMNs, while in control PMNs NOS inhibition had no effect. Augmentation of free radical generation from rat PMNs following LPS treatment seems to be regulated by NO.
Insights
Nitric oxide (NO) regulates free radical generation in rat polymorphonuclear leukocytes (PMNs). Lipopolysaccharide (LPS) treatment enhances this generation, which is inhibited by NO synthase inhibitors.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMNs) generate nitric oxide (NO) and free radicals, crucial in pathology.
- Understanding the regulation of PMN free radical generation is vital for disease research.
Purpose of the Study:
- To investigate the regulation of NO synthesis and free radical generation in rat PMNs.
- To determine the role of NO in modulating free radical generation, especially after lipopolysaccharide (LPS) stimulation.
Main Methods:
- Flow cytometry utilized fluorescent probes (DCFDA, DHR, DAF) to measure reactive oxygen and nitrogen species.
- Assessed the effects of antioxidants (SOD, catalase), myeloperoxidase (MPO) inhibitor (ABH), and calcium levels on free radical generation.
- Investigated the impact of NO synthase (NOS) inhibitors (AG, NI) on arachidonic acid (AA)-induced free radical generation in control and LPS-treated PMNs.
Main Results:
- Superoxide dismutase (SOD) and catalase significantly reduced arachidonic acid (AA)-induced free radical generation.
- Myeloperoxidase (MPO) inhibition did not affect free radical generation; calcium levels modulated it.
- LPS treatment significantly enhanced AA-induced free radical generation in PMNs.
- NOS inhibitors (AG, NI) suppressed AA-induced free radical generation in LPS-treated PMNs but not in control PMNs.
Conclusions:
- NO plays a regulatory role in the augmentation of free radical generation from rat PMNs following LPS treatment.
- These findings highlight the involvement of NO in inflammatory processes mediated by PMNs.
