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Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: A flowcytometric study

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Advanced Flow Cytometry: Applications in Biological Research

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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 × 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.

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Abbreviations

ABAH:

4-Aminobenzoic acid hydrazide

AG:

Aminoguanidine

AA:

Arachidonic acid

CPA:

Cyclopiazonic acid

DCFDA:

2′,7′Dichlorodihydrofluorescein diacetate

DHR:

Dihydrorhodamine-123

DAF:

4,5-Diaminofluorescein diacetate

4–400:

Dowex 50WX

1077 and 1119:

Histopaque

LPS:

Lipopolysacharide

7NI:

7-Nitroindazole

L-NAME:

L-Nitroarginine methyl ester

TMB-8:

8-(N.N-Diethylamino) octyl 3,4,5-trimethyl benzoate

PMA:

Phorbol myristate acetate

SOD:

Superoxide dismutase

OZ:

Opsonised zymosan

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Authors and Affiliations

  1. Division of Pharmacology, Central Drug Research Institute, Lucknow, 226001, India

    Dr. Madhu Dikshit PhD & Prashant Sharma

Authors
  1. Dr. Madhu Dikshit PhD
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  2. Prashant Sharma
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Correspondence to Madhu Dikshit PhD .

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Editors and Affiliations

  1. Department of Bio-Technology, Panjab University, 160014, Chandigarh, India

    R. C. Sobti

  2. Department of Radiation Oncology, University of Miami Medical School, 33101, Miami, FL, USA

    Awtar Krishan

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© 2003 Springer Science+Business Media Dordrecht

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Dikshit, M., Sharma, P. (2003). Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: A flowcytometric study. In: Sobti, R.C., Krishan, A. (eds) Advanced Flow Cytometry: Applications in Biological Research. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-0623-0_11

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  • DOI: https://doi.org/10.1007/978-94-017-0623-0_11

  • Publisher Name: Springer, Dordrecht

  • Print ISBN: 978-90-481-6368-7

  • Online ISBN: 978-94-017-0623-0

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