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Related Experiment Videos

Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes

A M Miles1, M W Owens, S Milligan

  • 1Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport 71130, USA.

Journal of Leukocyte Biology
|November 1, 1995
PubMed
Summary
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Inflammation upregulates inducible nitric oxide synthase (iNOS) in rat polymorphonuclear leukocytes (PMNs) but not in human PMNs. This study characterizes iNOS in circulating vs. extravasated PMNs, revealing species-specific regulation.

Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • Inflammation is linked to increased nitric oxide (NO) production.
  • Regulation of NO synthase (NOS) in macrophages is well-studied, but less is known about NOS in polymorphonuclear leukocytes (PMNs).
  • Inducible NOS (iNOS) plays a key role in NO generation during inflammation.

Purpose of the Study:

  • To characterize the molecular and biochemical properties of inducible NO synthase (iNOS) in circulating versus extravasated rat and human PMNs.
  • To investigate the regulation of iNOS expression and activity in PMNs during inflammation.

Main Methods:

  • Purification of circulating PMNs from peripheral blood.
  • Elicitation of extravasated PMNs in rats (glycogen) and humans (peritonitis).

Related Experiment Videos

  • Quantification of iNOS mRNA (slot blot), protein (Western blot), and enzymatic activity (L-arginine conversion).
  • Measurement of nitrite accumulation as an indicator of NO production.

    • Main Results:

    • Circulating rat and human PMNs showed no detectable iNOS mRNA, protein, or activity, and produced minimal NO.
    • Elicited rat PMNs exhibited time-dependent increases in iNOS mRNA (up to 42-fold) and protein, with significant NOS activity.
    • Extravasated rat PMNs produced measurable nitrite, with 20-30% of iNOS activity being Ca(2+)-dependent.
    • Extravasated human PMNs lacked iNOS message, protein, and enzymatic activity.

    Conclusions:

    • Inflammation-induced extravasation upregulates iNOS transcription and translation in rat PMNs.
    • A portion (20-30%) of the total inducible NOS in rat PMNs is Ca(2+)-dependent.
    • Human PMN iNOS gene regulation differs significantly from that in rats, as neither circulating nor extravasated human PMNs express iNOS.