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Gene expression of endothelin-1 (ET-1) and release of mature peptide by activated human neutrophils
C Cambiaggi1, M Mencarelli, M Muscettola
1Institute of General Physiology, University of Siena, Siena, Italy.
Abstract:
Polymorphonuclear neutrophils (PMNs) are only regarded as being involved in the cleavage of exogenous big endothelin-1 (ET-1) to the active peptide. The aims of the present study were to investigate whether PMNs may themselves express mRNA for prepro-ET-1 (pp-ET-1, a long precursor of 212 amino acids) and to determine the capacity of several PMN stimulants to modulate mRNA expression and the release of ET-1 in culture medium. PMNs, isolated from seven healthy adult volunteers, were stimulated with lipopolysaccharide (LPS, 0.25-10 microg/ml), or LPS (1 microg/ml) + phorbol myristate acetate (PMA, 10 ng/ml) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (f-MLP, 10(-5) M) or tumour necrosis factor-alpha (TNF-alpha, 50 IU/ml). They were found to express pp-ET-1 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Enzyme immunoassay (EIA) revealed low levels of ET-1 in the culture supernatants of PMNs stimulated for 3 h with LPS (10 microg/ml) and with LPS + PMA. Control unstimulated PMNs did not express pp-ET-1 mRNA. The local production of ET-1 by PMNs in vivo has significant implications in inflammatory diseases.
Insights
Polymorphonuclear neutrophils (PMNs) express messenger RNA for prepro-endothelin-1 (pp-ET-1) and release endothelin-1 (ET-1) when stimulated. This local production of ET-1 by PMNs has implications for inflammatory diseases.
Area of Science:
- Immunology
- Molecular Biology
- Cardiovascular Research
Background:
- Polymorphonuclear neutrophils (PMNs) were traditionally considered solely responsible for cleaving exogenous big endothelin-1 (ET-1) into its active form.
- The potential for PMNs to synthesize their own ET-1 precursor was not well understood.
Purpose of the Study:
- To investigate if PMNs express messenger RNA (mRNA) for prepro-ET-1 (pp-ET-1).
- To determine if PMN stimulants can modulate pp-ET-1 mRNA expression and ET-1 release.
Main Methods:
- PMNs were isolated from healthy volunteers.
- PMNs were stimulated with lipopolysaccharide (LPS), phorbol myristate acetate (PMA), N-formyl-L-methionyl-L-leucyl-L-phenylalanine (f-MLP), or tumor necrosis factor-alpha (TNF-alpha).
- Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect pp-ET-1 mRNA, and enzyme immunoassay (EIA) measured ET-1 levels in culture supernatants.
Main Results:
- PMNs were confirmed to express pp-ET-1 mRNA.
- Stimulation with LPS (10 microg/ml) and LPS + PMA induced low levels of ET-1 release into the culture medium.
- Unstimulated PMNs did not express pp-ET-1 mRNA.
Conclusions:
- PMNs are capable of local endothelin-1 production.
- This finding suggests a novel role for PMNs in regulating ET-1 levels, particularly in inflammatory conditions.
- The local production of ET-1 by PMNs has significant implications for understanding and managing inflammatory diseases.
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