Prolonged endotoxin (LPS) exposure impairs thrombininduced calcium mobilization in bovine aortic myocytes: role of nitric oxide

Mark Wylam, P. T. Murray, J. G. Umans

Research output: Contribution to journalArticle

Abstract

We and others have shown that nitric oxide (NO) synthase inhibition only partially restores vasoconstrictor responses in vascular tissue from LPStreated animals, and in vessels exposed to LPS in vitro. We therefore explored the contribution of LPS-mduced NO synthesis to impairment of thrombin- (TB) induced myocyte calcium mobilization. We used microspectrofluorimctry and fura-2 to measure basal and peak TB (10 units/ml)-stimulated cytosolic calcium ([Ca+2]i) in confluent bovine aortic myocytes (passage 25). Cells were incubated overnight (24 hrs) in DMEM + 10% FBS + Lginine (L-ARG, 10-4M), +/- E. Coli LPS (Img/ml), and +/- L-NAME (10-4M). Group comparisons of nitrite accumulation in media (by chemical reduction and NO chemiluminescence) and of basal and peak TB-induced [Ca+2]i were made by ANOVA with Bonferroni correction. Results: LPS induced a 9-fold increase in nitrite accumulation (Control = 19.5 ±10.0, LPS = 174.1 ±0.7 17.2 pM/100μl, n = 8, p < 0001) which was attenuated ∼50% by L-NAME (91.6 ±10,3 pM/100μl, n = 8, p < 0.0005). There was no effect of LPS on basal [Ca+2]i. However, LPS did reduce TB peak [Ca+2]i by 49% (n=4 p<0.002), an effect which was not altered by LNAME (n=4). We conclude that prolonged incubation with LPS impairs TB-induced intracellular [Ca+2]i mobilization and induces NO synthesis in cultured bovine myocytes. However, a 50% reduction in LPS-stimuIated NO synthesis fails to restore TB-stimulated calcium mobilization. It remains to be determined whether more complete NO synthase inhibition would improve TB-calcium mobilization in these cells, or whether NO might affect other vasoconstrictors in a similar manner. Supported, in part, by HL48302 and by the PhRMA Foundation.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number3
StatePublished - Dec 1 1996
Externally publishedYes

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Endotoxins
Thrombin
Muscle Cells
Nitric Oxide
Calcium
NG-Nitroarginine Methyl Ester
Vasoconstrictor Agents
Nitrites
Nitric Oxide Synthase
Chemiluminescence
Fura-2
Analysis of variance (ANOVA)
Luminescence
Escherichia coli
Blood Vessels
Analysis of Variance
Animals
Tissue

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Prolonged endotoxin (LPS) exposure impairs thrombininduced calcium mobilization in bovine aortic myocytes : role of nitric oxide. / Wylam, Mark; Murray, P. T.; Umans, J. G.

In: FASEB Journal, Vol. 10, No. 3, 01.12.1996.

Research output: Contribution to journalArticle

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abstract = "We and others have shown that nitric oxide (NO) synthase inhibition only partially restores vasoconstrictor responses in vascular tissue from LPStreated animals, and in vessels exposed to LPS in vitro. We therefore explored the contribution of LPS-mduced NO synthesis to impairment of thrombin- (TB) induced myocyte calcium mobilization. We used microspectrofluorimctry and fura-2 to measure basal and peak TB (10 units/ml)-stimulated cytosolic calcium ([Ca+2]i) in confluent bovine aortic myocytes (passage 25). Cells were incubated overnight (24 hrs) in DMEM + 10{\%} FBS + Lginine (L-ARG, 10-4M), +/- E. Coli LPS (Img/ml), and +/- L-NAME (10-4M). Group comparisons of nitrite accumulation in media (by chemical reduction and NO chemiluminescence) and of basal and peak TB-induced [Ca+2]i were made by ANOVA with Bonferroni correction. Results: LPS induced a 9-fold increase in nitrite accumulation (Control = 19.5 ±10.0, LPS = 174.1 ±0.7 17.2 pM/100μl, n = 8, p < 0001) which was attenuated ∼50{\%} by L-NAME (91.6 ±10,3 pM/100μl, n = 8, p < 0.0005). There was no effect of LPS on basal [Ca+2]i. However, LPS did reduce TB peak [Ca+2]i by 49{\%} (n=4 p<0.002), an effect which was not altered by LNAME (n=4). We conclude that prolonged incubation with LPS impairs TB-induced intracellular [Ca+2]i mobilization and induces NO synthesis in cultured bovine myocytes. However, a 50{\%} reduction in LPS-stimuIated NO synthesis fails to restore TB-stimulated calcium mobilization. It remains to be determined whether more complete NO synthase inhibition would improve TB-calcium mobilization in these cells, or whether NO might affect other vasoconstrictors in a similar manner. Supported, in part, by HL48302 and by the PhRMA Foundation.",
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