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Cardiac inducible nitric oxide synthase negatively modulates myocardial function in cultured rat myocytes
K I Kinugawa1, O Kohmoto, A Yao
1Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Abstract:
Recent work has demonstrated that endotoxin or cytokines induce nitric oxide synthase in heart or cardiac myocytes. We investigated the functional significance of inducible nitric oxide synthase (iNOS) in indo 1-loaded beating myocytes with regard to intracellular Ca2+ concentration ([Ca2+]i) and cell contraction. Lipopolysaccharide (LPS; 10 micrograms/ml) time dependently induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. Nitrite concentration in the medium and intracellular guanosine 3',5'-cyclic monophosphate (cGMP) contents after 24-h exposure to LPS increased in proportion to the levels of iNOS induction in these cells. Myocytes treated with both NG-monomethyl-L-arginine and LPS for 24 h expressed iNOS protein, but nitrite production was significantly inhibited. Subsequent perfusion with 100-fold molar excess L-arginine of these myocytes elicited decreases in peak systolic [Ca2+]i (790 +/- 42 to 551 +/- 27 nM, P < 0.05), relative amplitude of cell contraction (100 to 72.4 +/- 5.5%, P < 0.05), and spontaneous beating rate (146 +/- 13 to 85 +/- 22 beats/min, P < 0.05). Pretreatment with methylene blue or KT-5823 inhibited these negative myocardial effects. These results suggest that LPS induces iNOS in cardiac myocytes and that the increased nitric oxide produced by iNOS has cardiac depressant effects through the activation of cGMP-dependent protein kinase.
Insights
Lipopolysaccharide (LPS) induces inducible nitric oxide synthase (iNOS) in cardiac myocytes. This leads to reduced intracellular calcium, cell contraction, and heart rate via nitric oxide signaling.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
Background:
- Endotoxin and cytokines are known to induce nitric oxide synthase (iNOS) in cardiac cells.
- The functional impact of iNOS in cardiac myocytes remains an area of investigation.
Purpose of the Study:
- To investigate the functional significance of inducible nitric oxide synthase (iNOS) in cardiac myocytes.
- To determine the effects of iNOS-derived nitric oxide on intracellular calcium concentration and cell contraction.
Main Methods:
- Primary neonatal rat cardiac myocytes were cultured and exposed to lipopolysaccharide (LPS).
- Inducible nitric oxide synthase (iNOS) mRNA and protein levels were assessed.
- Intracellular calcium ([Ca2+]i) and cell contraction were measured in indo 1-loaded beating myocytes.
- Nitrite production and cyclic guanosine monophosphate (cGMP) levels were quantified.
Main Results:
- LPS treatment time-dependently induced iNOS mRNA and protein in cardiac myocytes.
- Increased nitrite and intracellular cGMP levels correlated with iNOS induction.
- Inhibition of iNOS with NG-monomethyl-L-arginine blocked nitrite production.
- L-arginine perfusion in iNOS-expressing myocytes significantly decreased peak systolic [Ca2+]i, cell contraction amplitude, and spontaneous beating rate.
- Methylene blue and KT-5823 pretreatment attenuated these negative cardiac effects.
Conclusions:
- Lipopolysaccharide (LPS) induces iNOS expression in cardiac myocytes.
- The resulting nitric oxide production exerts cardiac depressant effects.
- These effects are mediated through the activation of cGMP-dependent protein kinase.