Intrathoracic and Extrathoracic Sources of Exhaled Nitric Oxide in Porcine Endotoxemic Shock

¹ From the Department of Anesthesiology and Critical Care Medicine, Tokyo Medical and Dental University, School of Medicine, Tokyo, Japan
² From the Critical Care and Respiratory Divisions, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada

Sabah Hussain, MD, Room L3.05, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Quebec, Canada H3A 1A1

Objectives: Nitric oxide (NO), a highly reactive species produced by the activity of NO synthases (NOS), is normally present in the exhaled air of humans and animals. Exhaled NO concentration increases significantly in humans with sepsis and animals, but neither the source nor NOS isoforms responsible for this rise in pulmonary NO production are known. The main objective of this study is to determine the sites and the mechanisms of enhanced NO production in the exhaled air of endotoxemic pigs.

Design: Randomized, controlled, animal study.

Setting: University-based animal research facility.

Subjects: Thirteen pathogen-free adult female pigs (22 to 27 kg).

Interventions: Anesthetized pigs were divided into two groups: control and lipopolysaccharides (LPS) (septic) groups. In both groups, extrathoracic (upper airways, nasal, and paranasal) and intrathoracic (bronchi, bronchioles, and alveoli) compartments were ventilated equally with two separate ventilators connected to two tracheal tubes. The LPS group received slow infusion (over 2 h) of Escherichia coli endotoxin (10 µg/kg/h), whereas saline solution was infused into the control group. Expired air of the two compartments was collected throughout the 2-h observation period. The animals were then killed and the lungs were quickly excised and frozen.

Measurements: Hemodynamic variables were measured in both groups. NO concentration in the exhaled air of both compartments was measured with a chemiluminescence analyser. Pulmonary NOS activity was evaluated by measuring the conversion of L-[2,3^H]-arginine to L-[2,3^H]-citrulline, and pulmonary expression of NOS was evaluated by immunoblotting.

Results: Baseline NO concentration in both groups was significantly higher in the extrathoracic vs intrathoracic compartment (average of 5.2 vs 3.4 parts per billion). Endotoxin infusion elicited a significant and early (after 45 min) rise in exhaled NO concentration in the extrathoracic compartment. Exhaled NO in the intrathoracic compartment also rose significantly but after 90 min of endotoxin infusion. Measurement of lung NOS activity showed a substantial rise in Ca⁺⁺/calmodulin-dependent activity in the LPS group with no rise in Ca⁺⁺/calmodulin-independent activity. Immunoblotting of lung tissue samples indicated the absence of the inducible isoform in both groups of animals. Moreover, LPS injection elicited no significant alterations in the pulmonary expression of the endothelial and the neuronal isoforms.

Conclusions: Both extrathoracic and intrathoracic compartments contribute to the rise in exhaled NO production in experimental septic shock. The rise in exhaled NO production is due to increased activity of constitutive NOS isoforms as a result of increased cofactor availability and/or downregulation of the endogenous inhibitors of NOS.

Key Words: cardiac output • cytokines • endotoxin • inflammation • nitric oxide • nitric oxide synthase • pigs • sepsis • septic shock

Submitted on July 23, 1997
Accepted on January 26, 1998

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