Local anaesthetics upregulate nitric oxide generation in cord blood and adult human neutrophils

Karolina I Kulinska,Krzysztof Sawinski,Krzysztof W Nowak,Michał Gaca,Hanna Billert,Krzysztof Kusza,Maria Siemionow,Katarzyna Czerniak,Maria Billert

doi:10.1038/s41598-018-37090-9

Abstract

Nitric oxide (NO) generation by systemic neonatal neutrophils is not clarified. It is also not known whether local anaesthetics (LAs) transferred to the fetal systemic circulation following maternal epidural blockade may affect this process. In the present study, NO generation was evaluated in neutrophils from cord blood (CB, n = 11) and adult blood (n = 10) following exposure to bupivacaine (0.0005, 0.005, 1 mM), lidocaine (0.002, 0.02, 4 mM) and ropivacaine (0.0007, 0.007, 1.4 mM) using flow cytometry, as well as indirectly by determining nitrite concentrations in cell incubation media. To determine the role of NO synthase (NOS) isoforms in NO generation following exposure to LAs, experiments were repeated in the presence of the NOS inhibitors, NG-nitro-L-arginine methyl ester and aminoguanidine; in addition, the expression of NOS isoforms was analysed. CB neutrophils produced less NO than adult neutrophils. LAs, especially ropivacaine and lidocaine, stimulated neutrophil NO generation, but in CB neutrophils this effect was negligible at clinically relevant drug concentrations. A mechanism involving NOS activity was responsible for the observed phenomena. In conclusion, LAs are able to upregulate neutrophil NO production, but in neonates this effect is likely to be clinically insignificant.

Highlights

Neutrophils represent most abundant systemic leukocyte population critical for host defense, especially for the initiation, coordination and resolution of the inflammatory response
The present study showed that: (1) Phorbol myristate acetate (PMA)-stimulated CB neutrophils produce less Nitric oxide (NO) than adult cells, and that NO production in CB neutrophils is mediated via a NOS2-independent mechanism; (2) Local anaesthetics (LAs) increase NO generation by neutrophils depending on the type of anaesthetic and concentration; (3) following exposure to LAs, CB neutrophils produce less NO than adult neutrophils; and (4) LAs affect NO generation by neutrophils by differentially upregulating NO synthase (NOS) activity and NOS isoform expression in CB and adult neutrophils
Upon PMA stimulation, intracellular NO generation in adult neutrophils increased by approximately one-fourth of the initial values, remarkably close to the results obtained in LPS-stimulated neutrophils[17]

Summary

Introduction

Neutrophils represent most abundant systemic leukocyte population critical for host defense, especially for the initiation, coordination and resolution of the inflammatory response These cells produce nitric oxide (NO), a pleiotropic radical gasotransmitter, predominantly in a process of oxidation of L-arginine to L-citrulline and NO, using NADPH and oxygen. Previous studies including our own have demonstrated that at clinically relevant concentrations, LAs are able to decrease the activities of neonatal neutrophils critical for host defense, such as chemotaxis and ROS generation[15,16], closely interlinked with intracellular NO production[1]. In the present study we compared NO generation in neonatal (cord blood, CB) versus adult circulating neutrophils and investigated the effects of the LAs bupivacaine, lidocaine and ropivacaine on NO generation, with regard to the putative role of NOS in both cell populations studied. The underlying upregulation of NOS activity and NOS isoform expression varied between neonatal and adult neutrophils

Methods

Results

Conclusion