Quantitative determination of nitric oxide production in haemocytes: Nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae

Abstract

The generation of nitric oxide by Galleria mellonella larvae haemocytes has been investigated. For this purpose, a fluorescent method, specific for detection of NO, has been developed. The method is based on the application of fluorescence probe DAF-FM diacetate and nitronyl nitroxyl radical, NNR, which accelerates the NO-dependent formation of fluorescence product, DAF-FM triazole. The key feature of the method is the registration and analysis of differential kinetics, namely, the difference of kinetics obtained in samples with NNR and without NNR. This approach allows us to exclude any other kinetic processes not related to triazole formation. The differential kinetics were calibrated versus NO generation rate and the resulting low limit of method sensitivity was obtained as about 0.4–0.5nM/min. The generation of nitric oxide by the haemocytes of insects treated with LPS in vivo has been detected at a rate of 0.5–0.7nM/min. However, the production of NO in haemocyte suspensions treated with both the substrate, l-arginine, and the inhibitor, l-NAME, of NOS, has not been detected within method sensitivity. These data provide only the upper level of NO generation by haemocytes but cannot be used to draw definite conclusions about NOS as a source of NO. Meanwhile, it is known, that NO can be formed by NOS independent mechanism. Indeed, we have observed a significant increase in NO generation in the samples of haemocytes intracellularly loaded with nitrite. Moreover, adding nitrite to lysed haemocytes results in the higher NO generation rate. After addition of 500μM nitrite, the rates of NO generation in the samples are determined to be 2 and 20–30nM/min, respectively. The nitrite/nitrate content of haemocytes and lymph were found to be 5 and 50μM, respectively. The detected nitrite reduction activity of haemocytes allowed us to estimate the generation rate of nitric oxide as 0.05–0.5nM/min from endogenous nitrite. It is thus assumed that the observed nitrite reduction activity in haemocytes is dominant in the increased NO production during immune response of the G. mellonella larvae.