Nitric oxide synthase is not expressed, nor up-regulated in response to cold acclimation in liver or muscle of threespine stickleback (Gasterosteus aculeatus)

Abstract

There are three isoforms of the enzyme nitric oxide synthase (NOS) in mammals: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). All three isoforms oxidize arginine to citrulline in a reaction producing nitric oxide (NO), which regulates multiple signaling pathways and physiological functions in mammals. Less is known about NOS in fishes, in which the existence of eNOS is controversial. Nevertheless, multiple adjustments occur during cold acclimation of fishes, several of which are known to be mediated by eNOS and NO in mammals, including mitochondrial biogenesis, vasodilation and angiogenesis. We hypothesized that if NOS was present, and NO stimulated these pathways in fishes, then the activity of NOS would increase during cold acclimation. To test this hypothesis, we measured the activity and mRNA levels of NOS in three tissues (liver, oxidative muscle, glycolytic muscle) known to undergo mitochondrial biogenesis and/or angiogenesis. Measurements were made in the threespine stickleback, Gasterosteus aculeatus acclimated to either warm (20°C) or cold (8°C) temperature for 9weeks. Cold-acclimated fish were harvested on days 1–3, and at weeks 1, 4 and 9 at 8°C, while warm-acclimated fish were harvested on day 0 and after 9 weeks at 20°C. Transcript levels of NOS were quantified using quantitative real-time PCR, and NOS activity was measured using a radiochemical assay, which detected the rate of catabolism of 14C-labeled arginine. Neither NOS activity nor transcripts were detected in oxidative muscle or glycolytic muscle of warm- or cold-acclimated stickleback, although transcript levels of nNOS and NOS activity were detected in brain. Arginine catabolism was detected in liver of animals held at 10°C and 20°C for 9weeks, but was due to arginase activity, rather than NOS. Consistent with this, NOS transcripts were undetectable in liver. The absence of NOS in liver and muscles of stickleback indicates that signaling molecules other than NO likely mediate physiological changes during cold acclimation in stickleback.