Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C56
Signalling pathways involved in the superoxide anion production in response to serotonin in rat intrapulmonary arteries
M. Billaud1, N. Khoyrattee1, R. Marthan1, J. Savineau1, C. Guibert1
Introduction: We previously showed that (1) serotonin (5-HT) produces superoxide anion (O2●-) in the smooth muscle and NO in the endothelium from rat intrapulmonary arteries (IPA); (2) O2●- diffuses through the myoendothelial junctions to decrease endothelial NO production and thus strengthens pulmonary vasoreactivity (Billaud et al. 2009). In the present study, we focused on (1) the sources of O2●- and (2) the role of calcium in the production of O2●- in response to 5-HT in the smooth muscle of normal rat IPA. Methods: Wistar male rats were humanely killed according to national guidelines. Levels of O2●- were recorded by electron paramagnetic resonance in half of the intrapulmonary arterial tree. Implication of 5 HT receptors and 5 HT transporter (5HTT) was assessed using blockers of the 5-HT1 and 2A receptors (GR 127935 and ketanserin) and citalopram, respectively. Various inhibitors of possible sources of O2●- were also used (namely apocynin for NADPH oxidases, rotenone and antimycin A for the complexe I and III of the mitochondrial respiratory chain respectively and allopurinol for the xanthine oxidase). An ELISA SOD Assay kit-WST was used to address the superoxide dismutase (SOD) activity, an enzyme which degrades O2●-. All results are expressed as the mean ± S.E.M. Significance was considered when P<0.05 and tested with a non parametric test (Mann-Whitney) for unpaired samples and n represents the number of rats. Results: 5-HT (100 µM) significantly increased O2●- level in rat IPA (8916.9 ± 850.4 arbitrary units per mg.ml-1 of proteins in response to 5-HT vs 4648.9 ± 793.2 in basal conditions, n = 8-10). Phenylephrine (10 µM), endothelin-1 (0.1 µM) and angiotensin II (10 µM) other pulmonary vasoconstrictors had no effect on O2●- level (n = 4-8). Whereas, ketanserin (1 µM) significantly decreased the amount of O2●- produced by 5-HT (n = 11), GR 127935 (1 µM) or citalopram (1 µM) had no effect (n = 11-12). Apocynin (30 µM) or rotenone (5 µM) strongly reduced the 5-HT-induced O2●- increase (n = 9-11) but antimycin A (10 µM), or allopurinol (50 µM) induced no significant inhibition (n = 10-13). 5-HT did not modify SOD activity (n = 8). Removal of extracellular calcium partially reduced O2●- level produced by 5-HT (6309.8 ± 585.8, p<0.05, n = 13) whereas thapsigargin 1 µM which depletes intracellular calcium stores had no significant effect (n = 10). Conclusion: The present study shows that production of O2●- by 5-HT involves 5-HT2A receptors and extracellular calcium. Among known sources of O2●-, NADPH oxidase and complexe I of the mitochondrial respiratory chain seem to be potentially good candidates for this production of O2●- by 5-HT.
Where applicable, experiments conform with Society ethical requirements