Diabetes-induced Expression of iNOS in Rabbit Parotid Artery

OBJECTIVES: Expression of iNOS is critical determinant in the development of diabetes complications in humans. Evidence suggests potential importance of iNOS in salivary gland hypofunction associated with Sjögren's syndrome (Correia et al., 2010) and diabetic periodontal disease (Pan et al., 2010), but there are no data concerning the involvement of iNOS in vascular dysfunction in diabetic salivary glands. METHOD: Diabetes was induced by a single i.v. injection of alloxan ( 100 mg/kg ) in physiological saline solution. After 6 weeks of alloxan or saline administration, animals were sacrificed and rabbit parotid artery, branch of external carotid artery that supplies parotid gland was immediatly frozen in liqiud nitrogen. Selection of appropriate endogenous control was made by TaqMan Real-time RT-PCR in arterial rings from diabetic and nondiabetic rabbits.Expression stability was analyzed by GeNorm and Normfinder software packages. Quantification of iNOS expression was measured on ABI 7500 Real-time RT-PCR system usimg Applied Biosystems Taqman gene expression assay for iNOS in a MicroAmp 96 well optical plate.Results were analyzed using the comparative 2-DDCt method where Ct is the cycle threshold number. RESULTS: The variability in expression of the four endogenous controls, assesed by real-time RT-PCR and analyzed by GeNorm and NormFinder, identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a gene with the lowest stability value.Quantitive real-time RT-PCR analysis showed hardly any iNOS mRNA in nondiabetic rings. However, in diabetic rings there was 2.06 fold increase in iNOS expression. CONCLUSIONS: GAPDH was identified as the most suitable reference for studying alteration in iNOS gene expression in diabetic rabbit parotid gland feeding arterial rings. In this arterial preparations, iNOS expression was increased and it could be speculated that iNOS is involved in the pathogenesis of salivary hypofunction during diabetes. Study was supported by Ministry of Science, Serbia No 175021