Selective inhibition of NADPH oxidase reverses the over contraction of diabetic rat aorta

Atif Ur Rehman,Elma Dugic,Chris Benham,Lisa Lione,Louise S Mackenzie

doi:10.1016/j.redox.2013.12.002

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Abnormal vascular responsiveness in diabetes has been attributed to a number of changes in contractile pathways, affected in part by the overproduction of reactive oxygen species (ROS). It has been reported that NADPH oxidase (NOX) is increased in diabetic (streptozotocin treated; STZ) rat arteries; however the pharmacological agents used to inhibit NOX activity are known to be unsuitable for in vitro studies and have a high level of non-selectivity. Here we have used the highly selective NOX inhibitor VAS2870 in diabetic rat aorta and compared its effects with apocynin, SOD, and allopurinol on phenylephrine and U46619 induced contraction.Male Wistar rats were injected intraperitoneally with 65mg/kg STZ and development of diabetes was confirmed by testing blood glucose levels. Rats were killed by CO2 asphyxiation, and the thoracic aorta removed and mounted in an organ bath under a tension of 1g. Diabetic rat aortas exhibit a greatly increased response to phenylephrine, which was reduced to a level consistent with control rat aorta by 10−5M VAS2870 and 150U/ml SOD. Incubation with VAS2870 led to an increase in normal rat aorta contraction, but led to a significant reduction in phenylephrine and U46619 induced tone in diabetic rat aorta, which indicates that ROS in diabetic rats directly contributes to these contractile responses. Apocynin and allopurinol had no effect on contraction in diabetic or normal rat aorta. This data is the first to show that selective inhibition of NOX reduces diabetic arterial contraction in direct comparison with inhibition of other known contributors of ROS.

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The study of reactive oxygen species (ROS) in vascular biology has been hampered in part by the lack of availability of selective NADPH oxidase (NOX) inhibitors
To ascertain whether the increase in contraction in diabetic rat aorta was due to ROS production, aorta were incubated with 150 U/ml superoxide dismutase (SOD), which did not affect contraction in normal rat aorta (Fig. 1C), but significantly reduced phenylephrine induced in diabetic rat aorta (Fig. 1D)
This study demonstrates that selective inhibition of NOX in the STZ model of diabetes reduces contraction by phenylephrine to a level consistent with control aortas

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Introduction

The study of reactive oxygen species (ROS) in vascular biology has been hampered in part by the lack of availability of selective NADPH oxidase (NOX) inhibitors. There are a range of non-selective modes of action attributed to apocynin, including inhibition of RhoA [13] and direct action as an antioxidant in vascular smooth muscle cells and endothelial cells [7], which taken together has called into question the use of this inhibitor [7,13]. Vascular damage induced by diabetes has been attributed to the increased production of ROS by NOX [11,17], the pharmacological evidence relies on NOX inhibition with apocynin. We used inhibitors of ROS production, VAS2870, apocynin, superoxide dismutase (SOD) and the xanthine oxidase inhibitor allopurinol on phenylephrine and U46619 induced contraction in diabetic and control rats

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