Generation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase isozymes in the perfused mouse hindlimb
AR Dover1,2, PWF Hadoke2, E Miller2, DE Newby1 & BR Walker2
Glucocorticoids (cortisol in man, corticosterone in rodents) can inhibit angiogenesis, alter contractile function and reduce the inflammatory response to injury in the vascular wall. These effects are regulated by the 11beta-hydroxysteroid dehydrogenases (11HSDs) which inter-convert active glucocorticoids and their inactive 11-keto metabolites (cortisone; 11-dehydrocorticosterone) within target tissues. 11HSD2 is a unidirectional, exclusive dehydrogenase which inactivates glucocorticoids whereas 11HSD1 is considered a predominant reductase in vivo. However, we have recently demonstrated significant 11HSD1 dehydrogenase activity in intact mouse aortic rings. We have developed a perfused mouse hindlimb preparation to determine whether 11HSD1 is a predominant reductase in an intact vascular bed in situ. The abdominal aorta of male, C57Bl6 mice was cannulated for perfusion with oxygenated Krebs-Henseleit solution (37 degC); perfusate was collected via the vena cava. 11beta-Reductase and dehydrogenase activities were determined by infusing 5nM [3H4]-11-dehydrocorticosterone or [3H4]-corticosterone, respectively, and measuring conversion to the relevant product. To determine the relationship between substrate concentration and rate of enzyme activity for 11beta-reductase, [3H4]-11-dehydrocorticosterone was infused at concentrations of 5 -500nM. After 25 minutes perfusion, both [3H4]-corticosterone (2.72plus/minus0.16pmol/min) and [3H4]-11-dehydrocorticosterone (0.24plus/minus0.04pmol/min) were generated. From kinetic studies, the calculated Km for 11beta-reductase activity under these conditions is 1.064 micromolar. Production of [3H4]-corticosterone was abolished in 11HSD1-deficient mice. These results demonstrate that 11HSD1 is the sole reductase under these conditions. Furthermore, the comparison of reductase and dehydrogenase activities indicates that 11HSD1 is acting predominantly in the reductase direction. This novel technique for measurement of 11HSD activity will allow future investigation of the influence of 11HSD1 activity on angiogenesis, vascular function and inflammation.