METABOLISM OF DEXAMETHASONE IN THE HUMAN KIDNEY - NICOTINAMIDE ADENINE DINUCLEOTIDE-DEPENDENT 11-BETA-REDUCTION

Recently, two distinct isoenzymes of 11 beta-hydroxysteroid-dehydrogen ase (11 beta-HSD) have been cloned and characterized in several specie s: The isoenzyme 11 beta-HSD-I is widely distributed, bidirectional, p refers NADP(H) and has a low substrate affinity. The isoenzyme 11 beta -HSD-II seems to exclusively oxidize physiological glucocorticoids, us es NAD as cosubstrate, has high substrate affinity, and is only found in mineralocorticoid target tissues and the placenta. Synthetic steroi ds fluorinated in position 9, however, sue rapidly reduced by human ki dney cortex slices. We attempted to find out which isoenzyme is respon sible for this unexpected reductase activity. We studied the 11 beta-H SD activity towards cortisol (F)/cortisone (E) and dexamethasone (D)/1 1-dehydro-dexamethasone (DH-D) in microsomes prepared from human kidne y cortex. For the reaction E to F (not for DH-D to D!), glucose-g-phos phate and glucose-6-phosphate-dehydrogenase had to be added as a NADH/ NADPH-regenerating system. Oxidation off to E:NAD was the exclusively used cosubstrate; the affinity [Michael's constant (K-m) for F = 25.5 nmol/L and the maximum velocity (V-max = 22.9 nmol/mg/min) were high. Reduction of E to F:Without the NADH/NADPH-regenerating system, this r eaction was very slow. With this system, the K-m value for E was in th e nanomolar range (80.6 nmol/L) and the V-max value was very low (0.88 nmol/mg/min). The reaction was clearly NADH-preferring. For the stero id pair F/E, the quotient V-max(oxidation)/V-max(reduction) (=26) demo nstrates an equilibrium far on the 11-keto side. Oxidation of D to DH- D: With NAD as the only used cosubstrate, the kinetic analysis is comp atible with the existence of two different NAD-dependent isoenzymes: K -m for D = 327 nmol/L, V-max = 53.5 nmol/mg/min and K-m For D = 81.2 n mol/L; V-max = 20.4 nmol/mg/min. Reduction of DH-D to D: The maximum v elocity was higher than that of all other reactions tested: V-max = 22 6.0 nmol/mg/min, The reaction was exclusively NADH-dependent; the K-m value for]DH-D was 68.4 nmol/L. Far D/DH-D, the ratio V-max(oxidation) /V-max(reduction) was 0.24, demonstrating a shift to reductase activit y with the reaction equilibrium far on the Il-hydroxy side. The reacti on F to E was inhibited by E, DH-D, and D in a concentration-dependent manner, In conclusion the cosubstrate dependence, the K-m value of th e oxidation off and the product inhibition are in good correspondence with data for the cloned human 11 beta-HSD-II. The NADH-dependent 11 b eta-reduction of E and especially of DH-D are inconsistent with the do gma of an unidirectional 11 beta-HSD-II, The preference of D for the r eductase reaction in human kidney slices is probably caused by the flu or atom in position 9, is catalyzed by 11 beta-HSD-II, and leads to an activation of II-DH-D to D in the human kidney.