11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone

The 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11βHSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11βHSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 ± 3.1 nM, and that for NAD+ was approximately 8 μM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity, 11α-Hydroxyprogesterone (11αOH-P) was an order of magnitude more potent a competitive inhibitor of the 11βHSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 0.9 vs. 15 nM). 11βOH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5α-pregnandione and 5β-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11αOH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11αOH-P was not metabolized by 11βHSD-2. We were unable to demonstrate the presence of 11αOH-P in human urine. In conclusion, a cell line stably transfected with the rat 11βHSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11αOH-P was found to be a potent relatively specific inhibitor of the 11βHSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11βHSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme.Fil:Cozza, E.N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Aldosterone biosynthesis in the rat brain

Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain mince, with [1,23H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 μM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.Fil:Cozza, E.N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

The DELPHI detector at LEP

DELPHI is a 4π detector with emphasis on particle identification, three-dimensional information, high granularity and precise vertex determination. The design criteria, the construction of the detector and the performance during the first year of operation at the large electron positron collider (LEP) at CERN are described. © 1991 - Elsevier Science Publishers B.V. (North-Holland)