Here we describe further experiments t
o supp
ort
our hyp
othesis that bidirecti
onal 11
x3b2;-HSD1-dehydr
ogenase in Leydig cells is a NADP(H) regenerating system. In the absence
of andr
ostenedi
one (AD), substrate f
or 17
x3b2;-HSD3, incubati
on
of Leydig cells with c
ortic
oster
one (B)
or several C
19- and C
21-11
x3b2;-OH-ster
oids, in the presence
of [
3H]-11-dehydr
o-c
ortic
oster
one (A), stimulated 11
x3b2;-HSD1-reductase activity. H
owever, in presence
of 30 μM AD, test
oster
one (Tes
o) synthesis is stimulated fr
om 4 t
o 197 pic
om
ole/25,000 cells/30 min and c
onc
omitantly inhibited 11
x3b2;-HSD1-reductase activity, due t
o c
ompetiti
on f
or the c
omm
on c
ofact
or NADPH needed f
or b
oth reacti
ons. Test
o pr
oducti
on was further significantly increased (p < 0.05) t
o 224–267 pic
om
ole/25,000 cells/30 min when 10 μM 11
x3b2;-OH-ster
oids (in additi
on t
o 30 μM AD) were als
o included. Similar results were
obtained in experiments c
onducted with l
ower c
oncentrati
ons
of AD (5 μM), and B
or A (500 nM).
Incubations of 0.3–6.0 μM of corticosterone (plus or minus 30 μM AD) were then performed to test the effectiveness of 17x3b2;-HSD3 as a possible NADP+ regenerating system. In the absence of AD, increasing amounts (3–44 pmol/25,000 cells/30 min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30 μM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3–5-fold producing 4–210 pmol/25,000 cells/30 min of 11-dehydro-corticosterone. We conclude that 11x3b2;-HSD1 is enzymatically coupled to 17x3b2;-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems.