Endocrinol.Japon.
1978,25(5),
Effects the
423-430
of Ethacrynic Acid and Furosemide Hormone-Mediated Adenylate Cyclase Activation of the Hamster Kidney FUMIAKI MARUMO
on
AND TAKAO MISHINA
Department of Medicine, of Medicine, Sagamihara,
Kitasato University, School Kanagawa-ken 228, Japan
Synopsis The effects of ethacrynic acid and furosemide on the adenylate cyclase activity of the hamster kidney and heart were examined. Ethacrynic acid and furosemide suppressed the vasopressin-mediated increment of adenylate cyclase activity much stronger than the basal activity of the renal medulla. Ethacrynic acid and furosemide strongly suppressed the parathyroid hormone-mediated increment of adenylate cyclase activity on the renal cortex, though furosemide could not inhibit the basal activity of the enzyme. Ethacrynic acid and furosemide suppressed the 1-adrenaline-mediated increment of adenylate cyclase activity of the cardiac muscle, while furosemide could not inhibit the basal activity. Ethacrynic acid and furosemide suppressed the NaF-mediated increment of adenylate cyclase activity of the renal medulla. Thus, these diuretics seem to inhibit the catalytic unit of the enzyme. Ethacrynic acid and furosemide strongly suppressed the hormone-mediated increment of the adenylate cyclase activity, while Ca** could only depress it to the same degree as it did the basal activity. This difference suggests that these diuretics inhibit not only the catalytic unit of the enzyme, but also other steps of the adenylate cyclase system. Ethacrynic acid and furosemide suppressed the effect of 5'-guanylylimido-diphosphate [Gpp(NH)p] on the adenylate cyclase activation and strongly inhibited the enzyme activity by using the combination of vasopressin and Gpp(NH)p. These results might suggest that ethacrynic acid and furosemide not only affect the catalytic unit of the adenylate cyclase, but also the GTP sensitive site of the enzyme system.
The adenylate cyclase system is a key control point in the action of biogenic amines, prostaglandins, and peptide hormones, such as vasopressin and parathyroid hormone. However, the adenylate cyclase system is sensitive not only to hormones, but also to other agents such as diuretics. Ethacrynic acid was reported to block the hydroosmotic effect of vasopressin, but not of exogenous cyclic AMP in the toad bladder membrane (Bentley, 1969) or in the microperfused collecting ducts of rabbits (Abramow, 1974). Recently, several obReceived
October
25, 1977.
servations indicating that ethacrynic acid inhibited the renal adenylate cyclase activity were reported (Barnes et al., 1975; Ebel, 1974). In the early stage of the present study, we found ethacrynic acid and furosemide could strongly inhibit the vasopressinmediated increment of adenylate cyclase activity of the renal medulla of hamster, while they only slightly inhibited the basal activity of the enzyme. Therefore, this study was planned to explain the reason why the diuretics gave different actions between the basal activity and vasopressin activation of the adenylate cyclase.
424
MARUMO
AND
Tris. assay.
Methods Female were
golden
used.
rated
after into
in
each
gently
50
mM
in
genizer
in
the
centrifuged tates
were
(pH
7.4)
binding
assay
was of
(1970). was
incubation
buffer
(pH
bovine
serum
ATP-2Na.
The
gation
pg
cubation
mixture
incubated
for
50
of
kinase 200ƒÊl
the
enzyme, 3H-cyclic
and
free
cyclic
cyclic
was
Lowry
et
was
the
found
little mM)
the
The
on
test and
mone
(Lilly
substances,
were enzyme
obtained
added
mixed.
a crude
extract
parathyroid
of
by
and
furosemide
then
adjusted
our
pH
dissolved
renal
7.4
by
but M
could (p