Vol.
179,
No.
September
2, 1991
16,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1991
979-984
Eleanor Davies, Christopher R.W. Edwards and Brent C. Williams Department Received
July
21,
of Medicine, Western General Hospital, Crewe Road, Edinburgh EH4 ZXU, U.K. 1991
To investigate the role of calcium as a second messenger in serotonmstimulated aldosterone secretion, radiolabelled calcium influx studies were carried out in purified rat adrenal zona glomerulosa cells using 45CaCl2. The results show that serotonin caused calcium influx within 45 seconds of addition and this continued for up to 105 seconds. Angiotensin II also caused calcium influx; however, the effect was significantly smaller than that of serotonin. Serotonin-stimulated calcium influx could be inhibited by the calcium antagonist verapamil and by methysergide, a selective serotonin receptor type-l/2 antagonist. The data indicate that serotonin directly stimulates calcium uptake in zona glomerulosa cells via calcium channels which are coupled to specific serotonin receptors. B 1991Academic Press, Inc.
The stimulatory known (1, 2). remains 5-HT2 AMP
action of 5-HT on aldosterone
However, the cellular mechanism
unclear, although receptors
secretion
serotonin-stimulated -chelating
several studies have indicated
(3, 4, 5, 6). aldosterone
Although
the presence of specific
cyclase and increase cyclic
the role of extracellular
secretion has been studied indirectly
agents such as EGTA and Ca*+
direct studies using radiolabelled
in vitro is well
by which the effect is mediated
which appear to activate adenylate
rat adrenal zona glomerulosa
secretion
Ca2+
antagonists
Ca2+
in
using Ca2+
such as verapamil
(7),
have not been carried out. Using purified
cells, this study investigates
the role of Ca2+ influx in
Abbreviations:
ANOVA; analysis of variance. 5-HT; serotonin. EDTA; 1,2-Di (2-aminoethoxy) ethane tetra-acetic acid. SEM; standard error of mean. Ca2+; calcium. PI; phosphatidylinositol. IP3; inositol 1,4,5, tri-phosphate. ACTH; adrenocorticotropic hormone. Ci; Curie. Cyclic AMP; adenosine 3’, 5’-cyclic monophosphate.
919
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All righrs of reproduction in any form reserved.
Vol.
179, No. 2, 1991
5HT
stimulated
BIOCHEMICAL
aldosterone
effect of 5HT
secretion
with that of angiotensin
secretion through activation from intracellular
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by using 45CaC12.
It also compares
the
II, which is known to increase aldosterone
of phospholipase
C and subsequent
release of Ca*+
storage sites (8). MATERIALS
AND METHODS
Rat adrenal zona glomerulosa cells were isolated from female Wistar rats by collagenase (Worthington Biochemical Corp, U.S.A.) digestion according to previously published methods (2). After isolation the zona glomerulosa cells were purified by percoll density gradient centrifugation according to the method of McNamara et a/ 1980 (9). Ca *+ influx was measured using a modified version of the method of Kojima and colleagues (10). Briefly, 285 ul of the cell suspension (106 cells) was added to 15 pl of 45CaCl2 (5 @i) (Amersham International, Aylesbury, U.K.), 100 ~1 of Krebs Ringer and 100 pl of either 5-HT (creatinine sulphate complex; Sigma Chemical Company Ltd, Poole, U.K.) or angiotensin II (Universal Biologicals, Cambridge, U.K.). Angiotensin II and 5-HT were dissolved in Krebs Ringer to the appropriate concentration. Control tubes contained 200 ul of Where utilised, 50 ul of Krebs Ringer in addition to the cells and 45CaCl2. verapamil (Abbott Laboratories Ltd, Kent, U.K.) or methysergide (Sandoz Pharmaceuticals, Middlesex, U.K) was pre-incubated with the cells for 30 minutes at 370C before addition of the stimulus and the volume of Krebs Ringer was adjusted accordingly so that the final volume of each incubation was 500 pl. The specific activity of the 45CaCl2 was lo-40 mCi/mg Ca*+. A 100 ul aliquot of the incubation was removed at 15, 45, 75 and 105 second intervals. This was immediately diluted in 4 ml of ice cold Tris washing solution, containing 144 mM NaCI, 5 mM CaC12, 5 mM Tris/HCI, pH 7.4, filtered through a Whatman GF/C glass-fibre filter (Whatman International Ltd, Maidstone, U.K.) and washed a further 3 times with the washing solution. The 45Ca*+ taken up by the cells, i.e. that associated with the filter, was counted in a p-counter using Cocktail-T liquid scintillation fluid (BDH Ltd, Poole, U.K.).
RESULTS All results are expressed expressed l&i.
as a % uptake of the total radioactivity
Statistical
Range Test. Fiaure
significance
was calculated
Radiolabelled
Ca*+
influx is
added to each incubation
i.e. 5
using 2-way ANOVA and Duncan’s
A P value of less than 0.05 was considered significant.
1 shows the uptake
unstimulated Compared
as mean + SEM.
cells (control)
of 45Ca*+
over a 105 second
and those stimulated
time
course
with 5-HT or angiotensin
to the control group, 5-HT caused a significant uptake of 45Ca*+
45 seconds of addition
(PcO.01).
The uptake continued
II. within
at 75 seconds (PeO.01)
and up to the final point analyzed which was 105 seconds (PcO.01). 980
by
Angiotensin
II
Vol. 179, No. 2, 1991
BIOCHEMICAL
--IF
Control
-O-
MiT(10.6Y)
-A-
Anglotensln
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
II (IO-%)
0.0 0
20
40
60
Time
60
100
120
(seconds)
Fiaure 1 shows the effect of no stimulus (control), 5-HT or angiotensin II on 45Ca2+ uptake in isolated purified rat adrenal zona glomerulosa cells. The stimuluswas applied at time 0. Values are means+ SEM (n=5 / group).
also caused a significant
increase
in 45Ca2+
much slower and was not statistically Fiaure
2 shows the uptake
unstimulated the presence
However, the effect was
significant until 105 seconds (PcO.05).
of 45Ca2+
cells (control), cells stimulated of the Ca2+ antagonist
uptake.
over a 105 second
time
with 5HT, cells stimulated
verapamil
and cells stimulated
course
with 5-HT in with 5-HT in
2.0 -
15-
--a-
Control
-c-
5HT(1C&)
-A+
5-MT (lOaM) 5-HT (lObY)
+ verapamll(lZ.5 + methysfqide
Time
PM) (106M)
(seconds)
Figure 2 shows the effect of no stimulus (control, n=5 , 5-HT (n=5), 5-HT plus verapamil (n=3) and 5-HT plus methysergide(n=3) on b5Ca2+ uptake in isolated purified rat adrenal zona glomerulosacells. The stimuluswas applied at time 0, verapamil and methysergide were pre-incubated with the cells for 30 minutes before addition of the stimulus. Values are meansf SEM. 981
by
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the presence of the 5-HTt/2 receptor antagonist methysergide. control group, 5HT
caused a significant uptake of 45Ca2+
significance at 75 (P