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

Serotonin stimulates calcium influx in isolated rat adrenal zona glomerulosa cells.

To investigate the role of calcium as a second messenger in serotonin-stimulated aldosterone secretion, radiolabelled calcium influx studies were carr...
358KB Sizes 0 Downloads 0 Views