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[DES-ASP1] angiotensin II in the sheep: Blood levels and its effect on plasma renin concentration
Life Sciences, Vol. 27, PP. 1495-1502 Printed in the U.S.A.
Persamon Press
[DES-ASpI]ANGIOTENSIN II IN THE SHEEP: BLOOD LE~VELS AND ITS EFFECT ON PLASMA RENIN CONCENTRATION ] David T. W. Fez, Wllllam F. Graham, John G. McDougal Bruce A. Scogglns, John P. Coghlan. Howard Florey Instltute of Experimental Physlology and Medlclne, Unlverslty of Melbourne, PARKVILLE, VIC. 3052 AUSTRALIA (~eceived in final form
AuEust
/i, 1980)
Summary The present study describes an improved method for measurlng anglotensln III in arterial blood. This was accomplished by SE-sephadex column to separate angzotensln II from anglotensln III prior to radlolmmunoassay. The arterial concentration of anglotensln III measured before and after 24 to 48 hours sodium depletlon by acute cannulatlon of parotld gland was 12.4 ± 1.7 fmol/ml (SEM, n=7) and 49.8 ± 10.3 fmol/ml (SEM, n=7) respectlvely. The arterial concentratlon of Val~-anglotensln III obtained from continuous infusaon of Val%-anglotensln III at rates of 24 and 48 nmol/h in sodium deficient sheep were 245 ± 32.5 fmol/ml (n=6) and 330 Z 11.4 fmol/ ml (n=7) respectlvely. The clearance rate of exogenous Val~-anglo tensin III in sodium deflclent sheep after correction for endogenous level was calculated to be 140 ± 13.6 L/h (SEM, n=13). This was in the same order as IleS-anglotensln II and Ile~-anglotensln III reported earlier in sodium replete sheep Prolonged intravenous infusion of Val~-anglotensan III at a rate of 48 nmol/h an sodzumdef~clent sheep suppressed plasma renin concentration to the same extent as equimolar infusions of anglotensln II. Thls suggests that anglotensln III may ~nhLblt renln secretion by a slmilar mechanlsm to ang~otensln II. It has been suggested that [des-Aspl]anglotensln II (anglotensln III) may be an important mediator of various responses to angiotensln II (1-4). Both peptldes were known to have steroldogenlc and pressor activity (5). In sheep it has been quantitatlvely demonstrated that substantial production of both anglotensln II and anglotensln III occurs in the perlpheral vascular bed and that the level of anglotensln III zn arterial blood is about 42% of that of anglotensln II (6). Thls level of anglotensln III, albeit lower than anglotensin II, may be of physlologlcal Importance, especially for cells such as those In the adrenal zona glomerulosa whlch have been reported to be more sensltlve to anglotensln III than anglotensln II In aldosterone productlon during sodium deflclency (7). Thls study reports the effects of anglotensln III on plasma renln concentration in consclous, sodium replete and sodlum deplete sheep and compares thls wlth the effect of anglotens~n II Earlier ~mmunologlcal evldence
1.
Thls work was supported by the Natlonal Heart Foundatlon of Australla.
0024-3205/80/421495-08502.00/0 Copyrlght
(c) 1980 Pergamon Press Ltd.
1496
Angiotensln III in Arterial Blood
V01. 27, No.
16, 1980
(8[ has shown that in sheep the amino acid in posltlon 5 of anglotensln II is vallne Hence, ValS-anglotensln II and Val~-anglotensln III were used as standards and for lnfuslon in the present study Materlals and Methods Experlments were performed on Ii mature, conscious Merino crossbred sheep, weighing 33 to 40 kg. All animals were prepared wlth exteriorized carotld arteries. Four had a unilateral parotld fistula (9) Animals were rendered sodium deflclent by uncompensated loss of saliva for 24 to 48 hours Sodium repletlon was monitored by measurlng the potassium concentratlon in sallva, a value of 6 mmol/l or less being taken as representative of sodium repletlon. Arterial samples were taken from carotld artery for determlnatlon of anglotensln III before and after sodium depletion. Incremental infuslons of Va!~-anglotensln III Seven sheep were sodlum-depleted by acute cannulat~on of the right parotld sallvary duct 24 to 48 hours before the experlment (i0). Val~-angloten sln III (Peninsular Lab ) was infused through polyethylene chlorlde cannula (internal diameter of 1 mm) wlth its tlp posltzoned in the 3ugular veln, at 24 and 48 nmol/h, each for 30 minutes Blood samples were taken from the carotid artery for determination of Val4-anglotensln III and plasma renln concentration (PRC) at 0 and 30 minutes after the commencement of each infuslon rate. Prolonged infusion of ValS-anglotensln II and Val~-anglotensln III In a second series of experiments, four sheep which were prepared wlth a unilateral parotld flstula were sodlum-depleted by wlthholdlng access to sodlum blcarbonate solutlon for 24 to 48 hours. These animals were then infused intravenously wlth ValS-anglotensln II for 4 hours at a rate of 48 nmol/h. Blood samples were taken into precooled sequestered tubes contalnlng 50 mM EDTA at times 0, 15, 30, 60, 120, 180 and 240 minutes after the commencement of infuslons for determination of PRC. Two weeks after the flrst experiment the identlcal protocol was repeated on the same 4 anlmals uslng Val~-anglotensln III as infusate. Determination of plasma renln concentratlon Plasma renln concentration was determined by radlolmmunoassay uslng the approach of antibody capture method described by Poulsen and Jorgensen (ii). Thls involved the addltlon of 50 ~i of exogenous renln substrate, obtained from nephrectomlzed sheep plasma (wlth an anglotensln I concentration of 2,300 pmol/ml). 30 ~i of unknown plasma sample was then added together wlth 20 ~i of a concentrated anglotensln I antlbody and incubated at 37°C for 30 minutes At the end of incubatlon, 1.5 ml of izSI-anglotensln I (5,000 cpm) Was added The final antibody dllutlon was 1 40,000. The anglotensln I generated was determined by radlolmmunoassay and was expressed as pmol of anglotensln generated per hour per ml of plasma The antibody used in the present study had negligible cross-reactlvlty wlth the exogenous renln substrate and minimally wlth anglotensln II and its smaller metabolItes at 1 pmol (<0.2%) Determlnatlon of blood Val~-an~lotensl ~ III Val%-anglotensln III was extracted from blood and determlned by radloimmunoassay as described in detail elsewhere (12), wlth two mod~flcatlons as reported recently (13). Blood was collected into absolute ethanol (V/V = 3 7) to stop instantaneously the degradatlon of anglotenslns by serum amlnopeptldases. About 1,000 cpm of trltlum-labelled anglotensln III (from New England
Vol.
27, No.
16, 1980
AngiotensiD
Ill in Arterial
Blood
1497
Nuclear Corp Boston, wlth speclflc actlvlty of 41,2 Cl/mmol ) was then added to each sample as internal marker to estlmate losses during extractlon. The ethanollc blood mlxture was centrlfuged at 3,000 rpm for I0 mlnutes. The supernatant was washed wlth ether twlce and acldlfled to pH 3 before passlng down SE-sephadex column (150 mm in hezght and 9 mm in dlameter) whlch was equlllbrated In 0.05M acetlc acld (pH = 3). Using 0.1M phosphate buffer (pH = 7.4) as eluent, ang~otensln III was eluted and radlolmmunoassayed by means of an anglctensln II antlbody. After substractlng the mass of tracer and correctlng for losses durlng extractlon, the concentration of endogenous anglotensln III was then quantltatlvely determined. Statlstlcal
analysls
Unless otherwlse stated, all results were expressed as mean ± standard error of the mean. Student's test for palred observatlons was used for statlstlcal comparlsons. Results PRC assay The intra-assay and inter-assay var~atlons in the determlnatlon of one ~I U.W.H.O human renln (obtalned from the Instltute for Medlcal Research, Holy HIll, London) were 2.7% (C.V., n=27) and 11.8% (C.V., n=28) respectlvely. The generation of anglotensln I over a perlod of two hours at 37°C by varylng
WHO Renin /~IU
o
100
0 t
0
,
i
i
3O
60
I
I
120
Incubation- minutes Fig.
1
Reactlon rate of sheep renan substrate wlth known amount of WHO human renln. The generatlon of anglotensln I wlth varylng amount of WHO human renln (ranglng from 0.3 to 0.8 ~ I.U.) over a perlod of two hours at 37°C was obtalned. Each polnt represents the mean of 2 tubes.
1498
Vol. 27, No. 16, 1980
Angiotensin III in Arterial Blood
amounts of the W.H.O. human renzn (ranglng from 0 3 to 0.8 ~ I.U.) zs shown zn Fig. i. One ~I.U.W.H.O. renln whlch was used as an internal standard generated 191 ± 5 fmol anglotensln I/hour (n=28). Determination of Val~-angzotensln III in blood The elutlon pattern of Val~-angzotenszn III and 3H-anglotenszn III zn the SE-sephadex column is shown zn Fig. 2. Val~-anglotenszn III, wzth zts slower elutlon rate was suffzclently separated from ValS-anglotensln II przor to radzozmmunoassay. When known amounts of Val4-ang~otenszn III (rangzng from 200 to 1,500 fmol) was added to sheep blood, the measured concentratzon after correctlon of losses durzng extractzon bears a good correlatzon to what was added, wlth correlatlon co-effzclent of 0.98. The zntra-assay and Inter-assay varlatlon in determlnlng 500 fmol Val~-angzotensln III zn sheep blood was 10.7% (C.V., n=10) and 12.6% (C.V., n=7) respectlvely.
150
1500 SE - Sephadex
E O
~100
Va=; Aa
l Val!A.T
I
IOO0 .c_ ~ =4 o
I 5O0
z
, D
e"
I
/o ,
0
2
4
8
6
, ~'-~t~- ---,~--- 0
10
12
14
Elution Volume ml Fig. 2 The elutzon pattern of i, 000 cpm of 3H-angzotenszn III (4 4) and 500 fmol of each of Val 5-anglotenszn II and Val -anglotenszn III wlth the SE-sephadex column. The condltlons of elutlon are descrlbed in the methods.
Vol.
27, No.
Effect
16,
1980
Anglotensln
III in Arterzal
Blood
1499
of sodlum d e p l e t l o n
The m e a n sodlum d e f l c l t after 24 to 48 hours u n c o m p e n s a t e d loss of sallva by acute p a r o t l d c a n n u l a t l o n was 352 ± 45 mmol (n=7) Plasma renln c o n c e n t r a t l o n (PRC) d e t e r m l n e d In 7 sodlum-replete sheep was 1.5 ± 0 16 pmol AI/h/ml. A f t e r s o d l u m depletlon, PRC was elevated to 8.2 ± 1.4 p m o l / h / m l (n=7) w l t h range from 4 to 23 p m o l / h / m l The arterial V a l 4 - a n g l o t e n s l n III c o n c e n t r a t i o n was also slgn l f l c a n t l y e l e v a t e d from 12.4 ± 1.7 fmol/ml (n=7) to 49 8 ± 10.3 fmol/ml (n=7) after s o d l u m d e p l e t l o n Incremental
infuslon
of Val
-anglotens&n
III
C o n t l n u o u s intravenous infus~on of Val - a n g l o t e n s l n III to 7 sodlumdeflclent, n o n - f l s t u l a t e d sheep resulted in a slgnlflcant decrease of p l a s m a renln c o n c e n t r a t l o n shown in Fig 3 At an infuslon rate of 24 nmol/h, PRC taken 30 m l n u t e s after the c o m m e n c e m e n t of infusion was d e c r e a s e d from 8.2 ±
~
4OO
24 22
300
B
\
14 12
Arterial Val4-A gl 2O0 fnloI/ml
PRC pmol/h/ml
10 8 6
100 4 2
t Na-replete J
0
30 Time (m,n)
60
Fig.
0
30 6O Time (minutes)
3
The arterlal level of V a l ~ - a n g l o t e n s l n III (A) and p l a s m a renln c o n c e n t r a t l o n (B) d u r l n g intravenous infus~on of V a l 4 - a n g l o t e n s l n III at 24 and 48 nmol/h ~n 6 s o d l u m - d e p l e t e sheep. A n l m a l s were s o d l u m - d e p l e t e d by u n c o m p e n s a t e d sallvary loss t h r o u g h acute c a n n u l a t ~ o n of a p a r o t l d gland.
1500
Angiotensln
III in A r t e r i a l
Blood
Vol.
27, No.
16, 1980
1 4 p m o l / h / m l to 5.3 ± 0 95 p m o l / h / m l , a d e c r e a s e of 34 ± 5.6% from the pre• n f u s l o n values I n c r e a s l n g the I n f u s l o n rate to 48 nmol/h, PRC was d e c r e a s e d further to 3.8 ± 0 63 pmol/h/ml, a drop of 54 ± 4.4% from the p r e - l n f u s ~ o n values. 4 The arterial level of Val - a n g ~ o t e n s l n III m e a s u r e d by the p r e s e n t m e t h o d shown In Flg. 3, was 245 ± 32 5 fmol/ml (n=7) at low dose and 330 ± ii 4 fmol/ml 4 (n=7) at h ~ g h dose of Val - a n g ± o t e n s l n III ~nfuslon A f t e r c o r r e c t l o n of endogenous level of Val - a n g l o t e n s l n III uslng the formula of J o h n s t o n and c o l l e a g u e s (14) IR MCR = PRCI C - CO i PRC 0 where
IR C o C
~s the c o n s t a n t - l n f u s l o n rate ( in pmol/h) is the c o n c e n t r a t l o n of b l o o d Val - a n g l o t e n s l n III (in pmol/L) at 4 c o m m e n c e m e n t of Val - a n g l o t e n s l n III infuslon, is the e q u l l l b r l u m c o n c e n t r a t l o n of b l o o d Val - a n g l o t e n s l n III (in pmol/L) d u r l n g Val - a n g l o t e n s ~ n III infus~on, PRC and PRC are the p l a s m a renln c o n c e n t r a t l o n (in pmol/h/ml) at 0 and 4 o30 m ~ n u t ~ s after c o m m e n c e m e n t of Val - a n g l o t e n s ~ n III ~nfuslons, the m e t a b o l l c c l e a r a n c e rate of e x o g e n o u s V a l 4 - a n g l o t e n s l n III in sod±umd e p l e t e sheep was c a l c u l a t e d to be 140 ± 13.6 L/h (n=13). Prolon@ed
Infuslon of V a l S - a n g l o t e n s l n
II and Val - a n g l o t e n s l n
III
To c o m p a r e the e f f e c t of c l r c u l a t l n g Val - a n g l o t e n s l n III on PRC w i t h 5 that of Val - a n g l o t e n s l n II, an i d e n t ~ c a l p r o t o c o l was a d o p t e d Each peptlde was i n f u s e d i n t r a v e n o u s l y at a rate of 48 n m o l / h for four hours to four sodlumdeflclent, f l s t u l a t e d sheep Table I shows the PRC results after c o m m e n c e m e n t of a n g l o t e n s ~ n infuslons. TABLE
I
Plasma Renln C o n c e n t r a t l o n (pmol/h/ml) after C o m m e n c e m e n t of Infuslon of Val 4a n g l o t e n s l n III and V a l S - a n g l o t e n s l n II in S o d l u m - D e f l c l e n t , F l s t u l a t e d Sheep 4 Val - a n g l o t e n s z n III lnfuslon (48 nmol/h) Time of Infuslon (mlnutes) Anlmal 1 2 3 4 ValS-anglotensln 1 2 3 4
0
12.2 11.8 31.5 75.2 II infusion 13.8 13.7 6.4 15.5
15
30
60
120
7.7 7 8 20.4 75.2
7.5 5 9 19 6 58 9
3.7 3 4 13.5 52 2
2 6 1 5 12 7 30.7
8.9 4.2 1.8 6 5
5.7 4 3 3.4 4.4
180
0 5 i0 32
240
7 4 3 9
1.8 3 0 9.6 40 2
3.8 4 7 2 0 3 0
4 8 3 7 3.1 2 1
(48 nmol/h) I0 3 9.0 4.6 11.9
8.0 6 0 1.9 6.0
The extent of s u p p r e s s l o n of PRC by V a l S - a n g l o t e n s l n II and V a l 4 - a n g l o tensln III after 4 hours i n f u s l o n was i d e n t i c a l (69 ± 8% for V a l S - a n g l o t e n s l n II infuslon and 69 ± 7.3% for V a l 4 - a n g l o t e n s l n III infuslon) as shown in Fig S t a t l s t l c a l analysls shows no s l g n l f l c a n t d i f f e r e n c e of tne two curves.
Vol
27, No.
16,
1980
Anglotensln
III In Arterial
Blood
A
%or
1501
B
IO0
100
80
80
6O
% o f O0 I:~-fn~ Valuee
40
40
20
20
Pm Inlmmn PSC Vakum
T
T
]
0
1
2
3
4
0
1 Tim
Time [hoursl
F~g
2 (houm)
3
4
4
P e r c e n t suppresslon of p l a s m a renln c o n c e n t r a t l o n from p r e l n f u s ~ o n p r o l o n g e d infus~on of V a l ~ - a n g l o t e n s l n II (A) and V a l S - a n g l o t e n s l n 4 sodlum-deflclent, flstulated sheep
values by III (B) in
DISCUSSION The level of anglotensln III in arterial b l o o d relatlve to anglotensln II has been reported to be hlgh in rats (15) but low in dog and man (14, 16) Previous studies (6), uslng recovery m a r k e r and paper chromatography to separate V a l S - a n g l o t e n s l n II from V a l ~ - a n g l o t e n s l n III prior to radlolmmunoassay, have shown that in s o d i u m deflclent sheep the level of V a l ~ - a n g l o t e n s l n III In arterlal b l o o d is 42% of that of anglotensln II. The present study has improved the m e t h o d of m e a s u r l n g anglotensln III in blood reported p r e v l o u s l y (12). Instead of using paper c h r o m a t o g r a p h y to separate Val 4 - a n g l o t e n s l n III from V a l S - a n g l o t e n s l n II, S E - s e p h a d e x column was used to achieve the same end. Thls m o d i f i e d p r o c e d u r e is less tlme consuming but gives higher yield at the end of extraction. The final extract contained 45 to 55% of 3H-anglotensln III added initially, w h e n c o m p a r e d to 30% of yield in the prevlous method. In addition, a n g l o t e n s l n II can be s l m u l t a n e o u s l y d e t e r m l n e d an the same sample using 12sIa n g l o t e n s l n II as an internal m a r k e r which elutes in the same p o s i t i o n as Val 5a n g l o t e n s l n II. The p r e s e n t study conflrms the slgnzfzcant e l e v a t i o n of a n g l o t e n s l n III in arterial b l o o d after s o d l u m depletion. The clearance of exogenous V a l 4 - a n g l o tensln III (140 ± 13.5 L/h) was of the same order as I l e S - a n g l o t e n s l n II and 4 Ile - a n g l o t e n s l n III o b t a z n e d in s o d l u m - r e p l e t e sheep (i0). Thls is zn a~reem e n t w l t h e a r l i e r flndlngs in sheep w h e r e the a r t e r l o v e n o u s ratlo of Val -anglotensln III dld not change b e f o r e and after sodlum d e p l e t l o n (6).
1502
Angiotensln
III in A r t e r i a l
Blood
Vol.
27, No.
16, 1980
It has b e e n r e p o r t e d that a n g l o t e n s l n III is e q u l p o t e n t as a n g l o t e n s l n II on a l d o s t e r o n e s e c r e t i o n in vitro and in vlvo (18, 19). B o t h p e p t l d e s d e c r e a s e d renal b l o o d flow (4, 20) The h y p o t h e s i s has b e e n a d v a n c e d that a n g l o t e n s l n III m a y be a m e d i a t o r of the r e n l n - a n g l o t e n s l n system The p r e s e n t studies have shown that V a l 4 - a n g l o t e n s l n III at an infusion rate of 42 n m o l / h suppress renln s e c r e t i o n to the same extent as e q u l m o l a r i n f u s i o n s of V a l S - a n g l o t e n s l n II. W h e t h e r thls dose r e p r e s e n t s a m a x i m a l i n h i b i t i o n of renln a c t i v i t y and masks the a p p a r e n t d i f f e r e n c e of the two p e p t l d e s remains to be established. The data is in a g r e e m e n t w i t h e a r l i e r o b s e r v a t i o n s in rabbit (21) and dog (4). V a n d o g e n and c o l l e a g u e s (22) also o b s e r v e d a decrease in renln p r o d u c t i o n in the p e r f u s e d rat k i d n e y after a d d i t i o n of a n g l o t e n s l n III to the perfusate. The nature of thls i n h l b l t o r y actlon is not t h o r o u g h l y understood. Recently, C a m p b e l l and c o l l e a g u e s (23) have r e p o r t e d that the s t e r o l d o g e n l c actlon of a n g l o t e n s l n II and III in the adrenal c o r t e x was m e d l a t e d in p a r t by the p r o s t a g l a n d l n s W h e t h e r the release of renln in the kidney is m o d u l a t e d by the same m e c h a n i s m remalns to be l n v e s t l g a t e d REFERENCES i. 2. 3. 4. 5 6 7. 8. 9 i0. ii. 12. 13. 14 15. 16.
17 18. 19 20. 21 22 23.
J.R. BLAIR-WEST, J.P. COGHLAN, D.A. DENTON, J.R FUNDER, B.A SCOGGINS and R.D. WRIGHT, J. C l l n Endocrlnol. Metab , 3__22, 575-578 (1971). T. KONO, F. OSEKO, S. SHIMPO, M NANVO, and J. ENDO, J. Clln. Endo. and Metab., 41, 1174-1177 (1975) W S. SPIELM~N, J.O. DAVIS and R.H FREEMAN, Proc Soc Expt. Biol. Med., 151, 177-182 (1976) R H. FREEMAN, J.O DAVIS, T E. L O H M E I E R and W S. SPIELMAN, C l r c Res., 3 8 (Suppl II) 99-103 (1976) T L. G O O D F R I E N D and M.J PEACH, Clrc Res , 36/37 (Suppl I), 1-38-48 (1975) D.T.W. FEI, J P. COGHLAN, R.T. FERNLEY, B.A S C O G G I N S and G.W. TREGEAR, Clrc. Res , (in press) (1980) R.M CAREY, E.D. VAUGHAN, M J P E A C H and C.R AYERS, J. Clln. Invest., 61, 20-31 (1978). M.D. CAIN, K.J. CATT, J P COGHLAN, J R BLAIR-WEST, E n d o c r l n o l o g y , 86, 9 5 5 - 9 6 4 (1970). D.A. DENTON, Quart J. Exp P h y s l o l , 42, 72-94 (1975) S.F ABRAHAM, R M. BAKER, E.H BLAINE, D.A D E N T O N and M . J McKINLEY, J. C o m p a r a t l v e and P h y s l o l o g l c a l Psychology, 88, 503-518 (1975) K. P O U L S E N and J JORGENSEN, J. Clln. Endo. Metab., 39, 816-825 (1974). D . T W. FEI, J.P COGHLAN, R T. FERNLEY, B.A. SCOGGINS and G.W TREGEAR, Clln. Exp. Pharm. and P h y s l o l , 6, 129-137 (1979) J.P. COGHLAN, D T.W. FEI, T.J HUMPHERY, B.A. SCOGGINS and G.W. TREGEAR, E n d o c r l n e S o c l e t y of Australla, Perth (1979) C.I. JOHNSTON, F A O. M E N D E L S O H N and A E DOYLE, Circ. Res., 30-31 (Suppl II), I 1 2 0 3 - 2 1 2 (1972) P F. SEMPLE and J J MORTON, C i r c Res., 38 (Suppl II), I 1 1 2 2 - 1 2 6 (1976). A M. CARAVAGGI, G BIANCHI, J J. BROWN, A.F LEVER, J.J. MORTON, J.D. P O W E L L - J A C K S O N , J I.S. R O B E R T S O N and P F SEMPLE, Circ. Res , 38, 315-321 (1976). P F. SEMPLE. A S. BOYD, P M DAWS and J.J MORTON, Clrc Res., 39, 6 7 1 - 6 7 8 (1976). A.T. CHIU and M J. PEACH, Proc Natl Acad. Scl. U S.A., 71, 341-344 (1974). W.B. CAMPBELL, S.N BROOKS, W A. PETTINGER, Scl., 184, 9 9 4 - 9 9 6 (1974). S.L. BRITTON, W H. B E I E R W A L T E S , M.J F I K S E N - O L S E N and J. C A R L O S ROMERO, Clrc Res., 44 (5), 6 6 6 - 6 7 1 (1979) J.M. STEELE,--A J. N E N S Y and J. LOWENSTEIN, Circ. Res., 3 8 (Suppl II), i13-i16 (1976). R. VANDOGEN, W S. PEART, G.W BOYD, Am. J. P h y s l o l , 226, 277-282 (1974). W.B CAMPBELL, C E. G R O M E Z - S A N C H E Z , B.V. ADAMS, J.M. ITSKOVITZ, J. Clln Invest , 64, 1552-1557 (1979).
S C H M I T Z and H.D.
Persamon Press
[DES-ASpI]ANGIOTENSIN II IN THE SHEEP: BLOOD LE~VELS AND ITS EFFECT ON PLASMA RENIN CONCENTRATION ] David T. W. Fez, Wllllam F. Graham, John G. McDougal Bruce A. Scogglns, John P. Coghlan. Howard Florey Instltute of Experimental Physlology and Medlclne, Unlverslty of Melbourne, PARKVILLE, VIC. 3052 AUSTRALIA (~eceived in final form
AuEust
/i, 1980)
Summary The present study describes an improved method for measurlng anglotensln III in arterial blood. This was accomplished by SE-sephadex column to separate angzotensln II from anglotensln III prior to radlolmmunoassay. The arterial concentration of anglotensln III measured before and after 24 to 48 hours sodium depletlon by acute cannulatlon of parotld gland was 12.4 ± 1.7 fmol/ml (SEM, n=7) and 49.8 ± 10.3 fmol/ml (SEM, n=7) respectlvely. The arterial concentratlon of Val~-anglotensln III obtained from continuous infusaon of Val%-anglotensln III at rates of 24 and 48 nmol/h in sodium deficient sheep were 245 ± 32.5 fmol/ml (n=6) and 330 Z 11.4 fmol/ ml (n=7) respectlvely. The clearance rate of exogenous Val~-anglo tensin III in sodium deflclent sheep after correction for endogenous level was calculated to be 140 ± 13.6 L/h (SEM, n=13). This was in the same order as IleS-anglotensln II and Ile~-anglotensln III reported earlier in sodium replete sheep Prolonged intravenous infusion of Val~-anglotensan III at a rate of 48 nmol/h an sodzumdef~clent sheep suppressed plasma renin concentration to the same extent as equimolar infusions of anglotensln II. Thls suggests that anglotensln III may ~nhLblt renln secretion by a slmilar mechanlsm to ang~otensln II. It has been suggested that [des-Aspl]anglotensln II (anglotensln III) may be an important mediator of various responses to angiotensln II (1-4). Both peptldes were known to have steroldogenlc and pressor activity (5). In sheep it has been quantitatlvely demonstrated that substantial production of both anglotensln II and anglotensln III occurs in the perlpheral vascular bed and that the level of anglotensln III zn arterial blood is about 42% of that of anglotensln II (6). Thls level of anglotensln III, albeit lower than anglotensin II, may be of physlologlcal Importance, especially for cells such as those In the adrenal zona glomerulosa whlch have been reported to be more sensltlve to anglotensln III than anglotensln II In aldosterone productlon during sodium deflclency (7). Thls study reports the effects of anglotensln III on plasma renln concentration in consclous, sodium replete and sodlum deplete sheep and compares thls wlth the effect of anglotens~n II Earlier ~mmunologlcal evldence
1.
Thls work was supported by the Natlonal Heart Foundatlon of Australla.
0024-3205/80/421495-08502.00/0 Copyrlght
(c) 1980 Pergamon Press Ltd.
1496
Angiotensln III in Arterial Blood
V01. 27, No.
16, 1980
(8[ has shown that in sheep the amino acid in posltlon 5 of anglotensln II is vallne Hence, ValS-anglotensln II and Val~-anglotensln III were used as standards and for lnfuslon in the present study Materlals and Methods Experlments were performed on Ii mature, conscious Merino crossbred sheep, weighing 33 to 40 kg. All animals were prepared wlth exteriorized carotld arteries. Four had a unilateral parotld fistula (9) Animals were rendered sodium deflclent by uncompensated loss of saliva for 24 to 48 hours Sodium repletlon was monitored by measurlng the potassium concentratlon in sallva, a value of 6 mmol/l or less being taken as representative of sodium repletlon. Arterial samples were taken from carotld artery for determlnatlon of anglotensln III before and after sodium depletion. Incremental infuslons of Va!~-anglotensln III Seven sheep were sodlum-depleted by acute cannulat~on of the right parotld sallvary duct 24 to 48 hours before the experlment (i0). Val~-angloten sln III (Peninsular Lab ) was infused through polyethylene chlorlde cannula (internal diameter of 1 mm) wlth its tlp posltzoned in the 3ugular veln, at 24 and 48 nmol/h, each for 30 minutes Blood samples were taken from the carotid artery for determination of Val4-anglotensln III and plasma renln concentration (PRC) at 0 and 30 minutes after the commencement of each infuslon rate. Prolonged infusion of ValS-anglotensln II and Val~-anglotensln III In a second series of experiments, four sheep which were prepared wlth a unilateral parotld flstula were sodlum-depleted by wlthholdlng access to sodlum blcarbonate solutlon for 24 to 48 hours. These animals were then infused intravenously wlth ValS-anglotensln II for 4 hours at a rate of 48 nmol/h. Blood samples were taken into precooled sequestered tubes contalnlng 50 mM EDTA at times 0, 15, 30, 60, 120, 180 and 240 minutes after the commencement of infuslons for determination of PRC. Two weeks after the flrst experiment the identlcal protocol was repeated on the same 4 anlmals uslng Val~-anglotensln III as infusate. Determination of plasma renln concentratlon Plasma renln concentration was determined by radlolmmunoassay uslng the approach of antibody capture method described by Poulsen and Jorgensen (ii). Thls involved the addltlon of 50 ~i of exogenous renln substrate, obtained from nephrectomlzed sheep plasma (wlth an anglotensln I concentration of 2,300 pmol/ml). 30 ~i of unknown plasma sample was then added together wlth 20 ~i of a concentrated anglotensln I antlbody and incubated at 37°C for 30 minutes At the end of incubatlon, 1.5 ml of izSI-anglotensln I (5,000 cpm) Was added The final antibody dllutlon was 1 40,000. The anglotensln I generated was determined by radlolmmunoassay and was expressed as pmol of anglotensln generated per hour per ml of plasma The antibody used in the present study had negligible cross-reactlvlty wlth the exogenous renln substrate and minimally wlth anglotensln II and its smaller metabolItes at 1 pmol (<0.2%) Determlnatlon of blood Val~-an~lotensl ~ III Val%-anglotensln III was extracted from blood and determlned by radloimmunoassay as described in detail elsewhere (12), wlth two mod~flcatlons as reported recently (13). Blood was collected into absolute ethanol (V/V = 3 7) to stop instantaneously the degradatlon of anglotenslns by serum amlnopeptldases. About 1,000 cpm of trltlum-labelled anglotensln III (from New England
Vol.
27, No.
16, 1980
AngiotensiD
Ill in Arterial
Blood
1497
Nuclear Corp Boston, wlth speclflc actlvlty of 41,2 Cl/mmol ) was then added to each sample as internal marker to estlmate losses during extractlon. The ethanollc blood mlxture was centrlfuged at 3,000 rpm for I0 mlnutes. The supernatant was washed wlth ether twlce and acldlfled to pH 3 before passlng down SE-sephadex column (150 mm in hezght and 9 mm in dlameter) whlch was equlllbrated In 0.05M acetlc acld (pH = 3). Using 0.1M phosphate buffer (pH = 7.4) as eluent, ang~otensln III was eluted and radlolmmunoassayed by means of an anglctensln II antlbody. After substractlng the mass of tracer and correctlng for losses durlng extractlon, the concentration of endogenous anglotensln III was then quantltatlvely determined. Statlstlcal
analysls
Unless otherwlse stated, all results were expressed as mean ± standard error of the mean. Student's test for palred observatlons was used for statlstlcal comparlsons. Results PRC assay The intra-assay and inter-assay var~atlons in the determlnatlon of one ~I U.W.H.O human renln (obtalned from the Instltute for Medlcal Research, Holy HIll, London) were 2.7% (C.V., n=27) and 11.8% (C.V., n=28) respectlvely. The generation of anglotensln I over a perlod of two hours at 37°C by varylng
WHO Renin /~IU
o
100
0 t
0
,
i
i
3O
60
I
I
120
Incubation- minutes Fig.
1
Reactlon rate of sheep renan substrate wlth known amount of WHO human renln. The generatlon of anglotensln I wlth varylng amount of WHO human renln (ranglng from 0.3 to 0.8 ~ I.U.) over a perlod of two hours at 37°C was obtalned. Each polnt represents the mean of 2 tubes.
1498
Vol. 27, No. 16, 1980
Angiotensin III in Arterial Blood
amounts of the W.H.O. human renzn (ranglng from 0 3 to 0.8 ~ I.U.) zs shown zn Fig. i. One ~I.U.W.H.O. renln whlch was used as an internal standard generated 191 ± 5 fmol anglotensln I/hour (n=28). Determination of Val~-angzotensln III in blood The elutlon pattern of Val~-angzotenszn III and 3H-anglotenszn III zn the SE-sephadex column is shown zn Fig. 2. Val~-anglotenszn III, wzth zts slower elutlon rate was suffzclently separated from ValS-anglotensln II przor to radzozmmunoassay. When known amounts of Val4-ang~otenszn III (rangzng from 200 to 1,500 fmol) was added to sheep blood, the measured concentratzon after correctlon of losses durzng extractzon bears a good correlatzon to what was added, wlth correlatlon co-effzclent of 0.98. The zntra-assay and Inter-assay varlatlon in determlnlng 500 fmol Val~-angzotensln III zn sheep blood was 10.7% (C.V., n=10) and 12.6% (C.V., n=7) respectlvely.
150
1500 SE - Sephadex
E O
~100
Va=; Aa
l Val!A.T
I
IOO0 .c_ ~ =4 o
I 5O0
z
, D
e"
I
/o ,
0
2
4
8
6
, ~'-~t~- ---,~--- 0
10
12
14
Elution Volume ml Fig. 2 The elutzon pattern of i, 000 cpm of 3H-angzotenszn III (4 4) and 500 fmol of each of Val 5-anglotenszn II and Val -anglotenszn III wlth the SE-sephadex column. The condltlons of elutlon are descrlbed in the methods.
Vol.
27, No.
Effect
16,
1980
Anglotensln
III in Arterzal
Blood
1499
of sodlum d e p l e t l o n
The m e a n sodlum d e f l c l t after 24 to 48 hours u n c o m p e n s a t e d loss of sallva by acute p a r o t l d c a n n u l a t l o n was 352 ± 45 mmol (n=7) Plasma renln c o n c e n t r a t l o n (PRC) d e t e r m l n e d In 7 sodlum-replete sheep was 1.5 ± 0 16 pmol AI/h/ml. A f t e r s o d l u m depletlon, PRC was elevated to 8.2 ± 1.4 p m o l / h / m l (n=7) w l t h range from 4 to 23 p m o l / h / m l The arterial V a l 4 - a n g l o t e n s l n III c o n c e n t r a t i o n was also slgn l f l c a n t l y e l e v a t e d from 12.4 ± 1.7 fmol/ml (n=7) to 49 8 ± 10.3 fmol/ml (n=7) after s o d l u m d e p l e t l o n Incremental
infuslon
of Val
-anglotens&n
III
C o n t l n u o u s intravenous infus~on of Val - a n g l o t e n s l n III to 7 sodlumdeflclent, n o n - f l s t u l a t e d sheep resulted in a slgnlflcant decrease of p l a s m a renln c o n c e n t r a t l o n shown in Fig 3 At an infuslon rate of 24 nmol/h, PRC taken 30 m l n u t e s after the c o m m e n c e m e n t of infusion was d e c r e a s e d from 8.2 ±
~
4OO
24 22
300
B
\
14 12
Arterial Val4-A gl 2O0 fnloI/ml
PRC pmol/h/ml
10 8 6
100 4 2
t Na-replete J
0
30 Time (m,n)
60
Fig.
0
30 6O Time (minutes)
3
The arterlal level of V a l ~ - a n g l o t e n s l n III (A) and p l a s m a renln c o n c e n t r a t l o n (B) d u r l n g intravenous infus~on of V a l 4 - a n g l o t e n s l n III at 24 and 48 nmol/h ~n 6 s o d l u m - d e p l e t e sheep. A n l m a l s were s o d l u m - d e p l e t e d by u n c o m p e n s a t e d sallvary loss t h r o u g h acute c a n n u l a t ~ o n of a p a r o t l d gland.
1500
Angiotensln
III in A r t e r i a l
Blood
Vol.
27, No.
16, 1980
1 4 p m o l / h / m l to 5.3 ± 0 95 p m o l / h / m l , a d e c r e a s e of 34 ± 5.6% from the pre• n f u s l o n values I n c r e a s l n g the I n f u s l o n rate to 48 nmol/h, PRC was d e c r e a s e d further to 3.8 ± 0 63 pmol/h/ml, a drop of 54 ± 4.4% from the p r e - l n f u s ~ o n values. 4 The arterial level of Val - a n g ~ o t e n s l n III m e a s u r e d by the p r e s e n t m e t h o d shown In Flg. 3, was 245 ± 32 5 fmol/ml (n=7) at low dose and 330 ± ii 4 fmol/ml 4 (n=7) at h ~ g h dose of Val - a n g ± o t e n s l n III ~nfuslon A f t e r c o r r e c t l o n of endogenous level of Val - a n g l o t e n s l n III uslng the formula of J o h n s t o n and c o l l e a g u e s (14) IR MCR = PRCI C - CO i PRC 0 where
IR C o C
~s the c o n s t a n t - l n f u s l o n rate ( in pmol/h) is the c o n c e n t r a t l o n of b l o o d Val - a n g l o t e n s l n III (in pmol/L) at 4 c o m m e n c e m e n t of Val - a n g l o t e n s l n III infuslon, is the e q u l l l b r l u m c o n c e n t r a t l o n of b l o o d Val - a n g l o t e n s l n III (in pmol/L) d u r l n g Val - a n g l o t e n s ~ n III infus~on, PRC and PRC are the p l a s m a renln c o n c e n t r a t l o n (in pmol/h/ml) at 0 and 4 o30 m ~ n u t ~ s after c o m m e n c e m e n t of Val - a n g l o t e n s ~ n III ~nfuslons, the m e t a b o l l c c l e a r a n c e rate of e x o g e n o u s V a l 4 - a n g l o t e n s l n III in sod±umd e p l e t e sheep was c a l c u l a t e d to be 140 ± 13.6 L/h (n=13). Prolon@ed
Infuslon of V a l S - a n g l o t e n s l n
II and Val - a n g l o t e n s l n
III
To c o m p a r e the e f f e c t of c l r c u l a t l n g Val - a n g l o t e n s l n III on PRC w i t h 5 that of Val - a n g l o t e n s l n II, an i d e n t ~ c a l p r o t o c o l was a d o p t e d Each peptlde was i n f u s e d i n t r a v e n o u s l y at a rate of 48 n m o l / h for four hours to four sodlumdeflclent, f l s t u l a t e d sheep Table I shows the PRC results after c o m m e n c e m e n t of a n g l o t e n s ~ n infuslons. TABLE
I
Plasma Renln C o n c e n t r a t l o n (pmol/h/ml) after C o m m e n c e m e n t of Infuslon of Val 4a n g l o t e n s l n III and V a l S - a n g l o t e n s l n II in S o d l u m - D e f l c l e n t , F l s t u l a t e d Sheep 4 Val - a n g l o t e n s z n III lnfuslon (48 nmol/h) Time of Infuslon (mlnutes) Anlmal 1 2 3 4 ValS-anglotensln 1 2 3 4
0
12.2 11.8 31.5 75.2 II infusion 13.8 13.7 6.4 15.5
15
30
60
120
7.7 7 8 20.4 75.2
7.5 5 9 19 6 58 9
3.7 3 4 13.5 52 2
2 6 1 5 12 7 30.7
8.9 4.2 1.8 6 5
5.7 4 3 3.4 4.4
180
0 5 i0 32
240
7 4 3 9
1.8 3 0 9.6 40 2
3.8 4 7 2 0 3 0
4 8 3 7 3.1 2 1
(48 nmol/h) I0 3 9.0 4.6 11.9
8.0 6 0 1.9 6.0
The extent of s u p p r e s s l o n of PRC by V a l S - a n g l o t e n s l n II and V a l 4 - a n g l o tensln III after 4 hours i n f u s l o n was i d e n t i c a l (69 ± 8% for V a l S - a n g l o t e n s l n II infuslon and 69 ± 7.3% for V a l 4 - a n g l o t e n s l n III infuslon) as shown in Fig S t a t l s t l c a l analysls shows no s l g n l f l c a n t d i f f e r e n c e of tne two curves.
Vol
27, No.
16,
1980
Anglotensln
III In Arterial
Blood
A
%or
1501
B
IO0
100
80
80
6O
% o f O0 I:~-fn~ Valuee
40
40
20
20
Pm Inlmmn PSC Vakum
T
T
]
0
1
2
3
4
0
1 Tim
Time [hoursl
F~g
2 (houm)
3
4
4
P e r c e n t suppresslon of p l a s m a renln c o n c e n t r a t l o n from p r e l n f u s ~ o n p r o l o n g e d infus~on of V a l ~ - a n g l o t e n s l n II (A) and V a l S - a n g l o t e n s l n 4 sodlum-deflclent, flstulated sheep
values by III (B) in
DISCUSSION The level of anglotensln III in arterial b l o o d relatlve to anglotensln II has been reported to be hlgh in rats (15) but low in dog and man (14, 16) Previous studies (6), uslng recovery m a r k e r and paper chromatography to separate V a l S - a n g l o t e n s l n II from V a l ~ - a n g l o t e n s l n III prior to radlolmmunoassay, have shown that in s o d i u m deflclent sheep the level of V a l ~ - a n g l o t e n s l n III In arterlal b l o o d is 42% of that of anglotensln II. The present study has improved the m e t h o d of m e a s u r l n g anglotensln III in blood reported p r e v l o u s l y (12). Instead of using paper c h r o m a t o g r a p h y to separate Val 4 - a n g l o t e n s l n III from V a l S - a n g l o t e n s l n II, S E - s e p h a d e x column was used to achieve the same end. Thls m o d i f i e d p r o c e d u r e is less tlme consuming but gives higher yield at the end of extraction. The final extract contained 45 to 55% of 3H-anglotensln III added initially, w h e n c o m p a r e d to 30% of yield in the prevlous method. In addition, a n g l o t e n s l n II can be s l m u l t a n e o u s l y d e t e r m l n e d an the same sample using 12sIa n g l o t e n s l n II as an internal m a r k e r which elutes in the same p o s i t i o n as Val 5a n g l o t e n s l n II. The p r e s e n t study conflrms the slgnzfzcant e l e v a t i o n of a n g l o t e n s l n III in arterial b l o o d after s o d l u m depletion. The clearance of exogenous V a l 4 - a n g l o tensln III (140 ± 13.5 L/h) was of the same order as I l e S - a n g l o t e n s l n II and 4 Ile - a n g l o t e n s l n III o b t a z n e d in s o d l u m - r e p l e t e sheep (i0). Thls is zn a~reem e n t w l t h e a r l i e r flndlngs in sheep w h e r e the a r t e r l o v e n o u s ratlo of Val -anglotensln III dld not change b e f o r e and after sodlum d e p l e t l o n (6).
1502
Angiotensln
III in A r t e r i a l
Blood
Vol.
27, No.
16, 1980
It has b e e n r e p o r t e d that a n g l o t e n s l n III is e q u l p o t e n t as a n g l o t e n s l n II on a l d o s t e r o n e s e c r e t i o n in vitro and in vlvo (18, 19). B o t h p e p t l d e s d e c r e a s e d renal b l o o d flow (4, 20) The h y p o t h e s i s has b e e n a d v a n c e d that a n g l o t e n s l n III m a y be a m e d i a t o r of the r e n l n - a n g l o t e n s l n system The p r e s e n t studies have shown that V a l 4 - a n g l o t e n s l n III at an infusion rate of 42 n m o l / h suppress renln s e c r e t i o n to the same extent as e q u l m o l a r i n f u s i o n s of V a l S - a n g l o t e n s l n II. W h e t h e r thls dose r e p r e s e n t s a m a x i m a l i n h i b i t i o n of renln a c t i v i t y and masks the a p p a r e n t d i f f e r e n c e of the two p e p t l d e s remains to be established. The data is in a g r e e m e n t w i t h e a r l i e r o b s e r v a t i o n s in rabbit (21) and dog (4). V a n d o g e n and c o l l e a g u e s (22) also o b s e r v e d a decrease in renln p r o d u c t i o n in the p e r f u s e d rat k i d n e y after a d d i t i o n of a n g l o t e n s l n III to the perfusate. The nature of thls i n h l b l t o r y actlon is not t h o r o u g h l y understood. Recently, C a m p b e l l and c o l l e a g u e s (23) have r e p o r t e d that the s t e r o l d o g e n l c actlon of a n g l o t e n s l n II and III in the adrenal c o r t e x was m e d l a t e d in p a r t by the p r o s t a g l a n d l n s W h e t h e r the release of renln in the kidney is m o d u l a t e d by the same m e c h a n i s m remalns to be l n v e s t l g a t e d REFERENCES i. 2. 3. 4. 5 6 7. 8. 9 i0. ii. 12. 13. 14 15. 16.
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