Actions of Angiotensin II Antagonists upon Aldosterone Production by Isolated Adrenal Glomerulosa Cells S. SALTMAN, P. FREDLUND, AND K. J. CATT Section on Hormonal Regulation, Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 concentrations of [Sar1, Ile8]angiotensin II. The heptapeptide antagonist [Des-Asp1, Ile8]angiotensin II was much less potent than [Sar1, Ile8]angiotensin II as an inhibitor of the actions of both the heptapeptide and octapeptide agonists. The antagonist activity of six angiotensin II analogues at the adrenal level, determined by the concentration required for 50% inhibition of maximum aldosterone secretion, correlated well with their antagonist activity measured upon isolated smooth muscle. These observations demonstrate that the octapeptide antagonists are more effective than the heptapeptide antagonists upon angiotensin II-stimulated aldosterone production, and that angiotensin II receptors in smooth muscle and adrenal cortex exhibit generally similar responses to angiotensin II antagonists. Also, these results do not support the proposal that the [DesAsp1 ]heptapeptide is an important intermediate in the action of angiotensin II upon aldosterone production in the adrenal glomerulosa cells. The production of aldosterone by dispersed zona glomerulosa cells in vitro provides a highly sensitive and biologically appropriate response for evaluation of the agonist and antagonist properties of angiotensin II analogues upon the adrenal gland. (Endocrinology 98: 894, 1976)
ABSTRACT. The biological activities of angiotensin II antagonists upon basal and angiotensin II-stimulated aldosterone production were evaluated in an isolated canine glomerulosa cell preparation. The most potent competitive antagonist of angiotensin II-stimulated aldosterone production was the [Sar1, Ile8]derivative of angiotensin II. However, this peptide was also a partial agonist at concentrations required to inhibit the steroidogenic effect of angiotensin II on dog adrenal cells, and never reduced aldosterone production to basal levels. On a molar basis, the [Sar1, Ala8] and [Sar1, Gly8]derivatives of angiotensin II were relatively less potent as competitive inhibitors of angiotensin II-stimulated aldosterone production. However, the [Ala8] and [Gly8]analogues did not exhibit significant agonist activity and were therefore more effective antagonists of angiotensin II-stimulated aldosterone production. These results suggest that increased length of the aliphatic side chain at the C-terminus of angiotensin II antagonists is accompanied by enhanced affinity for the receptor site, but also by increased agonist activity upon aldosterone synthesis. The actions of angiotensin II and [Des-Asp1 ]angiotensin II upon aldosterone production were inhibited identically and completely by [Sar1, Ala8]angiotensin II, and identically, though incompletely, by lower
A
NUMBER of competitive antagonists of . angiotensin II have been synthesized and evaluated for activity upon the smooth muscle responses and blood pressure changes produced by angiotensin II (1-8). The binding activity of angiotensin II agonists and antagonists at the adrenal level has been recently studied in a radioligandreceptor assay using bovine adrenal cortex homogenate (9). Determination of the binding-inhibition potencies of angiotensin analogues by radioligand-receptor assay is of particular value as a measure of the relative binding affinities of agonist and antagonist peptides. However, the extent to which the binding affinity of peptide Received May 12, 1975.
analogues for angiotensin II receptors is associated with agonist or antagonist properties can only be determined by measurement of these biological activities upon an appropriate target cell response. In several species, angiotensin II antagonists have been used to evaluate the role of angiotensin II in blood pressure regulation and aldosterone secretion under certain pathologic and physiologic conditions (1012). In addition, angiotensin II antagonists have been employed to inhibit the actions of angiotensin II agonists upon aldosterone synthesis (13-14), and during comparisons of the properties of smooth muscle and adrenal receptors for angiotensin II (15-17). The development of a preparation of collagenase-dispersed canine adrenal cells
894
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
895
ANGIOTENSIN II ANTAGONISTS which responds to physiologic concentrations of angiotensin II (18) has provided a sensitive assay system for an analysis of the agonist and antagonist activities of angiotensin II analogues at the adrenal level. This method permits the abolition or control of other factors which are known to modulate aldosterone synthesis by the zona glomerulosa, so that the biological actions of angiotensin derivatives can be evaluated in the presence or absence of other determinants of aldosterone secretion. In the present report, the aldosterone response of enzymedispersed canine adrenal cells has been utilized for evaluation of the structure-function relationships of angiotensin II antagonists in vitro. Materials and Methods Synthetic [Asp1, Ile5]angiotensin II was purchased from Beckman Instruments, Inc., Palo Alto, Calif. The [Sar\ Ala8], [Sar1, Gly8], [Asp1, Ala8] and [D-Asp1, Ala8] derivatives of angiotensin II were obtained through the kindness of Dr. A. Castellion, Norwich Pharmaceutical Company, N. Y. The [He8] substituted angiotensin II analogues were generously provided by Dr. F. M. Bumpus, Research Division, Cleveland Clinic. Standard solutions of the angiotensin derivatives were prepared after weighing on a Mettler electrobalance by dissolving the individual peptides in 0.01M acetic acid; all peptide concentrations were calculated from the molar extinction coefficient for tyrosine of 1.34. Crude collagenase (Type 1) was obtained from Worthington, DNAse from Sigma, and bovine serum albumin (BSA) from Metrix. The procedures for collagenase dispersion and incubation of canine adrenal cells, and direct radioimmunoassay for aldosterone, have been reported in detail elsewhere (18). The minced capsular layer of canine adrenal glands was incubated at 37 C for 15 minutes with crude collagenase (2 mg/ml) and DNAse (25 /x,g/ml), followed by physical dispersion of the tissue fragments to give isolated glomerulosa cells. Following centrifugation at 100 x g for 15 minutes at 22 C, the cell pellet was resuspended in medium 199 containing 2 mg BSA per ml and 5.0 mM potassium, and 1 ml aliquots of the dispersed cells were incubated at 37 C under 95%
O2-5% CO2. After incubation for 2 hours, the cells were separated by centrifugation at 1500 x g for 15 minutes and direct radioimmunoassay of the incubation media for aldosterone was performed. Results
Inhibition of aldosterone angiotensin antagonists
synthesis
by
The angiotensin II analogues chosen for study were selected on the basis of their demonstrated activities as antagonists during previous studies in vivo and in vitro, and their known binding-inhibition activities in the adrenal radioligand-receptor assay (9). The inhibitory effects of the [Sar1, He 8 ], [Sar1, Ala8], and [Sar1, Gly8] derivatives upon angiotensin II-induced aldosterone synthesis by dispersed adrenal cells are shown in Fig. 1. The [Sar1, He8] analogue was the most potent inhibitor of aldosterone synthesis, producing 50% inhibition of the steroidogenic response to 10~9M angiotensin II at an antagonist-agonist dose ratio of 4:1. In contrast, [Sar1, Ala8]angiotensin II and [Sar1, Gly8]angiotensin II produced 50% inhibition of the aldosterone response at a dose ratio of 50:1, being therefore about 12 times less potent than the [Sar1, He8] derivative. The dose-response curves for inhibition of angiotensin II-stimulated aldosterone synthesis by [Asn1, Ala8]angiotensin II and [D-Asn1, Ala8]angiotensin are shown in Figs. 2 and 3. The dose ratios for 50% inhibition of the steroidogenic response by the D and L [Asn1] configurations were 170 and 150, respectively. Thus, no differential effect of the two.N-terminal aspartyl isomers upon the antagonist activity of [Ala8]angiotensin II was demonstrable. Also, this result illustrates that substitution of the N-terminal aspartyl residue with sarcosine causes a 3 4-fold increase in the antagonist potency of 8-alanine angiotensin II. The 8-thienylalanine derivative of angiotensin II has been shown to act as an antagonist of angiotensin II upon smooth
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
o
8
Endo i 1976 Vol 98 ( No 4
SALTMAN, FREDLUND AND CATT
896
[lieu5] Angiotensin n = 10"9M -
100
80 ^s^
I.D.50 = 5.0x10 8 M
60 I.D. 50= 4.3x10* M
IS < o
o
40
20
I.D. 50 = 5.0xl0' 8 M
Ui
a. 10
0.1 DOSE RATIO -
100
1000
ANTAGONIST/AGONIST
FIG. 1. Inhibition of the aldosterone response to 10~9M angiotensin II by varying concentrations of [Sar1, Ile8]angiotensin II, [Sar1, Gly8]angiotensin II and [Sar1, Ala8]angiotensin II. The ordinate represents the per cent of maximum aldosterone production, in comparison with the value obtained for cells incubated with 10~9M angiotensin II in the same experiment. The abscissa represents increasing antagonist concentrations expressed as a dose ratio to a constant angiotensin II concentration of 10~9M. (100% = 12 ng aldosterone/2 h; 0% = 2 h control = 3.7 ng aldosterone/2 h).
muscle, and exhibits a relatively high pA2* (9.36) on the rabbit aortic strip. Also, the binding-inhibition potency of the [Tal8]analogue was equal to that of angiotensin II in the adrenal radioligand-receptor assay (9). These observations indicate that [Tal8]angiotensin II should behave as a potent antagonist of the action of angiotensin II in the glomerulosa cell preparation. However, incubation of [Tal8]angiotensin II with adrenal cells exposed to 10~9M angiotensin II produced neither inhibition nor augmentation of the aldosterone response to angiotensin II, as shown in Fig. 4, which includes the inhibition of angiotensin II by [Sar1, Ala8]angiotensin II for comparison. When incubated with dispersed glomeru1
pA2 is the negative decimal logarithm of the concentration of antagonist which increases by two-fold the concentration of agonist needed for a selected effect. The pA2 values for antagonist peptides were provided by Dr. F. M. Bumpus and Dr. M. Hall, Cleveland Clinic, Ohio.
losa cells at a concentration of 10 9M in the absence of angiotensin II, [Tal8]angiotensin II displayed marked agonist activity upon aldosterone production and was almost as potent as angiotensin II in stimulating steroidogenesis (Fig. 5). However, the characteristic decrement in aldosterone production in the presence of supramaximal stimulating concentrations of angiotensin II (Fig. 6) was not observed with the [Tal8]derivative. Agonist properties of angiotensin II antagonists The agonist activities of the major angiotensin II antagonists upon aldosterone production by canine adrenal cells are illustrated in Fig. 6. It is apparent that antagonists prepared by C-terminal aliphatic substitution of angiotensin II with glycine or alanine do not possess significant agonist activity. In contrast, the antagonist peptide produced by C-terminal substitution with isoleucine
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
897
ANGIOTENSIN II ANTAGONISTS c^> [Asn! Ala 8 j AO + 1 0 9 M An A — a [Asn! Ala 8 ] A E
100
O ce
80 FIG. 2. Inhibition of the aldosterone response to 10~9M angiotensin II by increasing concentrations of [Asn1, Ala8]angiotensin II. Triangles represent agonist activity of increasing concentrations of [Asn1, Ala8]angiotensin II. (100% = 7.2 ng aldosterone/2 h; 0% = 2 h control = 2.2 ng aldosterone/2 h).
< z 60 >< Q < O
5 ex. LU
I.D. 5 0 1.5x 10" 7 M
40
20
O cc
UJ Q.
10 -9
10"8
10 -6
10"7
[Asn1 Ala 8 ] All CONCENTRATION (M)
displays significant agonist activity which is manifested over the concentration range necessary for inhibition of the action of angiotensin II. Similar agonist activity was demonstrated for the 1-guanidoacetic and 1-des-aspartyl analogues of [Ile8]angiotensin II. The agonist activity of [Des-Asp1, Ile 8 ]angiotensin II was less than that of the 1sarcosine and 1-guanidoacetic acid derivatives, in agreement with its lower binding affinity in the adrenal radioligand-receptor assay (9).
Action of antagonists upon responses to angiotensin II, [Des-Asp1]dSigiotensin II and [Sarl]angiotensin II In the collagenase-dispersed glomerulosa cell preparation, the dose-response curves for stimulation of aldosterone synthesis were identical for angiotensin II and the heptapeptide [Des-Asp1 ]angiotensin II, and the [Sar1] derivative of angiotensin II was about 10-fold more potent than angiotensin II (19). The antagonist activity of [Sar1, Ala8]I
UJ
0
inn
O
FIG. 3. Inhibition of the action of 10~9M angiotensin II by increasing concentrations of [r> Asn1, Ala8]angiotensin II. Triangles represent agonist activity of increasing concentrations of [D-Asn1, Ala 8 ]angiotensin II. (100% = 7.2 ng aldosterone/2 h; 0% = 2 h control = 2.2 ng aldosterone/2 h).
I
o[D-Asnf Ala8]An+109MAa A,a>n
80
\ O
60
I.D.50 = 1.7xl0"7M Q.
\
0
N.
40 o\
UJ
O
20 -
tr n^ 10"
"
*
10
-8
-^
' -7
10
10-6
[D-AsnUla8] AU CONCENTRATION (M)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1976 Vol 98 • No 4
SALTMAN, FREDLUND AND CATT
898
[Tal 8 ]AII + 10"9M All =>
100 6
CO
o
80
CO
O Q
60
40 [Sar 1 Ala 8 ]AII + 10'9M All
X
ABSTRACT. The biological activities of angiotensin II antagonists upon basal and angiotensin II-stimulated aldosterone production were evaluated in an isolated canine glomerulosa cell preparation. The most potent competitive antagonist of angiotensin II-stimulated aldosterone production was the [Sar1, Ile8]derivative of angiotensin II. However, this peptide was also a partial agonist at concentrations required to inhibit the steroidogenic effect of angiotensin II on dog adrenal cells, and never reduced aldosterone production to basal levels. On a molar basis, the [Sar1, Ala8] and [Sar1, Gly8]derivatives of angiotensin II were relatively less potent as competitive inhibitors of angiotensin II-stimulated aldosterone production. However, the [Ala8] and [Gly8]analogues did not exhibit significant agonist activity and were therefore more effective antagonists of angiotensin II-stimulated aldosterone production. These results suggest that increased length of the aliphatic side chain at the C-terminus of angiotensin II antagonists is accompanied by enhanced affinity for the receptor site, but also by increased agonist activity upon aldosterone synthesis. The actions of angiotensin II and [Des-Asp1 ]angiotensin II upon aldosterone production were inhibited identically and completely by [Sar1, Ala8]angiotensin II, and identically, though incompletely, by lower
A
NUMBER of competitive antagonists of . angiotensin II have been synthesized and evaluated for activity upon the smooth muscle responses and blood pressure changes produced by angiotensin II (1-8). The binding activity of angiotensin II agonists and antagonists at the adrenal level has been recently studied in a radioligandreceptor assay using bovine adrenal cortex homogenate (9). Determination of the binding-inhibition potencies of angiotensin analogues by radioligand-receptor assay is of particular value as a measure of the relative binding affinities of agonist and antagonist peptides. However, the extent to which the binding affinity of peptide Received May 12, 1975.
analogues for angiotensin II receptors is associated with agonist or antagonist properties can only be determined by measurement of these biological activities upon an appropriate target cell response. In several species, angiotensin II antagonists have been used to evaluate the role of angiotensin II in blood pressure regulation and aldosterone secretion under certain pathologic and physiologic conditions (1012). In addition, angiotensin II antagonists have been employed to inhibit the actions of angiotensin II agonists upon aldosterone synthesis (13-14), and during comparisons of the properties of smooth muscle and adrenal receptors for angiotensin II (15-17). The development of a preparation of collagenase-dispersed canine adrenal cells
894
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
895
ANGIOTENSIN II ANTAGONISTS which responds to physiologic concentrations of angiotensin II (18) has provided a sensitive assay system for an analysis of the agonist and antagonist activities of angiotensin II analogues at the adrenal level. This method permits the abolition or control of other factors which are known to modulate aldosterone synthesis by the zona glomerulosa, so that the biological actions of angiotensin derivatives can be evaluated in the presence or absence of other determinants of aldosterone secretion. In the present report, the aldosterone response of enzymedispersed canine adrenal cells has been utilized for evaluation of the structure-function relationships of angiotensin II antagonists in vitro. Materials and Methods Synthetic [Asp1, Ile5]angiotensin II was purchased from Beckman Instruments, Inc., Palo Alto, Calif. The [Sar\ Ala8], [Sar1, Gly8], [Asp1, Ala8] and [D-Asp1, Ala8] derivatives of angiotensin II were obtained through the kindness of Dr. A. Castellion, Norwich Pharmaceutical Company, N. Y. The [He8] substituted angiotensin II analogues were generously provided by Dr. F. M. Bumpus, Research Division, Cleveland Clinic. Standard solutions of the angiotensin derivatives were prepared after weighing on a Mettler electrobalance by dissolving the individual peptides in 0.01M acetic acid; all peptide concentrations were calculated from the molar extinction coefficient for tyrosine of 1.34. Crude collagenase (Type 1) was obtained from Worthington, DNAse from Sigma, and bovine serum albumin (BSA) from Metrix. The procedures for collagenase dispersion and incubation of canine adrenal cells, and direct radioimmunoassay for aldosterone, have been reported in detail elsewhere (18). The minced capsular layer of canine adrenal glands was incubated at 37 C for 15 minutes with crude collagenase (2 mg/ml) and DNAse (25 /x,g/ml), followed by physical dispersion of the tissue fragments to give isolated glomerulosa cells. Following centrifugation at 100 x g for 15 minutes at 22 C, the cell pellet was resuspended in medium 199 containing 2 mg BSA per ml and 5.0 mM potassium, and 1 ml aliquots of the dispersed cells were incubated at 37 C under 95%
O2-5% CO2. After incubation for 2 hours, the cells were separated by centrifugation at 1500 x g for 15 minutes and direct radioimmunoassay of the incubation media for aldosterone was performed. Results
Inhibition of aldosterone angiotensin antagonists
synthesis
by
The angiotensin II analogues chosen for study were selected on the basis of their demonstrated activities as antagonists during previous studies in vivo and in vitro, and their known binding-inhibition activities in the adrenal radioligand-receptor assay (9). The inhibitory effects of the [Sar1, He 8 ], [Sar1, Ala8], and [Sar1, Gly8] derivatives upon angiotensin II-induced aldosterone synthesis by dispersed adrenal cells are shown in Fig. 1. The [Sar1, He8] analogue was the most potent inhibitor of aldosterone synthesis, producing 50% inhibition of the steroidogenic response to 10~9M angiotensin II at an antagonist-agonist dose ratio of 4:1. In contrast, [Sar1, Ala8]angiotensin II and [Sar1, Gly8]angiotensin II produced 50% inhibition of the aldosterone response at a dose ratio of 50:1, being therefore about 12 times less potent than the [Sar1, He8] derivative. The dose-response curves for inhibition of angiotensin II-stimulated aldosterone synthesis by [Asn1, Ala8]angiotensin II and [D-Asn1, Ala8]angiotensin are shown in Figs. 2 and 3. The dose ratios for 50% inhibition of the steroidogenic response by the D and L [Asn1] configurations were 170 and 150, respectively. Thus, no differential effect of the two.N-terminal aspartyl isomers upon the antagonist activity of [Ala8]angiotensin II was demonstrable. Also, this result illustrates that substitution of the N-terminal aspartyl residue with sarcosine causes a 3 4-fold increase in the antagonist potency of 8-alanine angiotensin II. The 8-thienylalanine derivative of angiotensin II has been shown to act as an antagonist of angiotensin II upon smooth
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
o
8
Endo i 1976 Vol 98 ( No 4
SALTMAN, FREDLUND AND CATT
896
[lieu5] Angiotensin n = 10"9M -
100
80 ^s^
I.D.50 = 5.0x10 8 M
60 I.D. 50= 4.3x10* M
IS < o
o
40
20
I.D. 50 = 5.0xl0' 8 M
Ui
a. 10
0.1 DOSE RATIO -
100
1000
ANTAGONIST/AGONIST
FIG. 1. Inhibition of the aldosterone response to 10~9M angiotensin II by varying concentrations of [Sar1, Ile8]angiotensin II, [Sar1, Gly8]angiotensin II and [Sar1, Ala8]angiotensin II. The ordinate represents the per cent of maximum aldosterone production, in comparison with the value obtained for cells incubated with 10~9M angiotensin II in the same experiment. The abscissa represents increasing antagonist concentrations expressed as a dose ratio to a constant angiotensin II concentration of 10~9M. (100% = 12 ng aldosterone/2 h; 0% = 2 h control = 3.7 ng aldosterone/2 h).
muscle, and exhibits a relatively high pA2* (9.36) on the rabbit aortic strip. Also, the binding-inhibition potency of the [Tal8]analogue was equal to that of angiotensin II in the adrenal radioligand-receptor assay (9). These observations indicate that [Tal8]angiotensin II should behave as a potent antagonist of the action of angiotensin II in the glomerulosa cell preparation. However, incubation of [Tal8]angiotensin II with adrenal cells exposed to 10~9M angiotensin II produced neither inhibition nor augmentation of the aldosterone response to angiotensin II, as shown in Fig. 4, which includes the inhibition of angiotensin II by [Sar1, Ala8]angiotensin II for comparison. When incubated with dispersed glomeru1
pA2 is the negative decimal logarithm of the concentration of antagonist which increases by two-fold the concentration of agonist needed for a selected effect. The pA2 values for antagonist peptides were provided by Dr. F. M. Bumpus and Dr. M. Hall, Cleveland Clinic, Ohio.
losa cells at a concentration of 10 9M in the absence of angiotensin II, [Tal8]angiotensin II displayed marked agonist activity upon aldosterone production and was almost as potent as angiotensin II in stimulating steroidogenesis (Fig. 5). However, the characteristic decrement in aldosterone production in the presence of supramaximal stimulating concentrations of angiotensin II (Fig. 6) was not observed with the [Tal8]derivative. Agonist properties of angiotensin II antagonists The agonist activities of the major angiotensin II antagonists upon aldosterone production by canine adrenal cells are illustrated in Fig. 6. It is apparent that antagonists prepared by C-terminal aliphatic substitution of angiotensin II with glycine or alanine do not possess significant agonist activity. In contrast, the antagonist peptide produced by C-terminal substitution with isoleucine
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
897
ANGIOTENSIN II ANTAGONISTS c^> [Asn! Ala 8 j AO + 1 0 9 M An A — a [Asn! Ala 8 ] A E
100
O ce
80 FIG. 2. Inhibition of the aldosterone response to 10~9M angiotensin II by increasing concentrations of [Asn1, Ala8]angiotensin II. Triangles represent agonist activity of increasing concentrations of [Asn1, Ala8]angiotensin II. (100% = 7.2 ng aldosterone/2 h; 0% = 2 h control = 2.2 ng aldosterone/2 h).
< z 60 >< Q < O
5 ex. LU
I.D. 5 0 1.5x 10" 7 M
40
20
O cc
UJ Q.
10 -9
10"8
10 -6
10"7
[Asn1 Ala 8 ] All CONCENTRATION (M)
displays significant agonist activity which is manifested over the concentration range necessary for inhibition of the action of angiotensin II. Similar agonist activity was demonstrated for the 1-guanidoacetic and 1-des-aspartyl analogues of [Ile8]angiotensin II. The agonist activity of [Des-Asp1, Ile 8 ]angiotensin II was less than that of the 1sarcosine and 1-guanidoacetic acid derivatives, in agreement with its lower binding affinity in the adrenal radioligand-receptor assay (9).
Action of antagonists upon responses to angiotensin II, [Des-Asp1]dSigiotensin II and [Sarl]angiotensin II In the collagenase-dispersed glomerulosa cell preparation, the dose-response curves for stimulation of aldosterone synthesis were identical for angiotensin II and the heptapeptide [Des-Asp1 ]angiotensin II, and the [Sar1] derivative of angiotensin II was about 10-fold more potent than angiotensin II (19). The antagonist activity of [Sar1, Ala8]I
UJ
0
inn
O
FIG. 3. Inhibition of the action of 10~9M angiotensin II by increasing concentrations of [r> Asn1, Ala8]angiotensin II. Triangles represent agonist activity of increasing concentrations of [D-Asn1, Ala 8 ]angiotensin II. (100% = 7.2 ng aldosterone/2 h; 0% = 2 h control = 2.2 ng aldosterone/2 h).
I
o[D-Asnf Ala8]An+109MAa A,a>n
80
\ O
60
I.D.50 = 1.7xl0"7M Q.
\
0
N.
40 o\
UJ
O
20 -
tr n^ 10"
"
*
10
-8
-^
' -7
10
10-6
[D-AsnUla8] AU CONCENTRATION (M)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 15:09 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1976 Vol 98 • No 4
SALTMAN, FREDLUND AND CATT
898
[Tal 8 ]AII + 10"9M All =>
100 6
CO
o
80
CO
O Q
60
40 [Sar 1 Ala 8 ]AII + 10'9M All
X
