J Clin Endocrinol Metab 40: 530, 1975
ACTION OF [1-DES(ASPARTIC ACID), 8-ISOLEUCINE] ANGIOTENSIN II UPON THE PRESSOR AND STEROIDOGENIC ACTIVITY OF ANGIOTENSIN II. Emmanuel L. Bravo, Mahesh C. Khosla, and F. Merlin Bumpus, Research Division, The Cleveland Clinic Foundation, Cleveland, Ohio 44106. ABSTRACT. The vascular and steroidogenic responses to [1-Sarcosine, 8Isoleucine]-, and to [l-Des(Aspartic acid), 8-Isoleucine] angiotensin II were compared in bilaterally nephrectomized, ACTH-suppressed dogs receiving constant infusions of angiotensin II. Aldosterone secretion rate was significantly inhibited by pretreatment with 200 ng/Kg/min of the heptapeptide, [l-Des(Aspartic acid),8-Isoleucine] ang II, but not by similar doses of the octapeptide, [1-Sarcosine, 8-Isoleucine] ang II. In contrast, the pressor action of ang II was unaffected by [l-Des(Aspartic acid), 8-Isoleucine] ang II though significant inhibition occurred with relatively small doses of [1-Sarcosine, 8-Isoleucine] ang II. This study suggests that: (a) angiotensin receptors in adrenal cortex and vascular smooth muscle are functionally different, and (b) [l-Des(Aspartic acid), 8-Isoleucine] angiotensin II is a specific antagonist of steroidogenic effect of ang II. The octapeptide angiotensin II is a potent vasoconstrictor substance. In addition, it has significant aldosterone-stimulating activity. The (2-8) heptapeptide, [Des-Asp1] angiotensin II is a naturally occurring metabolite of angiotensin II, formed by the enzymatic hydrolysis of angiotensinase A. This heptapeptide has minimal pressor activity (10-30% of angiotensin II) but is at least equipotent to its octapeptide progenitor in stimulating aldosterone biosynthesis (1,2). Recently, there has been some speculation that this peptide may be the major stimulus to aldosterone secretion. In adrenal cortical cell suspensions from rabbits, Peach and coworkers (3) have shown that the heptapeptide is more potent than angiotensin II in stimulating the conversion of 3 H-cholesterol to ^H-aldosterone, has a more immediate effect than angiotensin in stimulating aldosterone biosynthesis, and has a much higher affinity for specific binding sites than angiotensin II. Similarly, Catt et a 1.(4,5) reported that the heptapeptide is almost equipotent with the octapeptide in binding-inhibition studies utilizing either beef or rat adrenal cortex particles. Since there
Submitted December 9, 1974
is little circulating heptapeptide in arterial blood (5), it has been proposed that angiotensin could act to stimulate aldosterone biosynthesis via local production of the heptapeptide (1, 3 ) . If this hypothesis were correct, the receptors for angiotensin II in the adrenal cortex and vascular smooth muscle should be different. Therefore, a competitive antagonist of the C-terminal heptapeptide should have potent inhibitory activity on aldosterone secretion but substantially less effect against the pressor action of the octapeptide (ang II). On the other hand, an antagonist directed against the octapeptide should possess extremely high affinity for vascular smooth muscle with minimal activity on adrenal glomerulosa cells. METHODS. The present investigation compared the vascular and steroidogenic responses to the octapeptide, [Sar^, Ile°] angiotensin II (6), and the heptapeptide, [Des-Asp1, lie 8 ] angiotensin II (7), during angiotensin II infusion. Experiments were performed in 17 bilaterally-nephrectomized, ACTH-suppressed male mongrel dogs
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RAPID COMMUNICATIONS
weighing 20-25 Kg. The isolated adrenal gland preparation used in these studies has been described previously (8). For the studies, the dogs were anesthetized with pentobarbital (20 mg/Kg). Timed specimens of adrenal venous effluent for aldosterone determinations were collected from the left lumboadrenal vein. All materials given by infusion were dissolved in normal saline and administered into peripheral veins through separate tubings at 1.0 ml per minute. Following two control periods [Des-Aspl, lie**]- or [Sari, H e 8 ] angiotensin II was Infused intravenously at the rate of 200 ng/Kg/m1n. This was followed 15 minutes later with angiotensin II infused at the constant rate of 20 ng/Kg/min. Measurements of aldosterone and cortisol were made at 15-minute intervals and arterial pressure was monitored continuously through a femoral arterial catheter connected to a Sanborn recorder. All blood removed for analysis was replaced with blood from donor dogs. Aldosterone was measured by radioimmunoassay method (8), and cortisol by a fluorometric method (8) modified slightly from that described by Mattingly (9). RESULTS AND DISCUSSION. Arterial pressure response is shown in Figure 1. 25
(n=5)
A-II,
20 ng/kg/min
20 CHANGE l 5 IN MAP , 0
plus [D«-Asp\lle 8 ] 200 ng/kg/min
mmHg
5 ^ plus [Sor1, I I . 8 ] A-H 200 ng/kg/min
0
15'
30'
45'
60'
Figure 1. Arterial pressure response to pretreatment with either [Des-Aspl, Ile°] angiotensin (closed circles) or [Sari, Ile8] (closed squares) in dogs receiving
531
constant doses of angiotensin II. Each value represents the mean ± S.E, of the change in mean arterial pressure. Angiotensin II increased arterial pressure by a mean of 20 mm Hg. Prior administration of the heptapeptide, [Des-Aspl, Ile8] angiotensin II, failed to prevent the pressor action of angiotensin II. In contrast, the octapeptide [Sari, Ile8] angiotensin II, specifically blocked this effect. The effects on aldosterone of both peptides is illustrated in Figure 2. plus [Sar.1 IU8] A-H I 200 ng/kg/min ~
*I
(n=6)
MINUTES
Figure 2. Change in aldosterone production in response to pre-_ treatment with either [Des-Asp , Ile8] angiotensin II (closed circles) or [Sar 1 , H e 8 ] angiotensin II (closed squares) in dogs receiving constant doses of angiotensin. Each value indicates mean ± S.E. of the change in aldosterone secretion. Angiotensin II-induced biosynthesis was significantly inhibited by preliminary infusion of [Des-Asp', Ile8] angiotensin II, but not by equivalent doses of [Sari, Ile8] angiotensin II. Cortisol secretion was essentially unchanged during these studies (8). Thus, the two antagonists differed strikingly in their ability to block the stimulating action of angiotensin II upon
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532
RAPID COMMUNICATIONS
aldosterone secretion by the adrenal gland and upon vascular smooth muscle. Aldosterone secretion was significantly inhibited by preliminary in- 1 fusion of 200 ng/Kg/min of [Des-Asp , H e 8 ] angiotensin II but1 not by equivalent doses of [Sar , lie 8 ] angiotensin II. Conversely, the pressor action of angiotensin II was unaffected by [Des-Aspl, Ile8] angiotensin II though significant inhibition occurred with relatively small doses of [Sar^, Ile8] angiotensin II.
JCE & M • 1975 Vol 40 • No 3
and Johnson used 30-fold higher doses (6^0 yq/Kg/min). We (8) and others (11-12) have found that these antagonists block pressor responses to angiotensin II in small amounts; only when given in large doses are they shown to inhibit aldosterone biosynthesis.
Taken together these observations indicate a measure of separation of the pressor action of the octapeptide and its ability to stimulate aldosterone biosynthesis. In particular, they lend further support to the conThese investigations suggest that: cept that receptors for angiotensin (a) angiotensin II receptors in adrenal II in adrenal cortex and vascular smooth muscle are functionally difcortex and vascular smooth muscle are ferent (8, 11-12). Although there are functionally different, and (b) [DesAspl, Ile8] ang II is a specific anta- indications that the heptapeptide, [Des-AspU ang II, plays a signifigonist of steroidogenic effect of ang cant role in the regulation of aldoII in adrenal zona glomerulosa. This sterone secretion, the possibility suggests the possibility that chain remains open that angiotensin II acts length of the peptide is important directly in the adrenal cortex to for stimulation of release or stimulate aldosterone biosynthesis. inhibition of aldosterone. Bravo This effect could be accomplished et al. (8) have recently demonstrated through receptors which are conformain dogs that the pressor response to tionally arranged so that a heptaangiotensin II was completely blocked by small doses (40 ng/Kg/min) of [Sari peptide antagonist can interfere either with binding of agonist or IleS] angiotensin II, whereas aldosecondary effects of binding. Much sterone biosynthesis could be inhimore information about the specific bited only at much higher doses nature of interaction of angiotensin (3,000 ng/Kg/min). Peach and Chiu analogs with adrenal cortical recep(3), using suspensions of isolated tors is required before a physiologicortical cells to measure aldosterone cal role for the heptapeptide can be responses obtained results indicating documented with certainty. that [Des-Aspl] angiotensin IIinduced steroidogenesis is not readily inhibited by [Sar^, H e 8 ] angiotensin ACKNOWLEDGEMENT. II. Thus, they showed that the maximum8 inhibitory peptide ratio of [SaH, Supported in part by a grant H e ] angiotensin II : [Des-AspU (HL-6835) from the National Heart angiotensin II was 50 : 1. and Lung Institute, U.S. Public Health Service, and a grant (3050R) from the Heart Association of Our findings conflict with Northeastern Ohio (American Heart those of Davis and Johnson (10) who Association). have shown that an angiotensin II antagonist, [SaH, Ala 8 ] ang II, REFERENCES. blocked both the vasoconstrictor and steroidogenic actions of Intravenously administered angiotensin II. This dis- 1. Blair-West, J.R., J.P. Coghlan, D.A. Denton, J.W. Funder, B.A. crepancy 1s readily explained not by Scoggins, and R.D. Wright, J Clin the type but by the dosage of antaEndo Metab 32: 575 (1971). gonist employed in these studies. The present studies Infused the antagonist at 200 ng/Kg/min while Davis
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RAPID COMMUNICATIONS
533
2. Campbell, W.B., S.N. Brooks, and W.A. Pettinger, Science 184: 994 (1974).
7. Khosla, M.C., M.M. Hall, R.R. Smeby, and F.M. Bumpus, J Med Chem V7: 431 (1974).
3. Peach, M.J., and A.T. Chiu, Circ Res (Suppl I), 34 & 35.: 1-7 (1974).
8. Bravo, E.L., M.C. Khosla, and F.M. Bumpus, Amer J Physiol (In Press, 1974).
4. Glossman, H., A.J. Baukal, and K.J. Catt, J Biol Chem 249; 825 (1974).
9. Mattingly, D.A., J Clin Path 15: 374 (1962).
5. Catt, K.J., M.D. Cain, J.P. Coghlan, P.Z. Zimmett, E. Cran, and J.B. Best, Circ Res (Suppl II) 26 & 27: H-177 (1970). 6. Khosla, M.C., R.A. Leese, W.L. Maloy, A.T. Ferreira, R.R. Smeby, and F.M. Bumpus, J Med Chem ]S: 792 (1972).
10. Johnson, J.A., and J.O. Davis, Science 179: 906 (1973). 11. Williams, G.H., L.M. McDonnell, M.C. Raux, and N.K. Hollenberg, Circ Res 34: 384 (1974). 12. Steele, J.M., Jr., and J. Lowenstein, Circ Res 35; 592 (1974).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:42 For personal use only. No other uses without permission. . All rights reserved.
ACTION OF [1-DES(ASPARTIC ACID), 8-ISOLEUCINE] ANGIOTENSIN II UPON THE PRESSOR AND STEROIDOGENIC ACTIVITY OF ANGIOTENSIN II. Emmanuel L. Bravo, Mahesh C. Khosla, and F. Merlin Bumpus, Research Division, The Cleveland Clinic Foundation, Cleveland, Ohio 44106. ABSTRACT. The vascular and steroidogenic responses to [1-Sarcosine, 8Isoleucine]-, and to [l-Des(Aspartic acid), 8-Isoleucine] angiotensin II were compared in bilaterally nephrectomized, ACTH-suppressed dogs receiving constant infusions of angiotensin II. Aldosterone secretion rate was significantly inhibited by pretreatment with 200 ng/Kg/min of the heptapeptide, [l-Des(Aspartic acid),8-Isoleucine] ang II, but not by similar doses of the octapeptide, [1-Sarcosine, 8-Isoleucine] ang II. In contrast, the pressor action of ang II was unaffected by [l-Des(Aspartic acid), 8-Isoleucine] ang II though significant inhibition occurred with relatively small doses of [1-Sarcosine, 8-Isoleucine] ang II. This study suggests that: (a) angiotensin receptors in adrenal cortex and vascular smooth muscle are functionally different, and (b) [l-Des(Aspartic acid), 8-Isoleucine] angiotensin II is a specific antagonist of steroidogenic effect of ang II. The octapeptide angiotensin II is a potent vasoconstrictor substance. In addition, it has significant aldosterone-stimulating activity. The (2-8) heptapeptide, [Des-Asp1] angiotensin II is a naturally occurring metabolite of angiotensin II, formed by the enzymatic hydrolysis of angiotensinase A. This heptapeptide has minimal pressor activity (10-30% of angiotensin II) but is at least equipotent to its octapeptide progenitor in stimulating aldosterone biosynthesis (1,2). Recently, there has been some speculation that this peptide may be the major stimulus to aldosterone secretion. In adrenal cortical cell suspensions from rabbits, Peach and coworkers (3) have shown that the heptapeptide is more potent than angiotensin II in stimulating the conversion of 3 H-cholesterol to ^H-aldosterone, has a more immediate effect than angiotensin in stimulating aldosterone biosynthesis, and has a much higher affinity for specific binding sites than angiotensin II. Similarly, Catt et a 1.(4,5) reported that the heptapeptide is almost equipotent with the octapeptide in binding-inhibition studies utilizing either beef or rat adrenal cortex particles. Since there
Submitted December 9, 1974
is little circulating heptapeptide in arterial blood (5), it has been proposed that angiotensin could act to stimulate aldosterone biosynthesis via local production of the heptapeptide (1, 3 ) . If this hypothesis were correct, the receptors for angiotensin II in the adrenal cortex and vascular smooth muscle should be different. Therefore, a competitive antagonist of the C-terminal heptapeptide should have potent inhibitory activity on aldosterone secretion but substantially less effect against the pressor action of the octapeptide (ang II). On the other hand, an antagonist directed against the octapeptide should possess extremely high affinity for vascular smooth muscle with minimal activity on adrenal glomerulosa cells. METHODS. The present investigation compared the vascular and steroidogenic responses to the octapeptide, [Sar^, Ile°] angiotensin II (6), and the heptapeptide, [Des-Asp1, lie 8 ] angiotensin II (7), during angiotensin II infusion. Experiments were performed in 17 bilaterally-nephrectomized, ACTH-suppressed male mongrel dogs
530 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:42 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS
weighing 20-25 Kg. The isolated adrenal gland preparation used in these studies has been described previously (8). For the studies, the dogs were anesthetized with pentobarbital (20 mg/Kg). Timed specimens of adrenal venous effluent for aldosterone determinations were collected from the left lumboadrenal vein. All materials given by infusion were dissolved in normal saline and administered into peripheral veins through separate tubings at 1.0 ml per minute. Following two control periods [Des-Aspl, lie**]- or [Sari, H e 8 ] angiotensin II was Infused intravenously at the rate of 200 ng/Kg/m1n. This was followed 15 minutes later with angiotensin II infused at the constant rate of 20 ng/Kg/min. Measurements of aldosterone and cortisol were made at 15-minute intervals and arterial pressure was monitored continuously through a femoral arterial catheter connected to a Sanborn recorder. All blood removed for analysis was replaced with blood from donor dogs. Aldosterone was measured by radioimmunoassay method (8), and cortisol by a fluorometric method (8) modified slightly from that described by Mattingly (9). RESULTS AND DISCUSSION. Arterial pressure response is shown in Figure 1. 25
(n=5)
A-II,
20 ng/kg/min
20 CHANGE l 5 IN MAP , 0
plus [D«-Asp\lle 8 ] 200 ng/kg/min
mmHg
5 ^ plus [Sor1, I I . 8 ] A-H 200 ng/kg/min
0
15'
30'
45'
60'
Figure 1. Arterial pressure response to pretreatment with either [Des-Aspl, Ile°] angiotensin (closed circles) or [Sari, Ile8] (closed squares) in dogs receiving
531
constant doses of angiotensin II. Each value represents the mean ± S.E, of the change in mean arterial pressure. Angiotensin II increased arterial pressure by a mean of 20 mm Hg. Prior administration of the heptapeptide, [Des-Aspl, Ile8] angiotensin II, failed to prevent the pressor action of angiotensin II. In contrast, the octapeptide [Sari, Ile8] angiotensin II, specifically blocked this effect. The effects on aldosterone of both peptides is illustrated in Figure 2. plus [Sar.1 IU8] A-H I 200 ng/kg/min ~
*I
(n=6)
MINUTES
Figure 2. Change in aldosterone production in response to pre-_ treatment with either [Des-Asp , Ile8] angiotensin II (closed circles) or [Sar 1 , H e 8 ] angiotensin II (closed squares) in dogs receiving constant doses of angiotensin. Each value indicates mean ± S.E. of the change in aldosterone secretion. Angiotensin II-induced biosynthesis was significantly inhibited by preliminary infusion of [Des-Asp', Ile8] angiotensin II, but not by equivalent doses of [Sari, Ile8] angiotensin II. Cortisol secretion was essentially unchanged during these studies (8). Thus, the two antagonists differed strikingly in their ability to block the stimulating action of angiotensin II upon
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:42 For personal use only. No other uses without permission. . All rights reserved.
532
RAPID COMMUNICATIONS
aldosterone secretion by the adrenal gland and upon vascular smooth muscle. Aldosterone secretion was significantly inhibited by preliminary in- 1 fusion of 200 ng/Kg/min of [Des-Asp , H e 8 ] angiotensin II but1 not by equivalent doses of [Sar , lie 8 ] angiotensin II. Conversely, the pressor action of angiotensin II was unaffected by [Des-Aspl, Ile8] angiotensin II though significant inhibition occurred with relatively small doses of [Sar^, Ile8] angiotensin II.
JCE & M • 1975 Vol 40 • No 3
and Johnson used 30-fold higher doses (6^0 yq/Kg/min). We (8) and others (11-12) have found that these antagonists block pressor responses to angiotensin II in small amounts; only when given in large doses are they shown to inhibit aldosterone biosynthesis.
Taken together these observations indicate a measure of separation of the pressor action of the octapeptide and its ability to stimulate aldosterone biosynthesis. In particular, they lend further support to the conThese investigations suggest that: cept that receptors for angiotensin (a) angiotensin II receptors in adrenal II in adrenal cortex and vascular smooth muscle are functionally difcortex and vascular smooth muscle are ferent (8, 11-12). Although there are functionally different, and (b) [DesAspl, Ile8] ang II is a specific anta- indications that the heptapeptide, [Des-AspU ang II, plays a signifigonist of steroidogenic effect of ang cant role in the regulation of aldoII in adrenal zona glomerulosa. This sterone secretion, the possibility suggests the possibility that chain remains open that angiotensin II acts length of the peptide is important directly in the adrenal cortex to for stimulation of release or stimulate aldosterone biosynthesis. inhibition of aldosterone. Bravo This effect could be accomplished et al. (8) have recently demonstrated through receptors which are conformain dogs that the pressor response to tionally arranged so that a heptaangiotensin II was completely blocked by small doses (40 ng/Kg/min) of [Sari peptide antagonist can interfere either with binding of agonist or IleS] angiotensin II, whereas aldosecondary effects of binding. Much sterone biosynthesis could be inhimore information about the specific bited only at much higher doses nature of interaction of angiotensin (3,000 ng/Kg/min). Peach and Chiu analogs with adrenal cortical recep(3), using suspensions of isolated tors is required before a physiologicortical cells to measure aldosterone cal role for the heptapeptide can be responses obtained results indicating documented with certainty. that [Des-Aspl] angiotensin IIinduced steroidogenesis is not readily inhibited by [Sar^, H e 8 ] angiotensin ACKNOWLEDGEMENT. II. Thus, they showed that the maximum8 inhibitory peptide ratio of [SaH, Supported in part by a grant H e ] angiotensin II : [Des-AspU (HL-6835) from the National Heart angiotensin II was 50 : 1. and Lung Institute, U.S. Public Health Service, and a grant (3050R) from the Heart Association of Our findings conflict with Northeastern Ohio (American Heart those of Davis and Johnson (10) who Association). have shown that an angiotensin II antagonist, [SaH, Ala 8 ] ang II, REFERENCES. blocked both the vasoconstrictor and steroidogenic actions of Intravenously administered angiotensin II. This dis- 1. Blair-West, J.R., J.P. Coghlan, D.A. Denton, J.W. Funder, B.A. crepancy 1s readily explained not by Scoggins, and R.D. Wright, J Clin the type but by the dosage of antaEndo Metab 32: 575 (1971). gonist employed in these studies. The present studies Infused the antagonist at 200 ng/Kg/min while Davis
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RAPID COMMUNICATIONS
533
2. Campbell, W.B., S.N. Brooks, and W.A. Pettinger, Science 184: 994 (1974).
7. Khosla, M.C., M.M. Hall, R.R. Smeby, and F.M. Bumpus, J Med Chem V7: 431 (1974).
3. Peach, M.J., and A.T. Chiu, Circ Res (Suppl I), 34 & 35.: 1-7 (1974).
8. Bravo, E.L., M.C. Khosla, and F.M. Bumpus, Amer J Physiol (In Press, 1974).
4. Glossman, H., A.J. Baukal, and K.J. Catt, J Biol Chem 249; 825 (1974).
9. Mattingly, D.A., J Clin Path 15: 374 (1962).
5. Catt, K.J., M.D. Cain, J.P. Coghlan, P.Z. Zimmett, E. Cran, and J.B. Best, Circ Res (Suppl II) 26 & 27: H-177 (1970). 6. Khosla, M.C., R.A. Leese, W.L. Maloy, A.T. Ferreira, R.R. Smeby, and F.M. Bumpus, J Med Chem ]S: 792 (1972).
10. Johnson, J.A., and J.O. Davis, Science 179: 906 (1973). 11. Williams, G.H., L.M. McDonnell, M.C. Raux, and N.K. Hollenberg, Circ Res 34: 384 (1974). 12. Steele, J.M., Jr., and J. Lowenstein, Circ Res 35; 592 (1974).
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