Des-1-Asp-Angiotensin II POSSIBLE INTRARENAL ROLE IN HOMEOSTASIS IN THE DOG By Ronald H. Freeman, James O. Davis, and Thomas E. Lohmeier
ABSTRACT The relative effects of des-1-Asp-angiotensin II and angiotensin II on renal function, including renin secretion, were investigated in normal and sodium-depleted dogs. Intrarenal arterial infusion of the heptapeptide fragment into normal dogs at a rate which was calculated to increase blood levels by only 7 ng/100 ml decreased renal blood flow from 254 ± 8 ml/min to 220 ± 12 and 219 ± 12 ml/min (P < 0.01 for both values) after 10 and 30 minutes of infusion, respectively; renin secretion decreased from 502 ± 214 ng/min to 253 ± 109 and 180 ± 53 ng/min (P < 0.05 for both values). Infusion of angiotensin II at the same rate decreased renal blood flow from 251 ± 26 ml/min to 224 ± 22 and 220 ± 16 ml/min (P < 0.01 and 0.025, respectively) and decreased renin secretion from 374 ± 25 ng/min to 166 ± 76 and 131 ± 37 ng/min (P < 0.025 for both values). Neither peptide significantly changed mean arterial blood pressure, creatinine clearance, or excreted sodium in these dogs. Infusion of des-1-Asp-angiotensin II into sodium-depleted dogs decreased renin secretion from 1094 ±211 ng/min to 768 ± 132 and 499 ± 31 ng/min (P < 0.025 for both values) after 10 and 30 minutes of infusion. Angiotensin II infusion decreased renin secretion from 1102 ± 134 to 495 ±235 and 502 ± 129 ng/min in these dogs (P < 0.05 and 0.025, respectively). Neither peptide significantly altered renal blood flow, arterial blood pressure, creatinine clearance, or excreted sodium in the sodium-depleted dogs. The data demonstrated that these two peptides have similar effects on the renin secretory mechanism and the vascular receptor at the level of the renal arterioles.
• Although the pressor activity of des-1-Asp-angiotensin II is only 25-50% of the activity of its octapeptide precursor (1), the observation that this [2-8]-heptapeptide metabolite of angiotensin II is a potent stimulus to aldosterone biosynthesis (2-5) has prompted speculation that it might be a mediator of the renin-angiotensin system at the level of the adrenal receptor. Also, recent evidence (Lohmeier et al., unpublished observation) shows that angiotensin II and the heptapeptide produce essentially the same qualitative and quantitative changes in the steroid profile for aldosterone, corticosterone, and cortisol in the dog. The relative potency of angiotensin II and des-1-Asp-angiotensin II for specific receptor sites in the juxtaglomerular cells and the renal arterioles has not been studied previously, but both peptides appear to decrease renin production in the isolated perfused rat kidney (6). Therefore, the present study was undertaken to assess the relative effects of these two peptides on the rate of renin release and on glomerular filtration rate, renal blood flow, renal resistance, and renal sodium excretion in normal and sodium-depleted dogs. This study was initiFrom the Department of Physiology, University of Missouri School of Medicine, Columbia, Missouri 65201. Dr. Freeman is a Fellow of the National Kidney Foundation. Received November 25, 1974. Accepted for publication April 10, 1975.
30
ated to provide new information concerning a possible physiological role for des-1-Asp-angiotensin II as a mediator of the renin-angiotensin system. Methods Fourteen female mongrel dogs weighing 16-22 kg were used in this study. Seven dogs were fed a diet that provided 65 mEq of sodium and 55 mEq of potassium daily for at least 3 days prior to the acute experiment. The remaining seven dogs were sodium depleted by a combination of a low-sodium diet (less than 3 mEq of sodium daily for 4 days) and the administration of a mercurial diuretic (2 ml of Mercuhydrin, im, for the first 2 days). Water was available ad libitum. The dogs were fed in the late afternoon, and all acute experiments were performed with the dogs in the postabsorptive state. Experiment 1: Effects of an Intrarenal Arterial Infusion of Angiotensin II and Des-1-Asp-Angiotensin II on Renal Function in Five Normal Dogs.—On the day of the experiment, the dog was anesthetized with sodium pentobarbital (30 mg/kg, iv), and polyvinyl catheters (French no. 8) were placed in the femoral artery and vein. The left kidney was exposed via a flank incision, and a catheter (PE 160) was placed in the ureter for continuous collection of urine samples. A noncannulating electromagnetic flow probe (Carolina Medical Electronics) was placed on the left renal artery for the measurement of renal blood flow. An 18-gauge needle was placed in the left renal vein to obtain renal venous blood samples, and a 22-gauge needle was inserted in the left renal artery distal to the flow probe for intrarenal infusions. Circulation Research, Vol. 37, July 1975
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DES-1-ASP-ANGIOTENSIN II: INTRARENAL ACTION After surgical preparations were finished, an intrarenal infusion of 0.9% saline was begun at 0.6 ml/min. At this time also, a priming dose of creatinine was given intravenously, and a constant infusion of creatinine was begun via the femoral vein catheter. A postsurgery stabilization period of 60 minutes was allowed before the experiment was started. Following this stabilization period, two 20-minute control renal clearance periods were observed; infusion of 0.9% saline into the renal artery was then discontinued. Infusion of either angiotensin II (Hypertensin, Ciba) or des-1-Asp-angiotensin II (angiotensin II heptapeptide, Schwartz/Mann) was initiated at a rate which was calculated to increase the renal blood concentration by 7 ng/100 ml (0.6 ml/min in 0.9% saline), and two 20-minute experimental renal clearance periods were observed. Infusion of the peptide was then stopped and replaced with an infusion of 0.9% saline. Forty minutes later, two 20-minute recovery clearance periods were monitored. Subsequently, infusion of the other peptide into the renal artery was begun at a rate which was calculated to increase the renal blood concentration by 7 ng/100 ml. Again, two 20-minute experimental renal clearance periods were observed, and the infusion was then changed to 0.9% saline. Forty minutes later, two final recovery renal clearance periods were monitored. In three of the five dogs, angiotensin II was infused first and des-1-Asp-angiotensin II was infused second. In the other two dogs the order was reversed. Arterial and renal venous blood samples for the determination of plasma renin activity and creatinine were obtained at the midpoint of each control, experimental, and recovery period. All blood removed for sampling was replaced with an equal volume of fresh donor blood. The values for arterial blood pressure and renal blood flow presented in the results are those which were recorded immediately prior to blood sampling. Experiment 2: Effects of Intrarenal Arterial Infusion of Angiotensin II and Des-1-Asp-Angiotensin II on Renal Function in Five Sodium-Depleted Dogs.—To evaluate the effects of angiotensin II and des-1-Asp-angiotensin II in these dogs, an experimental protocol identical to that in experiment 1 was followed. In three of these dogs, des-1-Asp-angiotensin II was infused first and angiotensin II was infused second. The order of administration was reversed in the other two dogs. Experiment 3: Dose-Response Relationship of Angiotensin II and Des-1-Asp-Angiotensin II in Normal and Sodium-Depleted Dogs.—The four dogs used in this part of the study were prepared like the dogs in experiments 1 and 2 except that catheters were not placed in the ureter or the renal vein. After the 60-minute postsurgery stabilization period, control levels of renal blood flow and mean arterial blood pressure were recorded, and the responses to three or six doses (range of 50 to 500 ng) of exogenous angiotensin II or des-1-Asp-angiotensin II were measured; the two peptides were injected into the renal artery via the 22-gauge needle. The octapeptide and the heptapeptide were administered as single injections in a constant volume of 0.5 ml of 0.9% saline. Recovery periods of 8-15 minutes were allowed between injections. Analytical Methods.—The method for measurement of plasma renin activity has been reported previously (7). Briefly, 10-ml samples of arterial and renal venous blood
31 were collected in 0.1 ml of 10% ethylenediaminetetraacetic acid (EDTA) during each control, experimental, and recovery period. The samples were cooled at 4°C, and the plasma was removed after centrifugation. Plasma samples were stored frozen until they were processed for angiotensin generation. Samples (2 ml) of plasma were dialyzed against a phosphate buffer (pH 5.3) for 18 hours (three changes). After sodium chloride and diisopropylfluorophosphate were added, the samples were incubated for 3 hours at 37 °C. Following enzyme inactivation in a boiling water bath for 10 minutes, each sample was diluted at 4 ml with a phosphate buffer (pH 8.3), and the supernatant fluid was stored frozen until it could be assayed. Samples were assayed by the pressor response in the pentobarbital-anesthetized, pentolinium-blocked rat with angiotensin II (Hypertensin, Ciba) as the standard. Renin secretion (ng/min) was calculated by multiplying the renal plasma flow by the difference between renal venous and arterial plasma renin activity. Hematocrits were determined in duplicate by a microhematocrit method. Plasma and urinary levels of creatinine were determined by standard methods; urinary electrolytes were measured by flame photometry. Student's J-test for paired observations was used for statistical analysis of the data. Values are presented as means ± SE. Results Experiment 1.—Renin secretion and renal function data for normal dogs before, during, and after the intrarenal infusions of angiotensin II and des-1Asp-angiotensin II are summarized in Table 1. Infusion of either peptide at a rate which increased renal blood levels by 7 ng/100 ml consistently decreased both renal blood flow and renin secretion; the magnitude of the responses was not significantly different (P > 0.1) for either the decrease in renal blood flow or the decrease in renin secretion. Renal sodium excretion appeared to decline in three of the five dogs for both peptides, but the decrease was not significant for the group (P > 0.05) for either the octapeptide or the heptapeptide. Mean arterial blood pressure and creatinine clearance were not influenced by infusion of either peptide. Thus, it appears that angiotensin II and its metabolite, des-1-Asp-angiotensin II, exert qualitatively and quantitatively similar actions on renin secretion and renal blood flow in the normal dog when they are infused at the present dose level. Experiment 2.—Renin secretion and renal function data for sodium-depleted dogs before, during, and after the intrarenal infusions of angiotensin II and des-1-Asp-angiotensin II are summarized in Table 2. Infusion of either angiotensin II or the angiotensin II heptapeptide fragment resulted in a consistent decrease in the renin secretion rate.
Circulation Research, Vol. 37, July 1975
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FREEMAN. DAVIS. LOHMEIER TABLE 1
Arterial Blood Pressure, Renal Blood Flow, Creatinine Clearance, Sodium Excretion, and Renin Secretion by the Experimental Kidney before, during, and after Intrarenal Infusion of Angiotensin II and Des-1-Asp-Angiotensin II in Five Normal Dogs Control period 1
ABP(mmHg) RBF (ml/rain) CCT (ml/min) E Na (/iEq/min) RS (ng/min)
141 244 32 20 578
±9 ± 24 ± 5 ± 9 ± 242
ABP(mmHg) RBF (ml/min) Ccr (ml/min) E Na (^Eq/min) RS (ng/min)
136 245 32 27 395
±6 ± 14 ± 4 ±11 ± 113
Experimental period 2
Recovery period
1
Angiotensin II Infusion 140 ± 8 138 ± 7 137 251 ± 26 224 ± 22* 220 30 33 ± 6 36 ± 4 15 27 ± 13 19 ± 9 131 374 ± 25 166 ± 76f Des-1-Asp-Angiotensin II Infusion TX 137 136 ± 6 136 ± 6 219 254 ± 8 220 ± 12* 31 34 ± 3 30 ± 6 25 ± 11 18 ± 9 16 502 ± 214 253 ± 109^ 180
±7 ± 16t ± 6 ± 5 ± 37f ± ± ± ± ±
6 12* 4 9 53^:
136
±
269
±
35
±
32 441
±
137 250 32
8 22
136 269
4 13
34 28
±
± ±
8 19 4
±
14
±
125
569
±
256
±
5 20 5
137 256
± ±
6 23
± ±
18 ± 10 345 ± 122
31 ± 6 20 ± 11 358 ± 68
Values are means ± SE. ABP = arterial blood pressure, RBF = renal blood flow, Ccr = creatinine clearance, ENa = sodium excretion, and RS = renin secretion. Angiotensin II and des-1-Asp-angiotensin II were infused at a rate calculated to increase renal blood concentration by 7 ng/100 ml; values for experimental periods 1 and 2 are those after 10 and 30 minutes of peptide infusion respectively. * P < 0.01 compared with the mean of the two control periods. f P < 0.025 compared with the mean of the two control periods. %P < 0.05 compared with the mean of the two control periods.
Mean arterial blood pressure, renal blood flow, creatinine clearance, and renal sodium excretion were not detectably influenced by infusion of either peptide at the present dose level. Thus, as in the normal dog, it appears that both the octapeptide and its heptapeptide metabolite exert qualitatively
and quantitatively similar actions on renin secretion and renal function in the sodium-depleted dog. Experiment 3.—Figure 1 shows the decrease in renal blood flow following intrarenal arterial injection of exogenous des-1-Asp-angiotensin II and angiotensin II into two normal and two sodium-
TABLE 2
Arterial Blood Pressure, Renal Blood Flow, Creatinine Clearance, Sodium Excretion, and Renin Secretion by the Experimental Kidney before, during, and after Intrarenal Infusion of Angiotensin II and Des-1-Asp-Angiotensin II in Five Sodium-Depleted Dogs Control period
2
1
A B P ( m m Hg) R B F (ml/min) Ccr (ml/min)
137 225
±
11
±
32
±
E
40 3 4
6± 880 28
Na
(jiEq/min)
R S (ng/min)
Recovery period
Experimental period
1
138 233 31 7 1102
Angiotensin ± 11 139 ± 42 218 ±2 32 ±5 7 ± 134 495
137 213 30 5 1094
± 14 ± 42 ± 2 ± 2 ± 211
II Infusion ± 9 ± 28 ±2 ±4 ± 235*
1
2
133 ± 11 223 ± 32 33 ± 3 7 ±4 1033 ± 149
136 ± 8 228 ± 37 34 ± 4 7 ±5 1226 ± 205
135 224 33 8 905
136 220 31 10 1199
2
137 215 32 7 502
± 10 ± 27 ±3 ±4 ± 129f
Des-1 -Asp-Angiotensin II Infusion
A B P ( m m Hg) R B F (ml/min) Ccr (ml/min) E N a (^Eq/min) R S (ng/min)
137 213 28 5 1345
±
13 25
±
2
±
236
±
1
137 210 29 5 768
± 11 ± 38 ± 3 ± 3 ± 132t
139 203 29 5 499
± ± ± ± ±
11
32 2
2 31t
± 11 ± 39 ± 3 ± 4 ± 27
± ± ± ± ±
12
44 2 5 188
Values are means ± SE. ABP = arterial blood pressure, RBF = renal blood flow, Ccr = creatinine clearance, ENa = sodium excretion, "and RS = renin secretion. Angiotensin II and des-1-Asp-angiotensin II were infused at a rate calculated to increase renal blood concentration by 7 ng/100 ml; values for experimental periods 1 and 2 are those after 10 and 30 minutes of peptide infusion, respectively. * P < 0.05 compared with the mean of the two control periods. i P < 0.025 compared with the mean of the two control periods. Circulation Research, Vol. 37. July 1975
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DES-1-ASP-ANGIOTENSIN II: INTRARENAL ACTION
IOO
200
NANOGRAMS
300
400
500
OF PEPTIDE
FIGURE 1
Effects of single intrarenal arterial injections of angiotensin II (squares) and des-1-Asp-angiotensin II (circles) on renal blood flow in two normal and two sodium-depleted dogs.
depleted dogs. The responses to the heptapeptide and the octapeptide were similar in each dog; however, the dose-response curves to each peptide were shifted down and to the right in the sodiumdepleted dogs. The dose of des-1-Asp-angiotensin II or angiotensin II required to reduce renal blood flow by 50% was four times greater in the sodiumdepleted dogs (approximately 400 ng for both peptides) than it was in the normal dogs (100 ng for both peptides). Discussion The important new findings of the present study are the comparative effects of des-1-Asp-angiotensin II and angiotensin II on renal blood flow and renin secretion. Infusion of the heptapeptide into the renal artery of normal and sodium-depleted dogs at a rate which was calculated to increase renal blood levels by only 7 ng/100 ml resulted in a significant reduction in both renal blood flow and renin secretion in normal dogs (Table 1) and a significant reduction in renin secretion with no change in renal blood flow in sodium-depleted dogs (Table 2). Infusion of des-1-Asp-angiotensin II did not significantly alter creatinine clearance or sodium excretion in either group of dogs. In both normal and sodium-depleted dogs, infusion of angiotensin II at this same rate produced results which are comparable both qualitatively and quantitatively to those produced by the infusion of des-1Asp-angiotensin II. Both agonists also demonstrated comparable dose-response characteristics in normal and sodium-depleted dogs as judged by a reduction in renal blood flow following single intrarenal arterial injections (Fig. 1). Thus, the present
33
data suggest strongly that the octapeptide and its natural metabolite des-1-Asp-angiotensin II (8) exert qualitatively and quantitatively similar actions on the renin secretory mechanism and the renal arterioles. Although studies on the mechanism of action of des-1-Asp-angiotensin II in the kidney are lacking, the results of the present study agree with the earlier finding that angiotensin II inhibits renin secretion (9, 10). The precise nature of this inhibitory action is not thoroughly understood, but the present data are consistent with the suggestion that angiotensin II influences renin secretion via a vascular receptor in the renal arteriole or by direct action on the juxtaglomerular cells (9, 10). The earlier finding (10) of failure of renal blood flow to change in the sodium-depleted dog with a nonfiltering kidney points to a direct action of angiotensin II on the juxtaglomerular cells. Presumably, des-1-Asp-angiotensin II inhibits renin secretion by a similar mechanism. Recently, Hollenberg et al. (11) have found that normal human subjects on a sodium-restricted diet are less sensitive to angiotensin II and more sensitive to norepinephrine, as judged by a reduction in renal blood flow, than are subjects on a normal sodium intake. Because the changes in sensitivity to angiotensin II and norepinephrine were directionally opposite, it seems unlikely that they were due to nonspecific effects of sodium restriction on the vascular smooth muscle. A similar mechanism may provide the basis for the changes in sensitivity to both the octapeptide and the heptapeptide which were observed in the present study. Recent studies have suggested that the vascular and adrenal receptors for angiotensin differ (12, 13, Bravo and Bumpus, unpublished observation); the receptors at the two sites may have different affinities for the octapeptide, or, alternatively, two different agonists may mediate the pressor and steroidogenic responses (13). With regard to the latter possibility, the observation that des-1-Aspangiotensin II is a potent stimulus to aldosterone production (2-5) but possesses only 25-50% of the pressor activity of angiotensin II (1) has prompted speculation that it is an important mediator of the renin-angiotensin system at the adrenal receptor level via local tissue production from its octapeptide precursor (2-5). The data presented in this study indicate that the intrarenal receptor sites for angiotensin are influenced in a qualitatively and quantitatively similar manner by intrarenal arterial infusion of either des-1-Asp-angiotensin II or angiotensin II.
Circulation Research, Vol. 37. July 1975
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FREEMAN, DAVIS. LOHMEIER
Since the potency of des-1-Asp-angiotensin II, as judged by an increase in mean arterial blood pressure, is only 25-50% of that of angiotensin II (1), the present data imply that the renal vascular receptors and other vascular receptors for angiotensin differ. This observation could mean that des-1Asp-angiotensin II also is a mediator of the reninangiotensin system at the level of the renal arteriolar receptors via local production of this agonist in a manner analogous to that postulated to occur at the adrenal receptor site. Another possible explanation for differences among arteriolar receptors is that the availability of local angiotensinases for conversion of angiotensin II to the heptapeptide differs among tissues. Osborne et al. (14) have separated the [2-8]-heptapeptide from other metabolic products in the effluent of isolated rat kidneys perfused with l-Asp-4-[3H]Tyr-5-Val-angiotensin II. This concept of local production of des-1-Aspangiotensin II at the level of the renal arteriolar and adrenal receptor sites is an attractive one which can reconcile the observations of the present study with those of other investigations concerning the relative pressor and steroidogenic activities of des-1-Asp-angiotensin II and angiotensin II. The present study thus provides evidence that both angiotensin II and its metabolite des-1-Aspangiotensin II exert qualitatively and quantitatively similar actions on the renin secretory mechanism and the vascular receptor at the level of the renal arterioles. Since previous studies have indicated that des-1-Asp-angiotensin II has less pressor activity than does angiotensin II, the present data suggest that the renal vascular receptors and other vascular receptors for angiotensin are functionally different. It is hypothesized that local production of des-1-Asp-angiotensin II at the level of the renal arteriolar receptors may occur. Acknowledgment We thank Mr. John E. Thomas, Ms. Jane Green, and Ms. Rebecca Wright for their excellent technical work.
References 1. BUMPUS FM, SMEBY RR: Angiotensin. In Renal Hypertension, edited by IH Page and JW McCubbin. Chicago, Year Book Medical Publishers, Inc., 1968, p 80 2. BLAIR-WEST JR, COGHLAN JP, DENTON DA, FUNDER JW,
SCOGGINS BA, WRIGHT RD: Effect of the heptapeptide
(2-8) and hexapeptide (3-8) fragments of angiotensin II on aldosterone secretion. J Clin Endocrinol Metab 32:575-578, 1971 3. CHIU AT, PEACH MJ: Inhibition of induced aldosterone biosynthesis with a specific antagonist of angiotensin II. Proc Natl Acad Sci USA 71:341-344, 1974 4. CAMPBELL WB, BROOKS SN, PETTINGER WA: Angiotensin II-
and angiotensin Ill-induced aldosterone release in the rat. Science 184:994-996, 1974 5. SPIELMAN WS, DAVIS JO, FREEMAN RH, JOHNSON JA: Stimu-
lation of aldosterone by a heptapeptide fragment of angiotensin II in the rat (abstr). Fed Proc 32:254, 1974 6. VANDONGEN R, PEART WS, BOYD G W: Effect of angiotensin II
and its nonpressor derivatives on renin secretion. Am J Physiol 226:277-282, 1974 7. SCHNEIDER EG, ROSTORFER HH, NASH FD: Distribution
volume and metabolic clearance rate of renin in anesthetized nephrectomized dogs. Am J Physiol 215:1115-1122, 1968 8. CAIN MD, CATT KJ, COGHLAN JP: Effect of circulating
fragments of angiotensin II on radioimmunoassay in arterial and venous blood. J Clin Endocrinol Metab 29:1639-1643, 1969 9. BUNAG RD, PAGE IH, MCCUBBIN JW: Inhibition of renin
release by vasopressin and angiotensin. Cardiovasc Res 1:67-73, 1967 10. SHADE RE, DAVIS JO, JOHNSON JA, GOTSHALL RW, SPIELMAN
WS: Mechanism of action of angiotensin II and antidiuretic hormone on renin secretion. Am J Physiol 224:926-929, 1973 11. HOLLENBEBG N K , SOLOMON H S , ADAMS D F , ABRAMS H L ,
MERRILL JP: Renal vascular responses to angiotensin and norepinephrine in normal man: Effect of sodium intake. Circ Res 31:750-757, 1972 12. WILLIAMS GH, MCDONNELL LM, RAUZ MC, HOLLENBERG
NK: Evidence for two different angiotensin II receptors in rat adrenal glomerulosa and rabbit vascular smooth muscle cells. Circ Res 34:384-390, 1974 13. STEELE JM, JR, LOWENSTEIN J: Differential effects of an
angiotensin II analogue on pressor and adrenal receptors in the rabbit. Circ Res 35:592-600, 1974 14. OSBORNE MJ, D'AURIAC GA, MEYER P, WORCEL M: Mecha-
nism of extraction of angiotensin II in coronary and renal circulations. Life Sci [I] 9:859-867, 1970
Circulation Research, Vol. 37. July 1975
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Des-1-Asp-angiotensin II. Possible intrarenal role in homeostasis in the dog. R H Freeman, J O Davis and T E Lohmeier Circ Res. 1975;37:30-34 doi: 10.1161/01.RES.37.1.30 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1975 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
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ABSTRACT The relative effects of des-1-Asp-angiotensin II and angiotensin II on renal function, including renin secretion, were investigated in normal and sodium-depleted dogs. Intrarenal arterial infusion of the heptapeptide fragment into normal dogs at a rate which was calculated to increase blood levels by only 7 ng/100 ml decreased renal blood flow from 254 ± 8 ml/min to 220 ± 12 and 219 ± 12 ml/min (P < 0.01 for both values) after 10 and 30 minutes of infusion, respectively; renin secretion decreased from 502 ± 214 ng/min to 253 ± 109 and 180 ± 53 ng/min (P < 0.05 for both values). Infusion of angiotensin II at the same rate decreased renal blood flow from 251 ± 26 ml/min to 224 ± 22 and 220 ± 16 ml/min (P < 0.01 and 0.025, respectively) and decreased renin secretion from 374 ± 25 ng/min to 166 ± 76 and 131 ± 37 ng/min (P < 0.025 for both values). Neither peptide significantly changed mean arterial blood pressure, creatinine clearance, or excreted sodium in these dogs. Infusion of des-1-Asp-angiotensin II into sodium-depleted dogs decreased renin secretion from 1094 ±211 ng/min to 768 ± 132 and 499 ± 31 ng/min (P < 0.025 for both values) after 10 and 30 minutes of infusion. Angiotensin II infusion decreased renin secretion from 1102 ± 134 to 495 ±235 and 502 ± 129 ng/min in these dogs (P < 0.05 and 0.025, respectively). Neither peptide significantly altered renal blood flow, arterial blood pressure, creatinine clearance, or excreted sodium in the sodium-depleted dogs. The data demonstrated that these two peptides have similar effects on the renin secretory mechanism and the vascular receptor at the level of the renal arterioles.
• Although the pressor activity of des-1-Asp-angiotensin II is only 25-50% of the activity of its octapeptide precursor (1), the observation that this [2-8]-heptapeptide metabolite of angiotensin II is a potent stimulus to aldosterone biosynthesis (2-5) has prompted speculation that it might be a mediator of the renin-angiotensin system at the level of the adrenal receptor. Also, recent evidence (Lohmeier et al., unpublished observation) shows that angiotensin II and the heptapeptide produce essentially the same qualitative and quantitative changes in the steroid profile for aldosterone, corticosterone, and cortisol in the dog. The relative potency of angiotensin II and des-1-Asp-angiotensin II for specific receptor sites in the juxtaglomerular cells and the renal arterioles has not been studied previously, but both peptides appear to decrease renin production in the isolated perfused rat kidney (6). Therefore, the present study was undertaken to assess the relative effects of these two peptides on the rate of renin release and on glomerular filtration rate, renal blood flow, renal resistance, and renal sodium excretion in normal and sodium-depleted dogs. This study was initiFrom the Department of Physiology, University of Missouri School of Medicine, Columbia, Missouri 65201. Dr. Freeman is a Fellow of the National Kidney Foundation. Received November 25, 1974. Accepted for publication April 10, 1975.
30
ated to provide new information concerning a possible physiological role for des-1-Asp-angiotensin II as a mediator of the renin-angiotensin system. Methods Fourteen female mongrel dogs weighing 16-22 kg were used in this study. Seven dogs were fed a diet that provided 65 mEq of sodium and 55 mEq of potassium daily for at least 3 days prior to the acute experiment. The remaining seven dogs were sodium depleted by a combination of a low-sodium diet (less than 3 mEq of sodium daily for 4 days) and the administration of a mercurial diuretic (2 ml of Mercuhydrin, im, for the first 2 days). Water was available ad libitum. The dogs were fed in the late afternoon, and all acute experiments were performed with the dogs in the postabsorptive state. Experiment 1: Effects of an Intrarenal Arterial Infusion of Angiotensin II and Des-1-Asp-Angiotensin II on Renal Function in Five Normal Dogs.—On the day of the experiment, the dog was anesthetized with sodium pentobarbital (30 mg/kg, iv), and polyvinyl catheters (French no. 8) were placed in the femoral artery and vein. The left kidney was exposed via a flank incision, and a catheter (PE 160) was placed in the ureter for continuous collection of urine samples. A noncannulating electromagnetic flow probe (Carolina Medical Electronics) was placed on the left renal artery for the measurement of renal blood flow. An 18-gauge needle was placed in the left renal vein to obtain renal venous blood samples, and a 22-gauge needle was inserted in the left renal artery distal to the flow probe for intrarenal infusions. Circulation Research, Vol. 37, July 1975
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DES-1-ASP-ANGIOTENSIN II: INTRARENAL ACTION After surgical preparations were finished, an intrarenal infusion of 0.9% saline was begun at 0.6 ml/min. At this time also, a priming dose of creatinine was given intravenously, and a constant infusion of creatinine was begun via the femoral vein catheter. A postsurgery stabilization period of 60 minutes was allowed before the experiment was started. Following this stabilization period, two 20-minute control renal clearance periods were observed; infusion of 0.9% saline into the renal artery was then discontinued. Infusion of either angiotensin II (Hypertensin, Ciba) or des-1-Asp-angiotensin II (angiotensin II heptapeptide, Schwartz/Mann) was initiated at a rate which was calculated to increase the renal blood concentration by 7 ng/100 ml (0.6 ml/min in 0.9% saline), and two 20-minute experimental renal clearance periods were observed. Infusion of the peptide was then stopped and replaced with an infusion of 0.9% saline. Forty minutes later, two 20-minute recovery clearance periods were monitored. Subsequently, infusion of the other peptide into the renal artery was begun at a rate which was calculated to increase the renal blood concentration by 7 ng/100 ml. Again, two 20-minute experimental renal clearance periods were observed, and the infusion was then changed to 0.9% saline. Forty minutes later, two final recovery renal clearance periods were monitored. In three of the five dogs, angiotensin II was infused first and des-1-Asp-angiotensin II was infused second. In the other two dogs the order was reversed. Arterial and renal venous blood samples for the determination of plasma renin activity and creatinine were obtained at the midpoint of each control, experimental, and recovery period. All blood removed for sampling was replaced with an equal volume of fresh donor blood. The values for arterial blood pressure and renal blood flow presented in the results are those which were recorded immediately prior to blood sampling. Experiment 2: Effects of Intrarenal Arterial Infusion of Angiotensin II and Des-1-Asp-Angiotensin II on Renal Function in Five Sodium-Depleted Dogs.—To evaluate the effects of angiotensin II and des-1-Asp-angiotensin II in these dogs, an experimental protocol identical to that in experiment 1 was followed. In three of these dogs, des-1-Asp-angiotensin II was infused first and angiotensin II was infused second. The order of administration was reversed in the other two dogs. Experiment 3: Dose-Response Relationship of Angiotensin II and Des-1-Asp-Angiotensin II in Normal and Sodium-Depleted Dogs.—The four dogs used in this part of the study were prepared like the dogs in experiments 1 and 2 except that catheters were not placed in the ureter or the renal vein. After the 60-minute postsurgery stabilization period, control levels of renal blood flow and mean arterial blood pressure were recorded, and the responses to three or six doses (range of 50 to 500 ng) of exogenous angiotensin II or des-1-Asp-angiotensin II were measured; the two peptides were injected into the renal artery via the 22-gauge needle. The octapeptide and the heptapeptide were administered as single injections in a constant volume of 0.5 ml of 0.9% saline. Recovery periods of 8-15 minutes were allowed between injections. Analytical Methods.—The method for measurement of plasma renin activity has been reported previously (7). Briefly, 10-ml samples of arterial and renal venous blood
31 were collected in 0.1 ml of 10% ethylenediaminetetraacetic acid (EDTA) during each control, experimental, and recovery period. The samples were cooled at 4°C, and the plasma was removed after centrifugation. Plasma samples were stored frozen until they were processed for angiotensin generation. Samples (2 ml) of plasma were dialyzed against a phosphate buffer (pH 5.3) for 18 hours (three changes). After sodium chloride and diisopropylfluorophosphate were added, the samples were incubated for 3 hours at 37 °C. Following enzyme inactivation in a boiling water bath for 10 minutes, each sample was diluted at 4 ml with a phosphate buffer (pH 8.3), and the supernatant fluid was stored frozen until it could be assayed. Samples were assayed by the pressor response in the pentobarbital-anesthetized, pentolinium-blocked rat with angiotensin II (Hypertensin, Ciba) as the standard. Renin secretion (ng/min) was calculated by multiplying the renal plasma flow by the difference between renal venous and arterial plasma renin activity. Hematocrits were determined in duplicate by a microhematocrit method. Plasma and urinary levels of creatinine were determined by standard methods; urinary electrolytes were measured by flame photometry. Student's J-test for paired observations was used for statistical analysis of the data. Values are presented as means ± SE. Results Experiment 1.—Renin secretion and renal function data for normal dogs before, during, and after the intrarenal infusions of angiotensin II and des-1Asp-angiotensin II are summarized in Table 1. Infusion of either peptide at a rate which increased renal blood levels by 7 ng/100 ml consistently decreased both renal blood flow and renin secretion; the magnitude of the responses was not significantly different (P > 0.1) for either the decrease in renal blood flow or the decrease in renin secretion. Renal sodium excretion appeared to decline in three of the five dogs for both peptides, but the decrease was not significant for the group (P > 0.05) for either the octapeptide or the heptapeptide. Mean arterial blood pressure and creatinine clearance were not influenced by infusion of either peptide. Thus, it appears that angiotensin II and its metabolite, des-1-Asp-angiotensin II, exert qualitatively and quantitatively similar actions on renin secretion and renal blood flow in the normal dog when they are infused at the present dose level. Experiment 2.—Renin secretion and renal function data for sodium-depleted dogs before, during, and after the intrarenal infusions of angiotensin II and des-1-Asp-angiotensin II are summarized in Table 2. Infusion of either angiotensin II or the angiotensin II heptapeptide fragment resulted in a consistent decrease in the renin secretion rate.
Circulation Research, Vol. 37, July 1975
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32
FREEMAN. DAVIS. LOHMEIER TABLE 1
Arterial Blood Pressure, Renal Blood Flow, Creatinine Clearance, Sodium Excretion, and Renin Secretion by the Experimental Kidney before, during, and after Intrarenal Infusion of Angiotensin II and Des-1-Asp-Angiotensin II in Five Normal Dogs Control period 1
ABP(mmHg) RBF (ml/rain) CCT (ml/min) E Na (/iEq/min) RS (ng/min)
141 244 32 20 578
±9 ± 24 ± 5 ± 9 ± 242
ABP(mmHg) RBF (ml/min) Ccr (ml/min) E Na (^Eq/min) RS (ng/min)
136 245 32 27 395
±6 ± 14 ± 4 ±11 ± 113
Experimental period 2
Recovery period
1
Angiotensin II Infusion 140 ± 8 138 ± 7 137 251 ± 26 224 ± 22* 220 30 33 ± 6 36 ± 4 15 27 ± 13 19 ± 9 131 374 ± 25 166 ± 76f Des-1-Asp-Angiotensin II Infusion TX 137 136 ± 6 136 ± 6 219 254 ± 8 220 ± 12* 31 34 ± 3 30 ± 6 25 ± 11 18 ± 9 16 502 ± 214 253 ± 109^ 180
±7 ± 16t ± 6 ± 5 ± 37f ± ± ± ± ±
6 12* 4 9 53^:
136
±
269
±
35
±
32 441
±
137 250 32
8 22
136 269
4 13
34 28
±
± ±
8 19 4
±
14
±
125
569
±
256
±
5 20 5
137 256
± ±
6 23
± ±
18 ± 10 345 ± 122
31 ± 6 20 ± 11 358 ± 68
Values are means ± SE. ABP = arterial blood pressure, RBF = renal blood flow, Ccr = creatinine clearance, ENa = sodium excretion, and RS = renin secretion. Angiotensin II and des-1-Asp-angiotensin II were infused at a rate calculated to increase renal blood concentration by 7 ng/100 ml; values for experimental periods 1 and 2 are those after 10 and 30 minutes of peptide infusion respectively. * P < 0.01 compared with the mean of the two control periods. f P < 0.025 compared with the mean of the two control periods. %P < 0.05 compared with the mean of the two control periods.
Mean arterial blood pressure, renal blood flow, creatinine clearance, and renal sodium excretion were not detectably influenced by infusion of either peptide at the present dose level. Thus, as in the normal dog, it appears that both the octapeptide and its heptapeptide metabolite exert qualitatively
and quantitatively similar actions on renin secretion and renal function in the sodium-depleted dog. Experiment 3.—Figure 1 shows the decrease in renal blood flow following intrarenal arterial injection of exogenous des-1-Asp-angiotensin II and angiotensin II into two normal and two sodium-
TABLE 2
Arterial Blood Pressure, Renal Blood Flow, Creatinine Clearance, Sodium Excretion, and Renin Secretion by the Experimental Kidney before, during, and after Intrarenal Infusion of Angiotensin II and Des-1-Asp-Angiotensin II in Five Sodium-Depleted Dogs Control period
2
1
A B P ( m m Hg) R B F (ml/min) Ccr (ml/min)
137 225
±
11
±
32
±
E
40 3 4
6± 880 28
Na
(jiEq/min)
R S (ng/min)
Recovery period
Experimental period
1
138 233 31 7 1102
Angiotensin ± 11 139 ± 42 218 ±2 32 ±5 7 ± 134 495
137 213 30 5 1094
± 14 ± 42 ± 2 ± 2 ± 211
II Infusion ± 9 ± 28 ±2 ±4 ± 235*
1
2
133 ± 11 223 ± 32 33 ± 3 7 ±4 1033 ± 149
136 ± 8 228 ± 37 34 ± 4 7 ±5 1226 ± 205
135 224 33 8 905
136 220 31 10 1199
2
137 215 32 7 502
± 10 ± 27 ±3 ±4 ± 129f
Des-1 -Asp-Angiotensin II Infusion
A B P ( m m Hg) R B F (ml/min) Ccr (ml/min) E N a (^Eq/min) R S (ng/min)
137 213 28 5 1345
±
13 25
±
2
±
236
±
1
137 210 29 5 768
± 11 ± 38 ± 3 ± 3 ± 132t
139 203 29 5 499
± ± ± ± ±
11
32 2
2 31t
± 11 ± 39 ± 3 ± 4 ± 27
± ± ± ± ±
12
44 2 5 188
Values are means ± SE. ABP = arterial blood pressure, RBF = renal blood flow, Ccr = creatinine clearance, ENa = sodium excretion, "and RS = renin secretion. Angiotensin II and des-1-Asp-angiotensin II were infused at a rate calculated to increase renal blood concentration by 7 ng/100 ml; values for experimental periods 1 and 2 are those after 10 and 30 minutes of peptide infusion, respectively. * P < 0.05 compared with the mean of the two control periods. i P < 0.025 compared with the mean of the two control periods. Circulation Research, Vol. 37. July 1975
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DES-1-ASP-ANGIOTENSIN II: INTRARENAL ACTION
IOO
200
NANOGRAMS
300
400
500
OF PEPTIDE
FIGURE 1
Effects of single intrarenal arterial injections of angiotensin II (squares) and des-1-Asp-angiotensin II (circles) on renal blood flow in two normal and two sodium-depleted dogs.
depleted dogs. The responses to the heptapeptide and the octapeptide were similar in each dog; however, the dose-response curves to each peptide were shifted down and to the right in the sodiumdepleted dogs. The dose of des-1-Asp-angiotensin II or angiotensin II required to reduce renal blood flow by 50% was four times greater in the sodiumdepleted dogs (approximately 400 ng for both peptides) than it was in the normal dogs (100 ng for both peptides). Discussion The important new findings of the present study are the comparative effects of des-1-Asp-angiotensin II and angiotensin II on renal blood flow and renin secretion. Infusion of the heptapeptide into the renal artery of normal and sodium-depleted dogs at a rate which was calculated to increase renal blood levels by only 7 ng/100 ml resulted in a significant reduction in both renal blood flow and renin secretion in normal dogs (Table 1) and a significant reduction in renin secretion with no change in renal blood flow in sodium-depleted dogs (Table 2). Infusion of des-1-Asp-angiotensin II did not significantly alter creatinine clearance or sodium excretion in either group of dogs. In both normal and sodium-depleted dogs, infusion of angiotensin II at this same rate produced results which are comparable both qualitatively and quantitatively to those produced by the infusion of des-1Asp-angiotensin II. Both agonists also demonstrated comparable dose-response characteristics in normal and sodium-depleted dogs as judged by a reduction in renal blood flow following single intrarenal arterial injections (Fig. 1). Thus, the present
33
data suggest strongly that the octapeptide and its natural metabolite des-1-Asp-angiotensin II (8) exert qualitatively and quantitatively similar actions on the renin secretory mechanism and the renal arterioles. Although studies on the mechanism of action of des-1-Asp-angiotensin II in the kidney are lacking, the results of the present study agree with the earlier finding that angiotensin II inhibits renin secretion (9, 10). The precise nature of this inhibitory action is not thoroughly understood, but the present data are consistent with the suggestion that angiotensin II influences renin secretion via a vascular receptor in the renal arteriole or by direct action on the juxtaglomerular cells (9, 10). The earlier finding (10) of failure of renal blood flow to change in the sodium-depleted dog with a nonfiltering kidney points to a direct action of angiotensin II on the juxtaglomerular cells. Presumably, des-1-Asp-angiotensin II inhibits renin secretion by a similar mechanism. Recently, Hollenberg et al. (11) have found that normal human subjects on a sodium-restricted diet are less sensitive to angiotensin II and more sensitive to norepinephrine, as judged by a reduction in renal blood flow, than are subjects on a normal sodium intake. Because the changes in sensitivity to angiotensin II and norepinephrine were directionally opposite, it seems unlikely that they were due to nonspecific effects of sodium restriction on the vascular smooth muscle. A similar mechanism may provide the basis for the changes in sensitivity to both the octapeptide and the heptapeptide which were observed in the present study. Recent studies have suggested that the vascular and adrenal receptors for angiotensin differ (12, 13, Bravo and Bumpus, unpublished observation); the receptors at the two sites may have different affinities for the octapeptide, or, alternatively, two different agonists may mediate the pressor and steroidogenic responses (13). With regard to the latter possibility, the observation that des-1-Aspangiotensin II is a potent stimulus to aldosterone production (2-5) but possesses only 25-50% of the pressor activity of angiotensin II (1) has prompted speculation that it is an important mediator of the renin-angiotensin system at the adrenal receptor level via local tissue production from its octapeptide precursor (2-5). The data presented in this study indicate that the intrarenal receptor sites for angiotensin are influenced in a qualitatively and quantitatively similar manner by intrarenal arterial infusion of either des-1-Asp-angiotensin II or angiotensin II.
Circulation Research, Vol. 37. July 1975
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34
FREEMAN, DAVIS. LOHMEIER
Since the potency of des-1-Asp-angiotensin II, as judged by an increase in mean arterial blood pressure, is only 25-50% of that of angiotensin II (1), the present data imply that the renal vascular receptors and other vascular receptors for angiotensin differ. This observation could mean that des-1Asp-angiotensin II also is a mediator of the reninangiotensin system at the level of the renal arteriolar receptors via local production of this agonist in a manner analogous to that postulated to occur at the adrenal receptor site. Another possible explanation for differences among arteriolar receptors is that the availability of local angiotensinases for conversion of angiotensin II to the heptapeptide differs among tissues. Osborne et al. (14) have separated the [2-8]-heptapeptide from other metabolic products in the effluent of isolated rat kidneys perfused with l-Asp-4-[3H]Tyr-5-Val-angiotensin II. This concept of local production of des-1-Aspangiotensin II at the level of the renal arteriolar and adrenal receptor sites is an attractive one which can reconcile the observations of the present study with those of other investigations concerning the relative pressor and steroidogenic activities of des-1-Asp-angiotensin II and angiotensin II. The present study thus provides evidence that both angiotensin II and its metabolite des-1-Aspangiotensin II exert qualitatively and quantitatively similar actions on the renin secretory mechanism and the vascular receptor at the level of the renal arterioles. Since previous studies have indicated that des-1-Asp-angiotensin II has less pressor activity than does angiotensin II, the present data suggest that the renal vascular receptors and other vascular receptors for angiotensin are functionally different. It is hypothesized that local production of des-1-Asp-angiotensin II at the level of the renal arteriolar receptors may occur. Acknowledgment We thank Mr. John E. Thomas, Ms. Jane Green, and Ms. Rebecca Wright for their excellent technical work.
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(2-8) and hexapeptide (3-8) fragments of angiotensin II on aldosterone secretion. J Clin Endocrinol Metab 32:575-578, 1971 3. CHIU AT, PEACH MJ: Inhibition of induced aldosterone biosynthesis with a specific antagonist of angiotensin II. Proc Natl Acad Sci USA 71:341-344, 1974 4. CAMPBELL WB, BROOKS SN, PETTINGER WA: Angiotensin II-
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NK: Evidence for two different angiotensin II receptors in rat adrenal glomerulosa and rabbit vascular smooth muscle cells. Circ Res 34:384-390, 1974 13. STEELE JM, JR, LOWENSTEIN J: Differential effects of an
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Circulation Research, Vol. 37. July 1975
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Des-1-Asp-angiotensin II. Possible intrarenal role in homeostasis in the dog. R H Freeman, J O Davis and T E Lohmeier Circ Res. 1975;37:30-34 doi: 10.1161/01.RES.37.1.30 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1975 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
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