Offset of actions of angiotensin II, angiotensin III, and their analogue antagonists in renal and femoral vascular beds: Further evidence for different vascular angiotensin receptors

Pergamon Press

Life Sciences, Vol . 24, pp . 503-512 Printed in the U .S .A .

OFFSET OF ACTIONS OF ANGIOTENSIN II, ANGIOTENSIN III, AND THEIR ANALOGUE ANTAGONISTS IN RENAL AND FEMORAL VASCULAR BEDS : FURTHER EVIDENCE FOR DIFFERENT VASCULAR ANGIOTENSIN RECEPTORS William J .H . Caldicott and ~~orman K . Hollenberg Departments of Radiology and Medicine, Harvard Medical School, The Children's Hospital Medical Center, and Peter Bent Brigham Hospital, Boston, Mass . 02115 (Received in final form December 26, 1978)

SUMMARY Half-time of the offset of antagonist action was used to assess the possibility that factors which determine the duration of action of angiotensin antagonists were responsible thus, for for regional differences in their effectiveness : example, enhanced degradation of angiotensin III analogues in the limb circulation would reduce their effectiveness there despite an angiotensin receptor identical to that in the kidney . In the anesthetized dog blood flow in the renal and femoral vascular beds was measured with an electromagnet ic flowmeter ; the octapeptide analogue saralasin (1-Sar, 8-Ala AII) and a heptapeptide analogue (des-Asp, 8-Ile AII) were infused intravenously (1 ug/k9/min) for 10 minutes and, after stopping the infusion, the effectiveness of their blockade of angiotensin II was assessed over time . The half-time of offset of the antagonist action was determined from semilogarithmic plots of percent inhibition during recovery . Offset of heptapeptideinduced inhibition in the hindlimb would have been more rapid if increased rate of degradation was the explanation for its Indeed offset reduced effectiveness and such was not the case : was more rapid in the renal (5 .8 t 1 .1 min) than the femoral Saralasin showed vascular bed (11 .7 ± 2 .1 min) (p > 0 .05) . identical offsets in the two beds (renal 17 .2 ± 1 .5 min ; femoral 15 .1 ± 2 .9 min) (p > 0 .5) . Consistent with these observations, the offset of the agonist action of angiotensin III was shorter in the kidney (0 .69 ± 0 .06 min) than in the limb (1 .46 t 0 .13 min ; p < 0 .001) . This study has conf firmed the relatively greater efficacy of heptapeptide analogues in the renal than in the femoral vascular bed and has ruled out degradation as accounting for that difference : The difference is most likely to lie in a different angiotensin receptor in the two regions . The observation that heptapeptide analogues of angiotensin III are systematically less effective antagonists in the hindlimb than in the kidney (1) potentially supports earlier suggestions that the renal and systemic vascular angiotensin receptors differ (2-6) . If heptapeptide analogues were degraded more effectively in the hindlimb than in the kidney, however, the difference in their relative effectiveness in the 0300-9653/79/0205-050302 .00/0 Copyright (c) 1979 Pergamon Press

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two vascular beds could have been accounted for without a necessity for postulating a difference in the angiotensin vascular receptors . The present study of the offset of the actions of angiotensins II and III, and of an octapeptide and a heptapeptide analogue reveals differences which are most compatible with regional differences in the angiotensin receptors . Methods Experiments were performed on 25 adult mongrel dogs which weighed between 12 and 25 kg . The model has been described in detail (7) . Briefly, anesthesia was induced with an initial intravenous dose of sodium pentobarbital (30 mg/kg) and maintained either with intermittent bolus injections of sodium pentobarbital or with a constant intravenous infusion at the rate of 75 mg/hr . Because of the instability of femoral blood flow during experiments, muscular paralysis and ganglionic blockade were produced by a constant intravenous infusion of succinylcholine chloride (8 .5 mg/hr ; Burroughs Wellcome Co .) and a single subcutaneous dose of pentolinium tartrate (7 mg ; Ansolysen, Wyeth) . Respiration was controlled with a Harvard respirator pump and a cuffed endotracheal tube . The arteriovenous anastomoses in the paw of the limb to be studied were excluded by a tourniquet at the ankle . Blood flow to the kidney and hindlimb were measured with an electromagnetic flow meter (Statham) . The left renal and femoral arteries were exposed through a retroperitoneal flank incision and an incision immediately below the inguinal ligament and appropriately-sized probes were placed on the arteries . Zero flow was established electrically and by clamping the artery distal to the probe . Probe calibrations were performed at the end of each experiment on the femoral artery . Arterial pressure was measured from a catheter in the abdominal aorta with a transducer (Statham P23Db) and blood flows and pressure were monitored on a Polygraph recorder (Grass Instrument Co .) . A pre-curved polyethylene catheter (0 .25 Blue Formocath, Becktin Dickinson) was introduced into the renal artery under fluoroscopic control . Injections into the femoral artery were made either through a 22 gauge Angiocath (Deseret Pharmaceutical Co .), introduced into the femoral artery immediately distal to the flow probe or proximal to the probe through a catheter attached to a 22 gauge needle . Studies of of fset of ag o nist action Bolus injections of angiotensin II and angiotensin III in the dose range of 1 to 100 ng/kg were made into the renal artery (9 dogs) or the femoral artery (10 dogs) . The agents were diluted in 0 .9X sodium chloride and given as a 0 .5 ml . bolus and washed into the artery with a constant infusion of 0 .9~ sodium chloride (1 ml/min) . Half-times of the offset of the vasoconstrictor response induced by both agents in both vascular beds was estimated from semilogarithmic plots of recovery of blood flow after the maximal vasoconstrictor response against time . The semilogarithmic plots of change in blood flow against time resulted in straight lines, fitted by eye, consistent with monoexponential recovery .

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Studies of offset of antagonist action Studies were made in the vascular beds of both the kidney and hindlimb in 6 dogs . Patency of the arterial lines was maintained with constant infusions of 0 .9% sodium chloride at the rate of 0 .5 ml/min . Angiotensin II (Bachem Inc .) was injected directly into the renal and femoral arteries as a 0 .5 ml . bolus and immediately flushed into the artery by a further 0 .5 ml . bolus of 0 .9% sodium chloride . The dose of angiotensin II was selected to produce approximately 50% reduction in blood flow to these vascular beds . (The dose required ranged from 1 to 50 ng/kg for the kidney and 1 to 100 ng/kg for the hindlimb .) Renal and femoral blood flow responses to angiotensin II were defined before, during, and at 2 to 5 minute intervals after cessation of a 10 minute intravenous infusion of an angiotensin antagonist, The antagonists used were the octapeptide analogue 1-Sar, 8-Ala All (Saralasin ; Norwich Pharmacal Co .) and the heptapeptide analogue des-Asp, 8-Ile All (Bachem, Calif .) . Each analogue was diluted in 0 .9% sodium chloride so that an infusion at the rate of 1 ml/min delivered 1 ug/k'g/min of the agent . After the end of the analogue infusion, bolus injections of angiotensin II were made either until the responses returned to control or for up to 30 minutes . Each animal received both antagonists : at least 60 minutes was allowed before the response to angiotensin II was again defined prior to the use of the second antagonist . Inhibition of renal and femoral vascular responses to angiotensin II were expressed as percent inhibition calculated from the formual C-E/C (100), where C is the control response to angiotensin II and E represents the experimental response, modified by the antagonist . The half-time of the offset of the blocking action of each antagonist was assessed in individual animals for both vascular beds from semilogarithmic plots of percent inhibition against time . This resulted in straight lines when fitted by eye, consistent with monoexponential recovery . Results are expressed as the mean with the standard error of the mean (SEM) used as the index of dispersion . Data were analyzed using the Student's t test . The null .hypothesis was rejected when a p value of less than 0 .05 was achieved . Results Offset of agonist action (Table I) In the kidney there was no significant difference in the half-times of the offset of the vasoconstrictor responses to angiotensin II (0 .76 ± 0 .09 min) and angiotensin III (0 .69 t 0 .06 min) (p > 0 .3) . However in the hindlimb the half-time of the offset of the response to angiotensin II (2 .61 ± 0 .26 min) was greater than for angiotensin III (1 .46 ± 0 .13 min) in each animal (p < 0 .005) . Offset of antagonist action The reduction in renal and femoral blood flow induced by intra-arterial bolus injections of angiotensin II were 58 .5 ± 3 .1 percent and 53 .8 t 3 .1 percent, respectively . The antagonists saralasin and des-Asp, 8-Ile All were mild agonists at the infusion rate of 1 ug/kg/min, increasing arterial pressure and

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reducing renal and femoral blood flow : changes from baseline flows and pressure were in the range of 6 to 10% (Figure 1) . TABLE I Half-time of offset of responses to All & AIII (minutes) Kidney

Mean S .E .M .

_ H ndlimb

AII

AIII

AII

AIII

0 .80 0 .77 1 .27 0 .80 1 .00 U .40 0 .50 0 .70 0 .60

0 .60 0 .73 0 .90 0 .67 0 .67 0 .45 0 .60 1 .00 0 .60

2 .80 1 .70 1 .60 2 .50 2 .25 3 .75 3 .75 2 .00 3 .50 2 .25

2 .00 1 .10 1 .10 1 .00 1 .70 1 .75 1 .00 1 .25 2 .00 1 .69

0 .76* ±0 .09

0 .69* ±0 .06

2 .61** ±0 .26

*p > 0 .3

0 m~in

-

**p<0.00';

1 .46** ±U .13

Paireâ t test)

HrDLMB OONiROL

SARI4_I~41N

~Inl

7

ß

T7

is

-~- .- .,,~--- .~---~--1~-'

Ko~r FIG . 1 Blood flow reductions produced by bolus injections of angiotensin II made directly into the femoral and renal arteries before (control), during (saralasin) and after a 10 minute intravenous infusion of saralasin (1 u9/kg/min) . Flow responses were inhibited during and recovered with time after cessation of the saralasin infusion . The small decrease in baseline flows during saralasin infusion indicates its partial agonist affect .

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As in previous studies saralasin and des-Asp, 8-Ile All were equally effective blockers of the renal vascular response to angiotensin II (1, 6) . When given as an intravenous infusion at the rate of 1 ug/kg/min saralasin induced 86 .9 ± 3 .1 percent inhibition and des-Asp, 8-Ile All 85 .4 t 1 .6 percent inhibition of angiotensin-induced renal vasoconstriction (Table II) . Also as we have reported before (1), both agents were less effective blockers of angiotensin-induced vasoconstriction in the hindlimb than in the kidney (Figure 1), and the reduction in potency in the hindlimb was greater for the heptapeptide analogue des-Asp, 8-Ile AII, which induced 44 .6 ± 9 .7 percent inhibition, than for the octapeptide saralasin which induced 66 .6 ± 4 .4 percent inhibition (Table II) . TABLE II Perçent inhibition of response to a ngi otens in II

Kidney Hindlimb

Saralasin*

des-Asp, 8-Ile AII*

86 .9 t 3 .1** 66 .6 ± 4 .4**

85 .4 t 1 .6*** 44 .6 ± 9 .1***

1 .3 Ratio Va ues represent means t SE . * 1 .0 ug/kg/min ** p < 0 .05

1 .9 *** p

<

0 .02 (Paired t test)

Figure 1 illustrates a typical experiment showing control renal and femoral blood flow responses to a bolus of angiotensin II, inhibition of those responses during an infusion of saralasin, and recovery with time after cessation of the saralasin infusion . Figure 2 shows individual data points for saralasin and des-Asp, 8-Ile All in the kidney for all 6 animals and clearly demonstrates the more rapid recovery from inhibition in the case of des-Asp, 8-Ile AII . The half-time of offset assessed from semilogarithmic plots of percent inhibition against time for individual experiments are shown in Figure 3 . In the kidney, des-Asp, 8-Ile All had a considerably shorter half-time (5 .8 t 1 .1 min) than saralasin (17 .2 t 1 .5 min) ; p < 0 .005 . In the hindlimb both antagonists had a longer half-time than did des-Asp, 8-Ile All in the kidney and although on average the half-time of des-Asp, 8-Ile All (11 .7 ± 2 .1 min) was shorter than for saralasin (15 .1 t 2 .9), the difference did not achieve significance (P > 0 .2) . DISCUSSION The purpose of this study was to assess the possibility that the greater effectiveness of heptapeptide analogues as antagonists in the renal than in the femoral vascular bed was not due to differences in the affinity of the receptors in those two vascular beds for the heptapeptide molecule, but rather to other factors . The effectiveness of an antagonist must be a function of its concentration in the biophase, the affinity of

508

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Action of Angiotensin Antagonists

the antagonist for the receptor, and the rate at which the molecule leaves the receptor and the receptor becomes available for activation . The concentration in the biophase at any infusion rate, in turn, must be a function 'of local and systemic degradation . We measured the offset of antagonist action as an index of two of these factors - the rate of degradation or the rate at which the molecule leaves the receptor . A shortened half-time of offset could have reflected either enhanced The latter, of degradation or a short sojourn on the receptor . course, reflects a fundamental difference in the receptor, consonant with the hypothesis of regional differences in receptors . The former, enhanced degradation, does not . There were reasons for suspecting that enhanced degradation might have contributed to the differences in the two vascular beds . For example aminopeptidase action may be more effective in the Our relatively sluggish circulation through skeletal muscle . working premise was that an enhanced degradation sufficiently important to influence the relative effectiveness of the blocker in the hindlimb circulation would be evident with this index, although a shortened half-time would not have proved that enhanced degradation was present .

"s~u~sOr ooES,~ev a-r.~ ~, 0 0

0 0 0

0

0 0

0

0 0 0 0 0

0

TOME pdN

FIG . 2 Recovery from antagonist-induced inhibition of renal vascular responses to angiotensin II after cessation of intravenous infusions of saralasin and des-Asp, 8-Ile AII . During antagonist infusion (0 min) both agents produced identical inhibition . Recovery from inhibition was more rapid for des-Asp, 8-Ile AII . Percent inhibition was calculated from the formula (C-E)/C(100) . Control renal blood flow response to angiotensin = C . Response during or after antagonist infusion = E .

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509

90 FIIPDIIN~

KIDPEY

0

0

10

~

0 10 SARALA3IN (t~ mN

20

3D

FIG . 3 Comparison of the half-times of offset of inhibition induced by saralasin and des-Asp, 8-Ile All in the renal and femoral vascular beds . In the kidney saralasin had a much In the hindlimb the longer half-time than des-Asp, 8-Ile AII . half-times were similar . This study No evidence of enhanced degradation was found . did confirm the previous finding that octapeptide and heptapeptide analogues produce less inhibition of angiotensininduced vasoconstriction in the hindlimb than in the kidney and that the reduction is greater for the heptapeptides (1) . Different protocols were used in the two studies : in the earlier study, both angiotensin and the analogues were given directly into the renal and femoral arteries ; in the present study inhibition of intra-arterial bolus injections of angiotensin was assessed during intravenous infusions of the analogues . The half-life of angiotensin II in the circulation has been reported to be in the range of 15 seconds to 1 minute (8-12), and that of angiotensin III to be the same (9, 10), or less by a factor of 2-3 times (13) . In the rat and dog, approximately 70 percent of injected angiotensin II was degraded in a single passage through either the kidney or hindlimb (8, 14, 15) . A comparable study of angiotensin III in the kidney showed 89 percent degradation (15) : Data for angiotensin III and the hindlimb are lacking . In the kidney we have found no significant difference in the half-times of offset of the vasoconstriction induced either by angiotensin II (0 .76 min) or angiotensin III (0 .69 min) . In the hindlimb both had a prolonged rate of offset, reater for angiotensin II (2 .61 min) than angiotensin III g(1 .46 min) . Of interest is the parallel equipotency of angiotensin II and angiotensin III in the kidney (5, 6) and their similar rates of offset, whereas there is a reduced constrictor

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effect of intravenously injected angiotensin III and a reduced rate of offset, relative to angiotensin II, in the hindlimb . The precise meaning of these observations remains obscure . However the present results are inconsistent with the finding that angiotensin II and angiotensin III are equally effective when injected directly into the femoral artery (16) . Furthermore the reduced offset of angiotensin II in the kidney does not explain the increased sensitivity of the renal vasculature to that agent (2) . Little is known of the half-lives of the analogues of angiotensin, and studies of the duration of their influence in individual vascular beds were previously lacking . The half-life of saralasin in the systemic circulation of the rat has been reported to be 6 .4 minutes (10) . We have shown the half-time of offset of saralasin to be 17 .2 and 15 .1 minutes in the vascular beds of the kidney and hindlimb, respectively . The heptapeptide analogue, des-Asp, 8-Ile AII, which is as potent an antagonist as saralasin in the kidney had a markedly reduced half-time in the renal vascular bed (5 .8 min) . In the hindlimb, where des-Asp, 8-Ile All is a less potent antagonist than in the kidney, the half-time was increased (11 .7 min), and only slightly less than that of saralasin (15 . 1 min) . In a previous study (1) we have shown that both octapeptide and heptapeptide analogues were less effective blocking agents in the hindlimb than in the kidney and that the decrease in effectiveness was greater for the heptapeptides ; and the same was true for saralasin and des-Asp, 8-Ile All in the present study . For these differences to have been caused by differences in offset, it would have been necessary for both agents to have had lesser rates of offset in the hindlimb than in the kidney and for the reduction to have been greater for des-Asp, 8-Ile AII ; and such was not the case . Hence the differences in the responses of the renal and femoral vascular beds to antagonists cannot be explained by enhanced degradation in the limb . The similarity in the offset of angiotensin II and angiotensin III in the renal vascular bed supports the previous report that aspartic acid in the N-terminal position does not The protection from protect angiotensin from degradation (17) . degradation resulting from the N-terminal sarcosine in saralasin may be due to increased affinity of receptors for agents with the sarcosine substitution and not relative immunity to breakdown by angiotensinases (16) . The present study has shown that the differences in the responses of the renal and femoral vascular angiotensin receptors to octapeptide and heptapeptide analogues (1) could not have been due to regional differences in offset of these agents . Taken together with the increased sensitivity of the renal vasculature both to angiotensin II (2) and the agonist effect of saralasin (3, 4), the equipotency of angiotensins II and III in the kidney (5, 6, 16) despite angiotensin III's reduced pressor activity, and the relative renal specificity of a heptapeptide analogue in a model in which the renin-angiotensin system was activated (6), these findings provide convincing evidence for differences between renal and systemic vascular angiotensin receptors .

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Acknowledgements It is a pleasure to acknowledge the assistance of Andrea KeTton, Michael Kuber, and Henry Syms and to express our gratitude to Donald Sucher for art work and photography and to Doctor G . Denning of Norwich Pharmacol Company for supplying the saralasin . This work was done during the tenure of a research grantin-aid from the American Heart Association, Greater Boston Massachusetts Division - #13-517-778 and was supported by grants from the Charles fl . Hood Foundation, N .A .S .A ., and the National Institute of Health (9M 18674, HL 14944, HL 11668, HE 05832) . Please mail reprint requests to William J .H . Caldicott, M .B ., B .S ., Kresge Foundation Laboratory, Department of Radiology, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115 . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .

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