Brain Research, 514 (1990) 5-10 Elsevier
5
BRES 15358
Intracerebroventricularly infused [D-Argl]angiotensin III, is superior to [D-Aspl]angiotensin II, as a pressor agent in rats John W. Wright, Kim A. Roberts, Vickie I. Cook, Cathy E. Murray, Michael E Sardinia and
Joseph W. Harding Departments of Psychology and Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164 (U.S.A.)
(Accepted 12 September 1989) Key words: [D-Asp~]Angiotensin II; [D-Argl]Angiotensin III; Angiotensin II; Angiotensin III; Amastatin; Bestatin; Sarthran; Blood pressure; Intracerebroventricular infusion; Rat
Two D-amino acid substitution angiotensin analogues were compared against native angiotensin II (AII) and angiotensin III (AIII) for their resistance to brain tissueqnduced degradation and for pressor potency when intracerebroventricularly (i.c.v.) infused in Sprague-Dawiey rats. The in vitro results indicate that [D-Aspl]AII was very resistant to degradation, AII and [D-Argl]AIII were degraded at similar rates, while AIII was the most rapidly degraded. In vivo results revealed that AII, AIII and [D-Arg~]AIII produced greater pressor responses than [D-Aspl]AII. Intracerebroventricular pretreatment with the aminopeptidase A inhibitor, amastatin, siginificantly reduced the subsequent pressor response to i.c.v, infused [D-Asp~]AII presumably by inhibiting its conversion to AIII. In contrast, pretreatment with the aminopeptidase B inhibitor, bestatin, potentiated the subsequent pressor response to i.c.v, infused [D-Arg~]AIII, presumably by inhibiting the conversion of [D-Arg~]AIII to the less active hexapeptide AII(3-8). Next, i.c.v, pretreatment with the specific angiotensin receptor antagonist, [Sar 1, ThrS]AII (Sarthran) was found to greatly diminish the subsequent pressor responses to i.c.v, infused [D-Aspl]AII and [D-Argl]AIII, suggesting that these analogues are having their effect at the same brain angiotensin receptor site. These results support the hypothesis that AIII, or AIII-like ligands, may serve as the active form of brain angiotensin. INTRODUCTION The brain renin-angiotensin system has become one of the most thoroughly characterized neuropeptide systems, and appears to contain the precursor molecules and enzymatic activity necessary to generate its own angiotensin peptides 4'6'8-1°,2°'23. Most studies have proceeded with the belief that the active form of angiotensin in the brain is angiotensin II (AII); however, the results of several recent investigations support an important role for the heptapeptide angiotensin III ( A I I I ) 14. For example, intracerebroventricular (i.c.v.) injections of A I I I are approximately equipotent to A l l with respect to pressor responses 7'32, drinking behavior 32,35, and salt appetite 3 in normotensive rats despite the increased lability of A I I I upon i.c.v, injection 13. Because of the apparent importance of aminopeptidases in the conversion of A I I to A I I I , and its degradation to less active fragments 1, aminopeptidase inhibitors have been employed in a n u m b e r of studies to delay conversion of A I I to A I I I , and A I I I to the less active hexapeptide A I I ( 3 - 8 ) . Several m a j o r observations may be made from this approach. First, the i.c.v, infusion of a cocktail of these inhibitors,
designed to protect both the N- and C-terminals of the angiotensin molecule, will indeed raise blood pressure and cerebrospinal fluid levels of angiotensin2; and i.c.v. pretreatment with the specific angiotensin receptor antagonist sarthran prevents the subsequent inhibitorinduced pressor effects suggesting an angiotensinergic mechanism of action. Second, i.c.v, pretreatment with amastatin (AM), a reasonably specific inhibitor of aminopeptidase A which is responsible for converting A I I to A I I I , significantly diminishes the subsequent blood pressure response to a bolus injection of A I I 3° and extends the half-life of i.c.v. [125I]AII as determined by microwave fixation 5. Third, A M selectively blocks the ability of A I I to stimulate angiotensin-sensitive neurons in the paraventricular nucleus of the hypothalamus of the rat, while the aminopeptidase B inhibitor, bestatin (BE), does not effect A I I activity 12. These results may suggest that conversion of A I I to A I I I is a prerequisite to angiotensin receptor activation. The present investigation employed two aminopeptidase-resistant angiotensin analogues in an attempt to provide additional evidence supporting the importance of A I I to A I I I conversion in determining physiological
Correspondence: J.W. Wright, Department of Psychology, Washington State University, Pullman, WA 99164, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
r e s p o n s i v e n e s s . To a c c o m p l i s h this task [ D - A s p l ] A I I and [ D - A r g l ] A I I I w e r e synthesized and e x a m i n e d for p r e s s o r p o t e n c y . T h e p r e s e n t results indicate that i.c.v, infused [ D - A s p l ] A I I is less p o t e n t as a p r e s s o r a g e n t t h a n A l l , A I I I and [ D - A r g l ] A I I I . P r e t r e a t m e n t with the a m i n o p e p tidase A i n h i b i t o r A M greatly r e d u c e d the s u b s e q u e n t activity of i.c.v,
infused
[D-Aspl]AII presumably
by
b l o c k i n g residual A I I I p r o d u c t i o n ; and p r e t r e a t m e n t with B E , an a m i n o p e p t i d a s e B i n h i b i t o r which i n t e r f e r e s with c o n v e r s i o n o f A I I I to its h e x a p e p t i d e A I I ( 3 - 8 ) , significantly e n h a n c e d the s u b s e q u e n t p r e s s o r r e s p o n s e i n d u c e d by t h e
i.c.v,
infusion
activity o f b o t h
of [ D - A r g l ] A I I I .
analogues
The
pressor
c o u l d be b l o c k e d by the
specific a n g i o t e n s i n r e c e p t o r a n t a g o n i s t [Sar 1, T h r S ] A I I (sarthran) suggesting that [ D - A s p l ] A I I and [D-ArgX]AIII are acting at a c o m m o n r e c e p t o r site.
MATERIALS AND METHODS Male Sprague-Dawley rats (300-420 g, Charles River derived) were bred and maintained in a AAALAC-approved vivarium. The animals used for in vitro metabolism were maintained in group cages. The rats utilized for alert testing were individually housed in separate cages for a minimum of 48 h before surgery on a 12:12 h light-dark cycle initiated at 07.00 h. Both food and water were available ad libitum except during testing which was conducted between 10.00 and 17.00 h. [Asp 1, IleS]AII and [des-Asp 1, IleS]AII were purchased from U.S. Biochemical. [D-Asp 1, IleS]AII and [D-Arg~, Ile4]AIII were synthesized in our laboratory using a Vega (Coupler 250) amino acid synthesizer, purified by reverse-phase high-performance liquid chromatography (HPLC), followed by amino acid analysis. The purities by weight of the compounds were determined to be 85, 82, 72, and 68%, respectively as measured by HPLC. Peptide purity ranged from 98 to 100% while acetate salts represented the major contributor to the decreased purity by weight. Amastatin and bestatin were purchased from Sigma Chemical. The purity by weight provided by Sigma was 85% for both inhibitors with a peptide content of 99%. The above values were used to adjust for the purity of each peptide so that the dose reflected actual peptide delivered in moles. In vitro metabolism of radioligands An initial experiment was conducted to determine the relative resistance of the compounds AII, [D-Aspl]AII, AIII and [oArgl]AIII, to metabolism by brain tissues. The metabolism of the angiotensins was monitored over a 2 h time period in which 15 time points were examined. Complete time courses for each peptide were developed in duplicate. Eight rats were decapitated, the brains were quickly removed and the tissue block consisting of the thalamus, hypothalamus, septum, and anteroventral third ventricle (AV3V) were dissected over ice. Tissue preparation entailed differential centrifugation, first with hypotonic buffer (pH 7.4 at 4 °C, 50 mM Tris) and then followed with an isotonic buffer (pH 7.4 at 23 °C, 110 mM NaC1, 5 mM KCI, 2 mM CaCl2, 1 mM MgCl2, 25 mM NaPO4). Monoiodinated [12SI]AII, [125I][D-Aspl]AII, [125I]AIII and [125I][DArgl]AIII (2176 Ci/mmoi by immunological self-displacement analysis) were obtained by an immobilized lactoperoxidase glucose oxidase method, using ~25Iodine (New England Nuclear nez-33h), Enzymobeads (Biorad). Assays were carried out at 22 °C in siliconized 12 x 75 mm borosilicate glass culture tubes. Of each iodinated compound 100/~l at a concentration of approximately 0.5 nM were incubated in 300 /~l of isotonic buffer. Tissue was added at consecutive time points
from 5 to 120 min. Incubation was terminated by adding 0.5 ml acetonitrile (ACN) to each tube and vortexing, the samples were then extracted through Baker Columns, dried by speed-vac, and stored frozen. HPLC analyses were carried out using a Partisphere C18 reverse-phase column and isocratic elution at a flow rate of 2 ml/min. The mobile phase consisted of 5 ml/l of H3PO 4 titrated to pH 3.0 with triethylamine plus 14.5% acetonitrile. Radioactivity was measured with a Beckman radioisotope detector (Model 170) and the percentage of intact peptide was calculated with a Hewlett Packard integrator (Model 3390A). Surgery and instrumentation Each animal used for alert testing was anesthetized with Equithesin (Jensen-Salsbury Laboratory, 3.5 ml/kg i.p.) and prepared with a right carotid catheter (PE-50, Clay Adams), inserted to a depth of 2-3.5 cm, plugged with a 1.5 cm length of stainless-steel wire, and externalized between the scapulae 32. The catheter was prefilled with heparinized saline (75 U/ml sterile 0.15 M NaCI). Each animal was also fitted with an i.c.v, guide cannula (PE-60) as previously described 29'32. Following surgery each animal received 0.1 ml i.m. procaine, penicillin G, in dihydrostreptomycin sulfate (200,000 U/ml, Combiotic, Pfizer, NY). Following a minimum of 48 h recovery from surgery, each animal was behaviorally tested in its home cage for the accuracy of the i.c.v, guide cannula placement. This was accomplished by placing a preloaded 24 g stainless-steel injector into the alert animal's guide such that it extended 2-3.5 mm beyond the tip of the guide, thus penetrating the roof of the lateral ventricle. The injector was attached to a 10/A Hamilton syringe by a 25 cm length of PE-20 tubing and contained AII (100 pmol in a total volume of 2 ~1 artificial cerebral spinal fluid [aCSF]2~). If a drinking response was not elicited within 5 min following the injection, the animal was replaced. Pressor responses A minimum of 24 h following behavioral confirmation of correct i.c.v, cannula placement, each animal was placed in a glass test jar (20.5 cm diameter × 20 cm tall) where attachments to a polygraph recorder (Grass Instruments, Model 7B) with Gould tranducers (Model P23ID) were available to monitor mean arterial blood pressure (MABP). The animal was allowed to adapt to the test chamber, and then a stable 5 min baselevel MABP was obtained before the i.c.v, administration of any compounds. All reported blood pressure changes were those that deviated from the established 5 min MABP baselevel. Four groups of rats (8 per group) were utilized to measure the pressor responses induced by the 5 rain i.c.v, infusion of 0, 0.1, 1, 10 and 100 pmol/min doses of All, [D-Aspl]AII, AIII, and [o-Argl]AIII. Each compound was corrected for purity and was prepared in aCSF and delivered at a rate of 2 /d/min (Sage Instruments, Model 355). The doses of each compound were counter-balanced within members of each group such that 4 animals received an ascending order and the other 4 rats a descending order. Sufficient time was allowed between doses to recover baslevel blood pressure. Two additional groups of animals were employed to test the effects of pretreatment with the aminopeptidase inhibitors AM or BE on the subsequent pressor responses induced by [D-Aspl]AII or [o-Argl]AIII. Specifically, four members of the first group received a 5 rain i.c.v, pretreatment infusion of AM (20 nmol/2/d aCSF/min) followed by a 10 rain delay and then a 5 min i.c.v, infusion of [D-Aspl]AII (100 pmol/2 ktl aCSF/min). Each animal was allowed a minimum of 30 min recovery and was next administered a 5 min i.c.v, infusion of aCSF (2/~l/min), followed by a 10 min delay and then a 5 min i.c.v, infusion of [D-Aspl]AII (100 pmol/2 /~l aCSF/min). The other 4 members of this group were tested similarly, but each received the aCSF pretreatment first and the AM pretreatment second. Members of the second group were handled equivalently however the aminopeptidase B inhibitor BE was substituted for AM, and [D-Argl]AIII was substituted for [DAspl]AII.
Pressor response inhibition by sarthran Two additional groups of rats were prepared as discribed above. Four members of the first group were i.c.v, pretreated with the specific angiotensin receptor antagonist [Sar~, ThrS]AII (sarthran, 20 nmol/2/zl/min) for 5 min. Following a 10 rain delay the animal received a 5 rain i.c.v, infusion of [D-ASpl]AII (100 pmol/2 /zl aCSF/min). Each animal was then allowed a minimun of 30 min to recover baselevel blood pressure, and the protocol was repeated, with aCSF replacing sarthran. The other 4 rats received these pretreatments in reversed order, i.e. aCSF followed by sarthran. Members of the second group were equivalently tested, however, [o-Arg~]AIII was substituted for [o-Asp~]AII.
incubation conditions where the metabolism of A I I ! is restricted, i.e. minus divalent cations, both A I I and [ o - A s p l ] A I I degradation resulted in the production of A I I I as the initial metabolite (data not shown). Comparison of complete time course data under the above conditions indicated that both A I I and [D-Aspl]AII are metabolized in an identical manner with the only difference being an overall reduction in degradation rate for [o-Aspl]AII.
Statistical analyses The data from the in vitro metabolism of radioligands was calculated as the percent of total counts applied as intact radioligand over time of incubation. The half-lives of disappearance of labeled angiotensins were calculated from the linear regression lines of the semilog plot of percent angiotensins remaining (ordinate) vs time (abscissa). With respect to the alert testing data sets, initial baselevel MABP were analyzed by one way analysis of variance (ANOVA). The magnitude of the pressor response was calculated by subtracting the corresponding MABP baselevel from the maximum pressor change induced by each treatment. The initial dose-response data set for AII, [D-Aspl]AII, AIII, and [D-Argl]AIII was analyzed by a 4 (Groups) × 5 (Doses) ANOVA with repeated measures on the second factor. The duration of each dose was defined as the time from termination of infusion until MABP returned to baselevel (i.e. nonstatistical differences from baselevel). This data set was also analyzed by a 4 (Groups) × 5 (Doses) ANOVA again with repeated measures on the second factor. The data set concerned with the influence of pretreatment with aCSF or the aminopeptidase inhibitors (AM or BE) upon subsequent pressor responses to [D-Aspl]AII and [o-Argl]AIII, was analyzed by a 2 (Groups) × 2 (Pretreatment condition) ANOVA with repeated measures on the second factor. The duration to return to baselevel MABP was evaluated by a 2 (Groups) × 2 (Pretreatment condition) with repeated measures on the second factor. Finally, the data set concerned with sarthran or aCSF pretreatment followed by [D-ASpl]AII or [D-Argl]AIII was also evaluated by a 2 (Groups) × 2 (Pretreatment condition) with repeated measures on the second factor as was the duration to recover baselevel MABP. Significant effects were further evaluated by Newman-Keuls post-hoe test with a level of significance set at 0.01.
Pressor dose-response curves There were no differences among the groups of the first pressor experiment with respect to baselevel blood pressure prior to treatment (F0.65, df 3,28, P > 0.10). Fig. 2 A presents the maximum changes in blood pressure in response to the infusion of each dose of A I I , [OA s p l ] A I I , A I I I , and [ o - A r g l ] A I I I , and there were significant overall differences among ligands (/74.59, df 3,28, P < 0.02). Post-hoe analyses indicated that [OA r g l ] A I I I , and A I I produced significantly greater pressor responses than [o-Aspl]AII, however [D-Argl]AIII and A I I did not differ, nor did [D-Aspl]AII and A I I I . As expected there was also a significant dose-response relationship (F91.74, df 4,112, P < 0.001); with the 100 pmol dose inducing the greatest elevations in blood pressure across ligands, 10 pmol the next greatest, followed by the 1 pmol dose, while the 0.1 pmol dose did not differ from aCSF infusion. Comparing the analogues at each dose, [ o - A s p l ] A I I revealed a reduced pressor response with respect to A I I and [D-Argl]AIII at the 100, 10 and 1 pmol doses, while at the 0.1 pmol dose the A I I I pressor response was significantly less than that produced by [ o - A s p l ] A I I and [D-Argl]AIII. Also the A I I pressor
RESULTS In vitro metabolism There were clear differences among the compounds utilized in this study concerning resistance to brain tissue-induced metabolism (Fig. 1). [D-Aspl]AII indicated the greatest resistance to metabolism, with a half-life of 251.2 min, while A I I (96.7 min) and [DA r g i ] A I I I (77.2 rain), were degraded at similar rates, and A I I I revealed the greatest degradation with a half-life of 17.3 min. The degradation of A I I I followed a multiphasic pattern with initially rapid degradation followed by a somewhat slower rate. Thus, the substitution of a D-isomer amino acid in position 1 of the A I I and A I I I molecules significantly decreased the rate of metabolism for each ligand, with the greatest contrast occurring between A I I I and its analogue [a-Argl]AIII. Utilizing
100 ¢' ~L~.O, __ ~ ~
g
"~
=
8- ~
~'o'--
I - - -o- -
All [D-Asp ]-All
. ---o--
AIII [D.Arg ]-AIII
o - - - O.
o
40 20 0
0
30 Incubation
60 Time
90
120
(min)
Fig. 1. Percent radiolabel [125I]AII, [125I][D-Aspl]AII, [125I]AIII, and [125I][D-Argl]AIIIremaining intact during a 120 min incubation period with brain tissue at 22 °C. Separate determinations were made in duplicate at 15 rain intervals.
response at the 0.1 pmol dose was reduced as compared with that of [D-ArgZ]AIII. Fig. 2B presents the mean (+ S.E.M.) durations of the pressor effects induced by each ligand at each dose. Although there were no overall differences in latencies comparing the 4 ligands, there was a dose-response effect (F79.32, df 4,112, P < 0.0001); the 100 pmol dose produced the longest latency to recover, 10 pmol the next longest, followed by the 1 pmol dose, while the 0.1 pmol dose and aCSF were not different. There was no interaction effect between ligands and doses. Pretreatment with amastatin or bestatin
The effect of i.c.v, pretreatment with aCSF upon the subsequent i.c.v, infusion of [D-Aspl]AII or [D-Argl]AIII may be compared with the i.c.v, pretreatment of AM followed by [D-Aspl]AII or BE followed by [o-Argl]AIII in Fig. 3A. There was a significant difference comparing the pressor effects induced by [D-Aspl]AII and [DArgl]AIII across pretreatment conditions (F17.95, df 1,14, P < 0.005). There was also a significant effect comparing
"A • 30 / []
All [D-Asp ]-All .
~.-
pretreatment conditions across analogues (F6.47 , df 1,14, P < 0.05); and there was a significant interaction effect of analogues x pretreatment conditions (F44.78, df 1,14, P < 0.005). Specifically, [D-Aspl]AII and [D-Argl]AIII did not differ in pressor activity following pretreatment with aCSF; however, the [D-Aspl]AII response was significantly diminished following pretreatment with AM, and the [D-ArgZ]AIII response was significantly elevated following pretreatment with BE. Fig. 3B presents the mean (+ S.E.M.) duration of the pressor effects induced by each i.c.v, pretreatment with aCSF or aminopeptidase inhibitors, followed by treatment with [D-Aspl]AII or [D-Argl]AIII. There was a significant difference between [o-Aspl]AII and [OArgl]AIII comparing the durations of their pressor effects (F18.12, df 1,14, P < 0.005); with [o-Argl]AIII indicating the greatest overall duration. There was also a significant difference due to pretreatment condition (F6.97 , df 1,14, P < 0.02), and the interaction effect was significant (F20.82, df 1,14, P < 0.001). Post-hoe tests indicated that although [o-Aspl]AII and [o-Argl]AIII
.
15
o~
0
aCSF
0.1
1.0
10
10
0
100
30 [ B
o)
25
80
Pretmt B
Trnt
Pretmt
Tmt
60 _
40-
7:
,,.
o
0
0 D o s e (pmol/2 ~tl a C S F / m i n )
Fig. 2. A: changes in mean arterial blood pressure (MABP -+ S.E.M.) from baselevel during the intracerebroventricular (i.c.v.) infusion of angiotensin II (AII), [D-ASpl]AII, angiotensin III (AIII), and [D-Argl]AIII at the indicated doses. Baselevel MABP were 128.1, 126.1, 121.7 and 125.2 mm Hg, respectively. B: mean (-+ S.E.M.) durations required to recover baselevel blood pressure following the termination of infusion for each analogue at each dose.
Pretmt
.
Tmt
,,,
Pretmt
Trot
"
Fig. 3. A: changes in MABP (+ S.E.M.) from baselevel during i.c.v, pretreatment with artificial cerebral spinal fluid (aCSF, 2 /~l/min) or the appropriate aminopeptidase inhibitor AM or BE (20 nmol/2 pl aCSF/min), followed 10 min later by i.c.v, infused [D-Asp1]AII or [D-Argl]AIII (100 pmol/2/~1 aCSF/min). The order of the pretreatments was counterbalanced among members of each group. Baselevel MABP were 128.1 and 122.5 mm Hg, for the [D-Aspl]AII and [D-Argl]AIII groups, respectively. B: mean (_+ S.E.M.) duration required to recover baselevel blood pressure following the termination of each infusion.
[] [D-Asp ]-All [] [D-Arg ]-AIII
30
.=
Pretmt
Tmt
h. J¢
I,.. J¢
m O~
C9
Pretmt
Tmt
Fig. 4. Changes in MABP (+ S.E.M.) from baselevel during i.c.v. pretreatment with aCSF (2 #llmin) or sarthran (20 nmol/2 #i aCSF/min) followed 10 min later by i.c.v, infused [D-Aspl]AII or [D-Argl]AIII (100 pmol/2/~1aCSF/min). The order of the pretreatments was counterbalanced among members of each group. Baselevel MABP were 133.1 and 128.1 mm Hg for the [o-Aspl]AII and [D-Argl]AIII groups, respectively.
did not differ in duration following the i.c.v, pretreatment with aCSF, [D-Argl]AIII revealed a considerably greater duration of pressor effects following BE than [D-Aspl]AII did following AM. Fig. 4 indicates the effects of i.c.v, pretreatment with aCSF or sarthran upon subsequent i.c.v, infusion of [D-Aspl]AII or [D-Argl]AIII-induced blood pressure effects. There was a significant difference comparing the pressor effects induced by [o-Aspl]AII and [D-Argl]AIII (Fsl.99, df 1,14, P < 0.001). However, there was no effect comparing pretreatment conditions across analogues; nor was there a significant interaction effect of analogues x pretreatment condition. Therefore, [o-Aspl]AII and [D-Argl]AIII did not differ in pressor activity following pretreatment with aCSF or sarthran. However, pretreatment with sarthran subsequently reduced the pressor activities induced by each analogue. Previous investigations have clearly demonstrated sarthran's ability to attenuate the pressor activity of i.c.v, administered AII and AIII TM. DISCUSSION [D-Asp] substitution in position 1 of angiotensin II has previously been reported to increase agonist activity, presumably via increased resistance to aminopeptidase activity26. Systematic substitution of D-amino acids in the interior positions 2-7 of the AII molecule produce weak agonists 17-19'22'27'31, while [D-Phea]AII is a weak antagonist24. The present investigation appears to be the first to employ [D-Argl]AII128. We determined that both [D-Aspl]AII and [o-Argl]AIII are agonists; however, under the present experimental conditions we did not confirm the previously reported enhancement of agonis-
tic activity of [D-Aspl]AI126. In fact [D-Aspl]AII was less active than AII, AIII, and [o-Argl]AIII. Nevertheless, pretreatment with a specific angiotensin receptor antagonist, sarthran, blocked the pressor activity induced by the subsequent i.c.v, infusion of both [D-Aspl]AII and [D-Argl]AIII, strongly suggests that these two ligands are acting at the same receptor site. Our laboratory has previously used the aminopeptidase A inhibitor amastatin to delay conversion of AII to AIII, and the aminopeptidase B inhibitor bestatin to delay conversion of AIII to the hexapeptide AII(3-8) 2,5,16,3o,33. The results of the present study demonstrate that i.c.v, pretreatment with AM greatly reduced the subsequent pressor response to [o-Aspl]AII. Additionally, it was shown that although [D-Aspl]AII exhibited enhanced resistance to metabolism, it was still slowly metabolized with the initial step being the production of AIII. These data suggest the possibility that the reduced activity of [D-Aspl]AII is due to the small amount of AIII that results from its degradation and encourage the speculation that AIII is indeed the centrally active form of angiotensin. This notion is strongly supported by the observation that amastatin can reduce the pressor activity of i.c.v, infused AII while having no effect on AIII 3°. These findings have been mirrored by electrophysiological studies which illustrate the greater potency of AII111, and the ability of amastatin to selectively block the action of iontophoretically applied AII ~2. The enhancement of [o-Argl]AIII's action by bestatin was fully expected and results most likely from reduced metabolism to less active fragments. Bestatin is known to reduce the metabolism of AII and AIII in vivo5 and to potentiate the potency of both angiotensins33. Additionally bestatin can dramatically augment the ability of both AII and AIII to stimulate neurons in the paraventricular nucleus of the rat 12. The present in vitro metabilism data confirms the earlier observation by Regoli eta]. 26 that t~-substitution in position 1 of angiotensin II stabilizes the molecule against aminopeptidase activity. A similar resistance to aminopeptidase activity was observed for [o-Argl]AIII, however [D-Argl]AIII only exhibited superior activity over AII and AIII at the lowest dose, 0.1 pmol. This observation might be expected if high affinity peptidases are involved in the in vivo metabolism of angiotensins. At higher concentrations these enzymes would become saturated and the advantage of degradation resistance would be lost. In summary, the present results offer support for the notion that AIII may be the active form of angiotensin in the mammalian brain, and encourage the continued use of D-amino acid substituted angiotensins in determining the identity of the active form(s) of central angiotensin.
10 Acknowledgements. Thanks are due to Mrs. Ruth Day for her excellent secretarial services. This investigation was supported by Grants HL 32063 and TW 01112 from NIH and Grant 831145 and
an Established Investigator Award to J.W.H. from the American Heart Association and 88 WA 5265 from its Washington Affiliate.
REFERENCES
904-906. 18 Khosla, M.C., Smeby, R.R. and Bumpus, F.M., Structureactivity relationship in angiotensin II analogues. In I.H. Page and EM. Bumpus (Eds.), Handbook of Experimental Pharmacology, Vol. 37, Springer, New York, 1974, pp. 126-160. 19 Khosla, M.C., Hall, M.M., Smeby, R.R. and Bumpus, F.M., Factors that influence the antagonistic properties of angiotensin II antagonists, J. Med. Chem., 16 (1973) 829-832. 20 Lynch, K.R., Simnad, V.I., Ben-Aft, E.T. and Garrison, J.C., Localization of preangiotensinogen messenger RNA sequences in the rat brain, Hypertension, 8 (1986) 540-543. 21 Malvin, R.L., Mouw, D. and Vander, A.J., Angiotensin: physiological role in water-deprivation-induced thirst of rats, Science, 197 (1977) 171-173. 22 Moore, G.J., Franklin, K.J., Nystrom, D.M. and Goghaft, M.H., Structure-desensitization relationships of angiotensin analogues in the rat isolated uterus, Can. J. Physiol. Pharmacol., 63 (1985) 966-971. 23 Ohkubo, H., Nakayama, K., Tanaka, T. and Nakanishi, S., Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity, J. Biol. Chem., 261 (1986) 319-323. 24 Regoli, D. and Park, W.K., The pressor and myotropic effects and the antagonistic properties of several analogues of angiotensin II, Can. J. Physiol. Pharmacol., 50 (1972) 99-112. 25 Regoli, D. Park, W.K. and Rioux, F., Pharmacology of angiotensin, Pharmacol. Rev., 26 (1974) 69-123. 26 Regoli, D., Rioux, F., Park, W.K. and Choi, C., Role of N-terminal amino acid for the biological activity of angiotensin and inhibitory analogues, Can. J. Physiol. Pharmacol., 52 (1974) 39-49. 27 Riniker, B. and Schwyzer, R., In I.H. Page and J.W. McCubbin (Eds.)., Renal Hypertension, Year Book Medical, Chicago, IL, 1968, pp. 80-82. 28 Samanen, J., Narindray, D., Adams, W., Cash, T., Vellin, T. and Regoli, D., Effects of D-amino acid substitution on antagonist activity of angiotensin analogues, J. Med. Chem., 31 (1988) 510-516. 29 Stone, E.A., Improved polyethylene intracerebroventricular cannulas for rats, Physiol. Behav., 20 (1978) 657-659. 30 Sullivan, M.J., Harding, J.W. and Wright, J.W., Differential effects of aminopeptidase inhibitors on angiotensin-induced pressor responses, Brain Research, 456 (1988) 249-253. 31 Visser, G.H., Schattenkerk, C., Kerling, K.E.T. and Havinga, E., Studies on polypeptides, Rec. Tray. Chim. Pays-Bas, 83 (1964) 684-688. 32 Wright, J.W., Morseth, S.L., Abhold, R.H. and Harding, J.W., Pressor action and dipsogenicity induced by angiotensin II and III in rats, Am. J. Physiol., 249 (1985) R514-R521. 33 Wright, J.W., Quirk, W.S., Hanesworth, J.M. and Harding, J.W., Influence of aminopeptidase inhibitors on brain angiotensin metabolism and drinking in rats, Brain Research, 441 (1988) 215-220. 34 Wright, J.W., Jensen, L.L., Roberts, K.A., Sardinia, M.E and Harding, J.W., Structure-function analyses of brain angiotensin control of pressor action in rats, Am. J. Physiol., in press. 35 Wright, J.W., Morseth, S.L., Mana, M.J., LaCross, E,, Petersen, E.E and Harding, J.W., Central angiotensin III induced dipsogenicity in rats and gerbils, Brain Research, 295 (1984) 121-126.
1 Abhold, R.H. and Harding, J.W., Metabolism of angiotensin II and AIII by membrane-bound peptidases from rat brain, J. Pharmacol. Exp. Ther. 245 (1988) 171-177. 2 Batt, C.M., Klein, E.W., Harding, J.W. and Wright, J.W., Pressor responses to amastatin, bestatin and plummer's inhibitors are suppressed by pretreatment with the angiotensin receptor antagonist sarthran, Brain Res. Bull., 21 (1988) 731-735. 3 Bredl, C.R. and Moe, K.E., The ability of angiotensin II vs angiotensin III to arouse a salt appetite, Soc. Neurosci. Abstr., 13 (1987) 1170. 4 Campbell, D.J., Bouhnik, J., Menard, J. and Corvol, E, Identity of angiotensinogen precursors of rat brain and liver, Nature (Lond.), 308 (1984) 206-208. 5 Dewey, A.L., Wright, J.W., Hanesworth, J.M. and Harding, J.W. Effects of aminopeptidase inhibition on the half-lives of [125I]angiotensins in the cerebroventricles of the rat, Brain Research, 448 (1988) 369-372. 6 Dzau, V.J., Ingelfinger, J., Pratt, R.E. and Ellison, K.E., Identification of renin and angiotensin messenger RNA sequences in mouse and rat brains, Hypertension, 8 (1986) 544-548. 7 Fink, G.D. and Bruner, C.A., Hypertension during chronic peripheral and central infusion of angiotensin III, Am. J. Physiol., 249 (1985) E201-E208. 8 Fisher-Ferraro, C., Nahmod, V.E., Golstein, D.J. and Finkelman, S., Angiotensin and renin in rat and dog brain, J. Exp. Med., 133 (1971) 353-361. 9 Ganten, D., Lange, R.E., Lehmann, P. and Unger, T., Brain angiotensin: on the way to becoming a well studied neuropeptide system, Biochem. Pharmacol., 33 (1984) 3523-3528. 10 Ganten, D., Minnich, J.L., Granger, P., Hayduk, K., Brecht, H.M., Barbeau, A., Boucher, R. and Genest, J. Angiotensinforming enzyme in brain tissue, Science, 173 (1971) 64-65. 11 Harding, J.W. and Felix, D., Angiotensin-sensitive neurons in the rat paraventricular nucleus: relative potencies of angiotensin II and angiotensin III, Brain Research, 410 (1987) 130-134. 12 Harding, J.W. and Felix, D. The effects of the aminopeptidase inhibitors amastatin and bestatin on angiotensin-evoked neuronal activity in rat brain, Brain Research, 424 (1987) 299-304. 13 Harding, J.W., Yoshida, M.S., Dilts, R.P., Woods, T.M. and Wright, J.W., Cerebroventricular and intravascular metabolism of [125I]angiotensins in rat, J. Neurochem., 46 (1986) 1292-1297. 14 Harding, J.W., Felix, D., Sullivan, M.J., Camara, C.A., Eftckson, J.B., Regulja, I., Abhold, R.E and Wright, J.W., The pivotal role of angiotensin III in the brain angiotensin system, Proc. West. Pharmacol. Soc., 30 (1987) 11-15. 15 Jensen, L.L., Harding, J.W. and Wright, J.W., Central effects of a specific angiotensin receptor antagonist sarthran ([Sar 1, Thra]AII) in normotensive and spontaneously hypertensive rat strains, Brain Research, 448 (1988) 359-363. 16 Jensen, L.L., Harding, J.W. and Wright, J.W., Increased blood pressure induced by central application of aminopeptidase inhibitors is angiotensinergic-dependent in normotensive and hypertensive rat strains, Brain Research, 490 (1989) 48-55. 17 Jorgensen, E.C., Rapaka, S.R., Windridge, G.C. and Lee, T.C., Angiotensin II analogues. VII. Stereochemical factors in the 5 positions influencing pressor activity, J. Med. Chem., 14 (1971)
5
BRES 15358
Intracerebroventricularly infused [D-Argl]angiotensin III, is superior to [D-Aspl]angiotensin II, as a pressor agent in rats John W. Wright, Kim A. Roberts, Vickie I. Cook, Cathy E. Murray, Michael E Sardinia and
Joseph W. Harding Departments of Psychology and Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164 (U.S.A.)
(Accepted 12 September 1989) Key words: [D-Asp~]Angiotensin II; [D-Argl]Angiotensin III; Angiotensin II; Angiotensin III; Amastatin; Bestatin; Sarthran; Blood pressure; Intracerebroventricular infusion; Rat
Two D-amino acid substitution angiotensin analogues were compared against native angiotensin II (AII) and angiotensin III (AIII) for their resistance to brain tissueqnduced degradation and for pressor potency when intracerebroventricularly (i.c.v.) infused in Sprague-Dawiey rats. The in vitro results indicate that [D-Aspl]AII was very resistant to degradation, AII and [D-Argl]AIII were degraded at similar rates, while AIII was the most rapidly degraded. In vivo results revealed that AII, AIII and [D-Arg~]AIII produced greater pressor responses than [D-Aspl]AII. Intracerebroventricular pretreatment with the aminopeptidase A inhibitor, amastatin, siginificantly reduced the subsequent pressor response to i.c.v, infused [D-Asp~]AII presumably by inhibiting its conversion to AIII. In contrast, pretreatment with the aminopeptidase B inhibitor, bestatin, potentiated the subsequent pressor response to i.c.v, infused [D-Arg~]AIII, presumably by inhibiting the conversion of [D-Arg~]AIII to the less active hexapeptide AII(3-8). Next, i.c.v, pretreatment with the specific angiotensin receptor antagonist, [Sar 1, ThrS]AII (Sarthran) was found to greatly diminish the subsequent pressor responses to i.c.v, infused [D-Aspl]AII and [D-Argl]AIII, suggesting that these analogues are having their effect at the same brain angiotensin receptor site. These results support the hypothesis that AIII, or AIII-like ligands, may serve as the active form of brain angiotensin. INTRODUCTION The brain renin-angiotensin system has become one of the most thoroughly characterized neuropeptide systems, and appears to contain the precursor molecules and enzymatic activity necessary to generate its own angiotensin peptides 4'6'8-1°,2°'23. Most studies have proceeded with the belief that the active form of angiotensin in the brain is angiotensin II (AII); however, the results of several recent investigations support an important role for the heptapeptide angiotensin III ( A I I I ) 14. For example, intracerebroventricular (i.c.v.) injections of A I I I are approximately equipotent to A l l with respect to pressor responses 7'32, drinking behavior 32,35, and salt appetite 3 in normotensive rats despite the increased lability of A I I I upon i.c.v, injection 13. Because of the apparent importance of aminopeptidases in the conversion of A I I to A I I I , and its degradation to less active fragments 1, aminopeptidase inhibitors have been employed in a n u m b e r of studies to delay conversion of A I I to A I I I , and A I I I to the less active hexapeptide A I I ( 3 - 8 ) . Several m a j o r observations may be made from this approach. First, the i.c.v, infusion of a cocktail of these inhibitors,
designed to protect both the N- and C-terminals of the angiotensin molecule, will indeed raise blood pressure and cerebrospinal fluid levels of angiotensin2; and i.c.v. pretreatment with the specific angiotensin receptor antagonist sarthran prevents the subsequent inhibitorinduced pressor effects suggesting an angiotensinergic mechanism of action. Second, i.c.v, pretreatment with amastatin (AM), a reasonably specific inhibitor of aminopeptidase A which is responsible for converting A I I to A I I I , significantly diminishes the subsequent blood pressure response to a bolus injection of A I I 3° and extends the half-life of i.c.v. [125I]AII as determined by microwave fixation 5. Third, A M selectively blocks the ability of A I I to stimulate angiotensin-sensitive neurons in the paraventricular nucleus of the hypothalamus of the rat, while the aminopeptidase B inhibitor, bestatin (BE), does not effect A I I activity 12. These results may suggest that conversion of A I I to A I I I is a prerequisite to angiotensin receptor activation. The present investigation employed two aminopeptidase-resistant angiotensin analogues in an attempt to provide additional evidence supporting the importance of A I I to A I I I conversion in determining physiological
Correspondence: J.W. Wright, Department of Psychology, Washington State University, Pullman, WA 99164, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
r e s p o n s i v e n e s s . To a c c o m p l i s h this task [ D - A s p l ] A I I and [ D - A r g l ] A I I I w e r e synthesized and e x a m i n e d for p r e s s o r p o t e n c y . T h e p r e s e n t results indicate that i.c.v, infused [ D - A s p l ] A I I is less p o t e n t as a p r e s s o r a g e n t t h a n A l l , A I I I and [ D - A r g l ] A I I I . P r e t r e a t m e n t with the a m i n o p e p tidase A i n h i b i t o r A M greatly r e d u c e d the s u b s e q u e n t activity of i.c.v,
infused
[D-Aspl]AII presumably
by
b l o c k i n g residual A I I I p r o d u c t i o n ; and p r e t r e a t m e n t with B E , an a m i n o p e p t i d a s e B i n h i b i t o r which i n t e r f e r e s with c o n v e r s i o n o f A I I I to its h e x a p e p t i d e A I I ( 3 - 8 ) , significantly e n h a n c e d the s u b s e q u e n t p r e s s o r r e s p o n s e i n d u c e d by t h e
i.c.v,
infusion
activity o f b o t h
of [ D - A r g l ] A I I I .
analogues
The
pressor
c o u l d be b l o c k e d by the
specific a n g i o t e n s i n r e c e p t o r a n t a g o n i s t [Sar 1, T h r S ] A I I (sarthran) suggesting that [ D - A s p l ] A I I and [D-ArgX]AIII are acting at a c o m m o n r e c e p t o r site.
MATERIALS AND METHODS Male Sprague-Dawley rats (300-420 g, Charles River derived) were bred and maintained in a AAALAC-approved vivarium. The animals used for in vitro metabolism were maintained in group cages. The rats utilized for alert testing were individually housed in separate cages for a minimum of 48 h before surgery on a 12:12 h light-dark cycle initiated at 07.00 h. Both food and water were available ad libitum except during testing which was conducted between 10.00 and 17.00 h. [Asp 1, IleS]AII and [des-Asp 1, IleS]AII were purchased from U.S. Biochemical. [D-Asp 1, IleS]AII and [D-Arg~, Ile4]AIII were synthesized in our laboratory using a Vega (Coupler 250) amino acid synthesizer, purified by reverse-phase high-performance liquid chromatography (HPLC), followed by amino acid analysis. The purities by weight of the compounds were determined to be 85, 82, 72, and 68%, respectively as measured by HPLC. Peptide purity ranged from 98 to 100% while acetate salts represented the major contributor to the decreased purity by weight. Amastatin and bestatin were purchased from Sigma Chemical. The purity by weight provided by Sigma was 85% for both inhibitors with a peptide content of 99%. The above values were used to adjust for the purity of each peptide so that the dose reflected actual peptide delivered in moles. In vitro metabolism of radioligands An initial experiment was conducted to determine the relative resistance of the compounds AII, [D-Aspl]AII, AIII and [oArgl]AIII, to metabolism by brain tissues. The metabolism of the angiotensins was monitored over a 2 h time period in which 15 time points were examined. Complete time courses for each peptide were developed in duplicate. Eight rats were decapitated, the brains were quickly removed and the tissue block consisting of the thalamus, hypothalamus, septum, and anteroventral third ventricle (AV3V) were dissected over ice. Tissue preparation entailed differential centrifugation, first with hypotonic buffer (pH 7.4 at 4 °C, 50 mM Tris) and then followed with an isotonic buffer (pH 7.4 at 23 °C, 110 mM NaC1, 5 mM KCI, 2 mM CaCl2, 1 mM MgCl2, 25 mM NaPO4). Monoiodinated [12SI]AII, [125I][D-Aspl]AII, [125I]AIII and [125I][DArgl]AIII (2176 Ci/mmoi by immunological self-displacement analysis) were obtained by an immobilized lactoperoxidase glucose oxidase method, using ~25Iodine (New England Nuclear nez-33h), Enzymobeads (Biorad). Assays were carried out at 22 °C in siliconized 12 x 75 mm borosilicate glass culture tubes. Of each iodinated compound 100/~l at a concentration of approximately 0.5 nM were incubated in 300 /~l of isotonic buffer. Tissue was added at consecutive time points
from 5 to 120 min. Incubation was terminated by adding 0.5 ml acetonitrile (ACN) to each tube and vortexing, the samples were then extracted through Baker Columns, dried by speed-vac, and stored frozen. HPLC analyses were carried out using a Partisphere C18 reverse-phase column and isocratic elution at a flow rate of 2 ml/min. The mobile phase consisted of 5 ml/l of H3PO 4 titrated to pH 3.0 with triethylamine plus 14.5% acetonitrile. Radioactivity was measured with a Beckman radioisotope detector (Model 170) and the percentage of intact peptide was calculated with a Hewlett Packard integrator (Model 3390A). Surgery and instrumentation Each animal used for alert testing was anesthetized with Equithesin (Jensen-Salsbury Laboratory, 3.5 ml/kg i.p.) and prepared with a right carotid catheter (PE-50, Clay Adams), inserted to a depth of 2-3.5 cm, plugged with a 1.5 cm length of stainless-steel wire, and externalized between the scapulae 32. The catheter was prefilled with heparinized saline (75 U/ml sterile 0.15 M NaCI). Each animal was also fitted with an i.c.v, guide cannula (PE-60) as previously described 29'32. Following surgery each animal received 0.1 ml i.m. procaine, penicillin G, in dihydrostreptomycin sulfate (200,000 U/ml, Combiotic, Pfizer, NY). Following a minimum of 48 h recovery from surgery, each animal was behaviorally tested in its home cage for the accuracy of the i.c.v, guide cannula placement. This was accomplished by placing a preloaded 24 g stainless-steel injector into the alert animal's guide such that it extended 2-3.5 mm beyond the tip of the guide, thus penetrating the roof of the lateral ventricle. The injector was attached to a 10/A Hamilton syringe by a 25 cm length of PE-20 tubing and contained AII (100 pmol in a total volume of 2 ~1 artificial cerebral spinal fluid [aCSF]2~). If a drinking response was not elicited within 5 min following the injection, the animal was replaced. Pressor responses A minimum of 24 h following behavioral confirmation of correct i.c.v, cannula placement, each animal was placed in a glass test jar (20.5 cm diameter × 20 cm tall) where attachments to a polygraph recorder (Grass Instruments, Model 7B) with Gould tranducers (Model P23ID) were available to monitor mean arterial blood pressure (MABP). The animal was allowed to adapt to the test chamber, and then a stable 5 min baselevel MABP was obtained before the i.c.v, administration of any compounds. All reported blood pressure changes were those that deviated from the established 5 min MABP baselevel. Four groups of rats (8 per group) were utilized to measure the pressor responses induced by the 5 rain i.c.v, infusion of 0, 0.1, 1, 10 and 100 pmol/min doses of All, [D-Aspl]AII, AIII, and [o-Argl]AIII. Each compound was corrected for purity and was prepared in aCSF and delivered at a rate of 2 /d/min (Sage Instruments, Model 355). The doses of each compound were counter-balanced within members of each group such that 4 animals received an ascending order and the other 4 rats a descending order. Sufficient time was allowed between doses to recover baslevel blood pressure. Two additional groups of animals were employed to test the effects of pretreatment with the aminopeptidase inhibitors AM or BE on the subsequent pressor responses induced by [D-Aspl]AII or [o-Argl]AIII. Specifically, four members of the first group received a 5 rain i.c.v, pretreatment infusion of AM (20 nmol/2/d aCSF/min) followed by a 10 rain delay and then a 5 min i.c.v, infusion of [D-Aspl]AII (100 pmol/2 ktl aCSF/min). Each animal was allowed a minimum of 30 min recovery and was next administered a 5 min i.c.v, infusion of aCSF (2/~l/min), followed by a 10 min delay and then a 5 min i.c.v, infusion of [D-Aspl]AII (100 pmol/2 /~l aCSF/min). The other 4 members of this group were tested similarly, but each received the aCSF pretreatment first and the AM pretreatment second. Members of the second group were handled equivalently however the aminopeptidase B inhibitor BE was substituted for AM, and [D-Argl]AIII was substituted for [DAspl]AII.
Pressor response inhibition by sarthran Two additional groups of rats were prepared as discribed above. Four members of the first group were i.c.v, pretreated with the specific angiotensin receptor antagonist [Sar~, ThrS]AII (sarthran, 20 nmol/2/zl/min) for 5 min. Following a 10 rain delay the animal received a 5 rain i.c.v, infusion of [D-ASpl]AII (100 pmol/2 /zl aCSF/min). Each animal was then allowed a minimun of 30 min to recover baselevel blood pressure, and the protocol was repeated, with aCSF replacing sarthran. The other 4 rats received these pretreatments in reversed order, i.e. aCSF followed by sarthran. Members of the second group were equivalently tested, however, [o-Arg~]AIII was substituted for [o-Asp~]AII.
incubation conditions where the metabolism of A I I ! is restricted, i.e. minus divalent cations, both A I I and [ o - A s p l ] A I I degradation resulted in the production of A I I I as the initial metabolite (data not shown). Comparison of complete time course data under the above conditions indicated that both A I I and [D-Aspl]AII are metabolized in an identical manner with the only difference being an overall reduction in degradation rate for [o-Aspl]AII.
Statistical analyses The data from the in vitro metabolism of radioligands was calculated as the percent of total counts applied as intact radioligand over time of incubation. The half-lives of disappearance of labeled angiotensins were calculated from the linear regression lines of the semilog plot of percent angiotensins remaining (ordinate) vs time (abscissa). With respect to the alert testing data sets, initial baselevel MABP were analyzed by one way analysis of variance (ANOVA). The magnitude of the pressor response was calculated by subtracting the corresponding MABP baselevel from the maximum pressor change induced by each treatment. The initial dose-response data set for AII, [D-Aspl]AII, AIII, and [D-Argl]AIII was analyzed by a 4 (Groups) × 5 (Doses) ANOVA with repeated measures on the second factor. The duration of each dose was defined as the time from termination of infusion until MABP returned to baselevel (i.e. nonstatistical differences from baselevel). This data set was also analyzed by a 4 (Groups) × 5 (Doses) ANOVA again with repeated measures on the second factor. The data set concerned with the influence of pretreatment with aCSF or the aminopeptidase inhibitors (AM or BE) upon subsequent pressor responses to [D-Aspl]AII and [o-Argl]AIII, was analyzed by a 2 (Groups) × 2 (Pretreatment condition) ANOVA with repeated measures on the second factor. The duration to return to baselevel MABP was evaluated by a 2 (Groups) × 2 (Pretreatment condition) with repeated measures on the second factor. Finally, the data set concerned with sarthran or aCSF pretreatment followed by [D-ASpl]AII or [D-Argl]AIII was also evaluated by a 2 (Groups) × 2 (Pretreatment condition) with repeated measures on the second factor as was the duration to recover baselevel MABP. Significant effects were further evaluated by Newman-Keuls post-hoe test with a level of significance set at 0.01.
Pressor dose-response curves There were no differences among the groups of the first pressor experiment with respect to baselevel blood pressure prior to treatment (F0.65, df 3,28, P > 0.10). Fig. 2 A presents the maximum changes in blood pressure in response to the infusion of each dose of A I I , [OA s p l ] A I I , A I I I , and [ o - A r g l ] A I I I , and there were significant overall differences among ligands (/74.59, df 3,28, P < 0.02). Post-hoe analyses indicated that [OA r g l ] A I I I , and A I I produced significantly greater pressor responses than [o-Aspl]AII, however [D-Argl]AIII and A I I did not differ, nor did [D-Aspl]AII and A I I I . As expected there was also a significant dose-response relationship (F91.74, df 4,112, P < 0.001); with the 100 pmol dose inducing the greatest elevations in blood pressure across ligands, 10 pmol the next greatest, followed by the 1 pmol dose, while the 0.1 pmol dose did not differ from aCSF infusion. Comparing the analogues at each dose, [ o - A s p l ] A I I revealed a reduced pressor response with respect to A I I and [D-Argl]AIII at the 100, 10 and 1 pmol doses, while at the 0.1 pmol dose the A I I I pressor response was significantly less than that produced by [ o - A s p l ] A I I and [D-Argl]AIII. Also the A I I pressor
RESULTS In vitro metabolism There were clear differences among the compounds utilized in this study concerning resistance to brain tissue-induced metabolism (Fig. 1). [D-Aspl]AII indicated the greatest resistance to metabolism, with a half-life of 251.2 min, while A I I (96.7 min) and [DA r g i ] A I I I (77.2 rain), were degraded at similar rates, and A I I I revealed the greatest degradation with a half-life of 17.3 min. The degradation of A I I I followed a multiphasic pattern with initially rapid degradation followed by a somewhat slower rate. Thus, the substitution of a D-isomer amino acid in position 1 of the A I I and A I I I molecules significantly decreased the rate of metabolism for each ligand, with the greatest contrast occurring between A I I I and its analogue [a-Argl]AIII. Utilizing
100 ¢' ~L~.O, __ ~ ~
g
"~
=
8- ~
~'o'--
I - - -o- -
All [D-Asp ]-All
. ---o--
AIII [D.Arg ]-AIII
o - - - O.
o
40 20 0
0
30 Incubation
60 Time
90
120
(min)
Fig. 1. Percent radiolabel [125I]AII, [125I][D-Aspl]AII, [125I]AIII, and [125I][D-Argl]AIIIremaining intact during a 120 min incubation period with brain tissue at 22 °C. Separate determinations were made in duplicate at 15 rain intervals.
response at the 0.1 pmol dose was reduced as compared with that of [D-ArgZ]AIII. Fig. 2B presents the mean (+ S.E.M.) durations of the pressor effects induced by each ligand at each dose. Although there were no overall differences in latencies comparing the 4 ligands, there was a dose-response effect (F79.32, df 4,112, P < 0.0001); the 100 pmol dose produced the longest latency to recover, 10 pmol the next longest, followed by the 1 pmol dose, while the 0.1 pmol dose and aCSF were not different. There was no interaction effect between ligands and doses. Pretreatment with amastatin or bestatin
The effect of i.c.v, pretreatment with aCSF upon the subsequent i.c.v, infusion of [D-Aspl]AII or [D-Argl]AIII may be compared with the i.c.v, pretreatment of AM followed by [D-Aspl]AII or BE followed by [o-Argl]AIII in Fig. 3A. There was a significant difference comparing the pressor effects induced by [D-Aspl]AII and [DArgl]AIII across pretreatment conditions (F17.95, df 1,14, P < 0.005). There was also a significant effect comparing
"A • 30 / []
All [D-Asp ]-All .
~.-
pretreatment conditions across analogues (F6.47 , df 1,14, P < 0.05); and there was a significant interaction effect of analogues x pretreatment conditions (F44.78, df 1,14, P < 0.005). Specifically, [D-Aspl]AII and [D-Argl]AIII did not differ in pressor activity following pretreatment with aCSF; however, the [D-Aspl]AII response was significantly diminished following pretreatment with AM, and the [D-ArgZ]AIII response was significantly elevated following pretreatment with BE. Fig. 3B presents the mean (+ S.E.M.) duration of the pressor effects induced by each i.c.v, pretreatment with aCSF or aminopeptidase inhibitors, followed by treatment with [D-Aspl]AII or [D-Argl]AIII. There was a significant difference between [o-Aspl]AII and [OArgl]AIII comparing the durations of their pressor effects (F18.12, df 1,14, P < 0.005); with [o-Argl]AIII indicating the greatest overall duration. There was also a significant difference due to pretreatment condition (F6.97 , df 1,14, P < 0.02), and the interaction effect was significant (F20.82, df 1,14, P < 0.001). Post-hoe tests indicated that although [o-Aspl]AII and [o-Argl]AIII
.
15
o~
0
aCSF
0.1
1.0
10
10
0
100
30 [ B
o)
25
80
Pretmt B
Trnt
Pretmt
Tmt
60 _
40-
7:
,,.
o
0
0 D o s e (pmol/2 ~tl a C S F / m i n )
Fig. 2. A: changes in mean arterial blood pressure (MABP -+ S.E.M.) from baselevel during the intracerebroventricular (i.c.v.) infusion of angiotensin II (AII), [D-ASpl]AII, angiotensin III (AIII), and [D-Argl]AIII at the indicated doses. Baselevel MABP were 128.1, 126.1, 121.7 and 125.2 mm Hg, respectively. B: mean (-+ S.E.M.) durations required to recover baselevel blood pressure following the termination of infusion for each analogue at each dose.
Pretmt
.
Tmt
,,,
Pretmt
Trot
"
Fig. 3. A: changes in MABP (+ S.E.M.) from baselevel during i.c.v, pretreatment with artificial cerebral spinal fluid (aCSF, 2 /~l/min) or the appropriate aminopeptidase inhibitor AM or BE (20 nmol/2 pl aCSF/min), followed 10 min later by i.c.v, infused [D-Asp1]AII or [D-Argl]AIII (100 pmol/2/~1 aCSF/min). The order of the pretreatments was counterbalanced among members of each group. Baselevel MABP were 128.1 and 122.5 mm Hg, for the [D-Aspl]AII and [D-Argl]AIII groups, respectively. B: mean (_+ S.E.M.) duration required to recover baselevel blood pressure following the termination of each infusion.
[] [D-Asp ]-All [] [D-Arg ]-AIII
30
.=
Pretmt
Tmt
h. J¢
I,.. J¢
m O~
C9
Pretmt
Tmt
Fig. 4. Changes in MABP (+ S.E.M.) from baselevel during i.c.v. pretreatment with aCSF (2 #llmin) or sarthran (20 nmol/2 #i aCSF/min) followed 10 min later by i.c.v, infused [D-Aspl]AII or [D-Argl]AIII (100 pmol/2/~1aCSF/min). The order of the pretreatments was counterbalanced among members of each group. Baselevel MABP were 133.1 and 128.1 mm Hg for the [o-Aspl]AII and [D-Argl]AIII groups, respectively.
did not differ in duration following the i.c.v, pretreatment with aCSF, [D-Argl]AIII revealed a considerably greater duration of pressor effects following BE than [D-Aspl]AII did following AM. Fig. 4 indicates the effects of i.c.v, pretreatment with aCSF or sarthran upon subsequent i.c.v, infusion of [D-Aspl]AII or [D-Argl]AIII-induced blood pressure effects. There was a significant difference comparing the pressor effects induced by [o-Aspl]AII and [D-Argl]AIII (Fsl.99, df 1,14, P < 0.001). However, there was no effect comparing pretreatment conditions across analogues; nor was there a significant interaction effect of analogues x pretreatment condition. Therefore, [o-Aspl]AII and [D-Argl]AIII did not differ in pressor activity following pretreatment with aCSF or sarthran. However, pretreatment with sarthran subsequently reduced the pressor activities induced by each analogue. Previous investigations have clearly demonstrated sarthran's ability to attenuate the pressor activity of i.c.v, administered AII and AIII TM. DISCUSSION [D-Asp] substitution in position 1 of angiotensin II has previously been reported to increase agonist activity, presumably via increased resistance to aminopeptidase activity26. Systematic substitution of D-amino acids in the interior positions 2-7 of the AII molecule produce weak agonists 17-19'22'27'31, while [D-Phea]AII is a weak antagonist24. The present investigation appears to be the first to employ [D-Argl]AII128. We determined that both [D-Aspl]AII and [o-Argl]AIII are agonists; however, under the present experimental conditions we did not confirm the previously reported enhancement of agonis-
tic activity of [D-Aspl]AI126. In fact [D-Aspl]AII was less active than AII, AIII, and [o-Argl]AIII. Nevertheless, pretreatment with a specific angiotensin receptor antagonist, sarthran, blocked the pressor activity induced by the subsequent i.c.v, infusion of both [D-Aspl]AII and [D-Argl]AIII, strongly suggests that these two ligands are acting at the same receptor site. Our laboratory has previously used the aminopeptidase A inhibitor amastatin to delay conversion of AII to AIII, and the aminopeptidase B inhibitor bestatin to delay conversion of AIII to the hexapeptide AII(3-8) 2,5,16,3o,33. The results of the present study demonstrate that i.c.v, pretreatment with AM greatly reduced the subsequent pressor response to [o-Aspl]AII. Additionally, it was shown that although [D-Aspl]AII exhibited enhanced resistance to metabolism, it was still slowly metabolized with the initial step being the production of AIII. These data suggest the possibility that the reduced activity of [D-Aspl]AII is due to the small amount of AIII that results from its degradation and encourage the speculation that AIII is indeed the centrally active form of angiotensin. This notion is strongly supported by the observation that amastatin can reduce the pressor activity of i.c.v, infused AII while having no effect on AIII 3°. These findings have been mirrored by electrophysiological studies which illustrate the greater potency of AII111, and the ability of amastatin to selectively block the action of iontophoretically applied AII ~2. The enhancement of [o-Argl]AIII's action by bestatin was fully expected and results most likely from reduced metabolism to less active fragments. Bestatin is known to reduce the metabolism of AII and AIII in vivo5 and to potentiate the potency of both angiotensins33. Additionally bestatin can dramatically augment the ability of both AII and AIII to stimulate neurons in the paraventricular nucleus of the rat 12. The present in vitro metabilism data confirms the earlier observation by Regoli eta]. 26 that t~-substitution in position 1 of angiotensin II stabilizes the molecule against aminopeptidase activity. A similar resistance to aminopeptidase activity was observed for [o-Argl]AIII, however [D-Argl]AIII only exhibited superior activity over AII and AIII at the lowest dose, 0.1 pmol. This observation might be expected if high affinity peptidases are involved in the in vivo metabolism of angiotensins. At higher concentrations these enzymes would become saturated and the advantage of degradation resistance would be lost. In summary, the present results offer support for the notion that AIII may be the active form of angiotensin in the mammalian brain, and encourage the continued use of D-amino acid substituted angiotensins in determining the identity of the active form(s) of central angiotensin.
10 Acknowledgements. Thanks are due to Mrs. Ruth Day for her excellent secretarial services. This investigation was supported by Grants HL 32063 and TW 01112 from NIH and Grant 831145 and
an Established Investigator Award to J.W.H. from the American Heart Association and 88 WA 5265 from its Washington Affiliate.
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