From the Department of Physiology, Michigan State University (East
Lansing) (P.F.D., R.S.R.-B.); and the Department of Research Chemistry, Amylin
Pharmaceuticals, Inc, San Diego, Calif (D.D.H.).
Two fundamentally different approaches to antisense peptide
design have been used to generate both Ang II antipeptides and
antisense peptides against other peptide targets. One, first suggested
theoretically by Mekler,16 was reinvented later
by Biro17 and Blalock and
Smith18 and experimentally was pioneered by
Blalock and colleagues.19 20 It uses the RNA
sequence that would be generated by the noncoding (-) or complementary
strand of the DNA encoding Ang II if it were read in the normal or
antiparallel reading frame (5'
RB6 peptide has much lower activity as an antagonist to Ang
II using the synaptic membrane assay
(Kd=5x10-6), but
direct evidence of antisense peptide binding to Ang II exists for this
peptide from all of the techniques mentioned
above.12 13 The binding constant obtained from
nuclear magnetic resonance studies of RB6 peptide binding to Ang II in
aqueous solution is somewhat lower, but it is consistent with
measured antagonist activity.13 We
note that this micromolar binding is more in line than nanomolar
affinity with what would be expected from peptide interactions in
aqueous solution.
We have compared our results with published data on the inhibition of
Ang II contractions with the nonpeptide agent losartan, which
reduces contraction of the rabbit aorta and has been found to lower
blood pressure after infusion into rats.31 32 33
Although this antagonism is not directly related to antisense peptides,
it provides a benchmark for functionality. Moreover, losartan
is known to antagonize Ang II activity due to allosteric binding to the
Ang II receptor, a model that should merit further consideration in
analyzing Ang II–antisense peptide activity.
Tissue Procedures
The tissues were prepared for mechanical measurements using the
procedures previously established for carotid
strips,34 35 36 with modification as described
below for the use of tissue rings. The aorta was debrided of excess
connective tissue, flushed of any remaining blood, and placed in fresh
PSS. Aortic rings of 3 mm were cut using a single-edge razor
blade, and the rings were placed in fresh PSS. The scissor-cut ends
were not used. A pair of stainless steel loops with a flat, straight
central section was passed through the lumen of each aortic ring. Upper
and lower loops were secured to Plexiglas–stainless steel clamps with
stainless steel screws. The lower clamp was attached to a
micrometer (Newport Corp) for length adjustment. The upper
clamp was connected to a 50-g force transducer (Kulite Semiconductor)
with a gold chain. The force transducers were interfaced with an
eight-channel Gould Instruments signal conditioner and
recorder.
The rings were immersed in 20-mL or 25-mL aerated, jacketed tissue
baths (Harvard Apparatus) and maintained at 37°C with a
Haake (Karlsruhe) circulator. After mounting, each ring was stretched
to 5 g and allowed to stress-relax for 2 hours before activation.
If stress-relaxation reached 0 g, the ring was restretched to
2 g and allowed to stress-relax until the passive force was
stable. The rings had a stretched linear length of 3 to 4 mm.
The tissues were activated with either K+
or Ang II. Modifications of the Ang II contractions were made with
different combinations and concentrations of Bl peptide, RB peptides,
and Ang II antiserum. The Bl and RB6 peptides were used alone to assess
their ability to produce contractions independent of Ang II. Individual
contractions were generated by replacing PSS in the tissue baths with
prewarmed stimulating PSS. Each contraction lasted 4 to 6 minutes,
followed by at least 15 minutes of relaxation before a following
contraction. Relaxation to baseline force typically took 10 minutes. At
the conclusion of the experiment, the rings were removed from the
baths, blotted dry as previously described,35 and
weighed on a Mettler balance to the nearest 0.1 mg. For the 29 rings in
these experiments, the average±SE weight was 5.9±0.3 mg. The tissue
force measurements were compared with a control 10 nmol/L Ang II
contraction on the same ring. To minimize error that can be introduced
by percentage comparisons in dose-response curves, the contractions
were also normalized to the weight of the ring (grams of force per
milligram of tissue).
In control experiments the passive force reached by the method
described above was compared with the force generated by
K+ contractions. For eight rings,
K+ contractions generated 1.18±0.10 g/mg
(mean±SE). Passive tension in these rings was 0.25±0.03 g/mg of the
K+ contractions. This placed the rings at
approximately Lo, the optimal length for force
development.34 The average normalized passive
force in all 29 rings was 0.22±0.02 g/mg. Postcontrol contractions
were made after high micromolar exposure to peptides to test for
peptide-generated tissue damage. Comparisons between different samples
was made by using Student's two-tailed t test. A value of
P<.02 was considered as indicating a significant
difference. Estimates of the Ang II force inhibition effects of
RB6+antiserum or Bl peptide+antiserum versus either peptide's
individual inhibition used the semi-logarithmic scale of the Ang II
individual dose-response curve.
Ang II contractions were generated using differing concentrations of
Ang II. The only experiments in which cumulative dose-response curves
were generated were comparisons of Ang II with and without Bl or RB6
peptides, to minimize the cost of the peptide use at high peptide
concentrations. All others used individual contractions.
Fig 2
Fig 3
The intriguing augmentation of Ang II contractions by RB6 peptide was
investigated further. In Fig 4
Fig 5
Inhibition of Ang II contractions with RB6 peptide is demonstrated in
Fig 6
The Bl peptide does not give the same result (Fig 6
Various modifications of RB6 peptide listed in Table 1
The most interesting and surprising result concerning RB6 peptide
activity was that at high nanomolar and low micromolar concentrations
it significantly increased the force of Ang II–induced contractions in
a dose-dependent manner (Figs 2 through 4
Clearly, two opposing types of activity must be present to develop
the inverting curve observed in Fig 4
Whatever the mechanism of RB6 activity, the nature of its dual
augmentation/antagonistic activity raises an interesting
possible application of this or related compounds to migraine
treatment. There are several theories of migraine production,
and no definitive mechanism has been determined as yet. In one of those
theories, migraines are thought to be characterized by an initial
decrease in cerebral vascular perfusion accompanied by focal
neurological deficits, followed by a phase in which there is an
increase in cerebral blood flow accompanied by
headache.42 A quickly degrading peptide given in
high concentrations in the decreased-blood-flow, neurological-deficit
phase would relax arterial blood vessels, increasing blood
flow. The lower concentrations of peptide available at a later time
would augment vascular contraction during the increased-blood-flow
headache phase.
The negative results obtained with sequence variants of RB6 are also
interesting (see Tables 1
Our results are also very important because they clarify a
long-standing debate over the activity and mechanisms of Ang II
antisense peptides. Elton et al6 have previously
reported that Bl peptide antagonizes Ang II activity with a
Kd of 58x10-9
mol/L, but assumed in their calculation is that all of the observed
activity was due to direct binding of Blalock peptide to Ang II. In
light of the failure of any physicochemical technique to validate this
assumption, their calculation is
suspect.5 8 9 11 12 13 27 30 Direct measurement of
activity gives a functional Kd on the order
of 2x10-6 mol/L, some 200 times lower than
previously reported (Fig 5
Mid-micromolar activity in conjunction with the lack of evidence for
direct binding to Ang II and the strong similarity between Bl peptide
and Ang II all suggest that Bl peptide is a sequence-variant
antagonist of the Ang II receptor. To test this
possibility, the effect of Ang II antiserum on Bl peptide activity was
assayed. If Bl peptide bound directly to Ang II and not at all to the
Ang II receptor, then one would expect that the effects of Ang II
antiserum would be additive with those of Bl peptide. Both would bind
up some proportion of Ang II, so that Ang II–induced muscle
contractions would be less than if only the antiserum or only the Bl
peptide were present. In fact, the effect of Ang II antiserum in
the presence of Bl peptide was not additive (Fig 6
We note that the same basic problems observed with Bl peptide in this
study exist for a very wide range of peptides derived by the
Mekler-Biro-Blalock approach.14 25 26 Calculated
binding constants often assume direct binding of the antipeptide to its
peptide in the absence of physicochemical evidence or despite direct
evidence to the contrary.43 44 45 46 47 48 In some
cases,45 49 the Root-Bernstein approach yields
active antisense peptides when the Mekler-Biro-Blalock approach does
not. Reported binding constants vary from nanomolar values to no
measurable binding by different research group, technique used, and the
particular antisense peptide being
studied.14 25 26
These experiments also negate any functional antisense-binding
contractile-inhibition effect of Ang II antipeptides generated in the
parallel reading frame. Although we were unable to complete a full
activity curve for Root-Bernstein peptide (this procedure would have
required approximately a gram of peptide to reach the high
concentrations necessary), the data available suggest that the
previously reported Kd of
To summarize, our data demonstrate that although neither parallel- nor
antiparallel-generated Ang II antipeptides have sufficient sensitivity
to be pharmacologically useful in inhibiting Ang II contractions, the
parallel antipeptide RB6 at high nanomolar to low micromolar
concentrations exhibits unique previously unreported properties in
augmenting Ang II contractions that may have great clinical and
investigative potential. Elucidating the mechanism of the augmentation
effects of RB6 may lead to a better understanding of the slow pressor
effect of chronic Ang II exposure and of migraine treatment. The dual
activity of RB6 on Ang II contractions could create a novel paradigm
for peptide therapies if other antisense peptide systems show similar
dual activities.
Received July 22, 1997;
first decision July 30, 1997;
accepted October 20, 1997.
2.
Clouston WM, Lloyd CJ, Richards R. I. Inducible
antisense RNA for angiotensinogen in stably transformed
hepatoma cell lines. J Mol Endocrinol. 1990;4:107–117.
3.
Schinke M, Bohm M, Bricca G, Ganten D, Bader M.
Permanent inhibition of angiotensinogen synthesis by
antisense RNA expression. Hypertension. 1996;27:508–513.
4.
Ruiz-Opazo N, Akimoto K, Herrera VLM. Identification
of a novel dual angiotensin II/vasopressin receptor on the
basis of molecular recognition theory. Nat Med. 1995;1:1074–1081.[Medline]
[Order article via Infotrieve]
5.
Soffer RL, Bandyopadhyay S, Rosenberg E, Hoeprich P,
Teitelbaum A, Brunck T, Colby CB, Gloff C. Unexpected binding of an
octapeptide to angiotensin II receptor. Proc Natl
Acad Sci U S A. 1987;84:9219–9222.
6.
Elton TS, Dion LD, Bost KL, Oparil S, Blalock JE.
Purification of an angiotensin II binding protein by using
antibodies to a peptide encoded by angiotensin II
complementary RNA. Proc Natl Acad Sci U S A. 1988;85:2518–2522.
7.
Elton TS, Oparil S, Blalock JE. The use of
complementary peptides in the purification of an
angiotensin II binding protein. J Hypertens
Suppl. 1988;6:S404–S407.[Medline]
[Order article via Infotrieve]
8.
Moore GJ, Ganter RC, Franklin KJ.
Angiotensin `antipeptides': (-) messenger RNA
complementary to human angiotensin II (+) messenger RNA
encodes an angiotensin receptor antagonist.
Biochem Biophys Res Commun. 1989;160:1387–1391.[Medline]
[Order article via Infotrieve]
9.
Weist SA, Gesellchen PD, Zimmerman K, Steinberg MI.
Binding and pharmacological properties of peptides derived from human
and rat angiotensin II (AII) mRNA. Biochem Biophys
Res Commun. 1990;170:462–469.[Medline]
[Order article via Infotrieve]
10.
Budisavljevic M, Bea ML, Bensoussan M, Laubie M, Chuong
PPV, Dussaule JC, Verroust PJ, Ronco PM. Antagonist effect
of a receptor-mimicking peptide encoded by human
angiotensin II complementary RNA. Hypertension. 1992;19:345–354.
11.
Jackson EK, Prakash C, Blair IA. Studies on the
peptides encoded by rat and human angiotensin II
complementary RNA. Hypertension. 1993;21:42–49.
12.
Loo JA, Holsworth DD, Root-Bernstein RS. Use of
electrospray ionization mass spectrometry to probe antisense peptide
interactions. Biol Mass Spect. 1994;23:6–12.
13.
Holsworth DD, Kiely JS, Root-Bernstein RS, Overhiser
RW. Antisense-designed peptides: a comparative study focusing on
possible complements to angiotensin II. Pept
Res. 1994;7:185–193.[Medline]
[Order article via Infotrieve]
14.
Root-Bernstein RS, Holsworth DD. Antisense peptides: a
critical mini-review. J Theor Biol. In press.
15.
Budisavljevic M, Bea ML, Bensoussan M, Laubie M, Chuong
PPV, Dussaule JC, Verroust PJ, Ronco PM. Antagonist effect
of a receptor mimicking peptide encoded by human
angiotensin II. Hypertension. 1992;19:345–354.
16.
Mekler LB. On the specific selective interaction
between amino acid residues of polypeptide chains.
Biofizika. 1969;14:581–584.[Medline]
[Order article via Infotrieve]
17.
Biro J. Comparative analysis of
specificity in protein-protein interaction. Med Hypotheses. 1981;7:969–1007.[Medline]
[Order article via Infotrieve]
18.
Blalock JE, Smith EM. Hydropathic anti-complementarity
of amino acids based on the genetic code. Biochem Biophys Res
Commun. 1984;121:203–207.[Medline]
[Order article via Infotrieve]
19.
Bost KL, Smith EM, Blalock JE. Similarity between the
corticotropin (ACTH) receptor and a peptide encoded by an RNA that is
complementary to ACTH mRNA. Proc Natl Acad Sci
U S A. 1985;82:1372–1375.
20.
Blalock JE, Bost KL. Binding of peptides that are
specified by complementary RNAs. Biochem J. 1986;234:679–683.[Medline]
[Order article via Infotrieve]
21.
Root-Bernstein RS. Amino acid pairing. J Theor
Biol. 1982;94:885–894.[Medline]
[Order article via Infotrieve]
22.
Root-Bernstein RS. On the origin of the genetic code.
J Theor Biol. 1982;94:895–904.[Medline]
[Order article via Infotrieve]
23.
Root-Bernstein RS, Westall FC.
Fibrinopeptide: a binds Gly-Pro-Arg-Pro. Proc
Natl Acad Sci U S A. 1984;81:4339–4342.
24.
Root-Bernstein RS, Westall FC. Bovine pineal
antireproductive tripeptide binds to luteinizing hormone-releasing
hormone: a model for peptide modulation by sequence specific peptide
interactions? Brain Res Bull. 1986;17:519–528.[Medline]
[Order article via Infotrieve]
25.
Tropsha A, Kizer JS, Chaiken IM. Making sense from
antisense: a review of experimental data and developing ideas on
sense-antisense peptide recognition. J Mol Recognit. 1992;5:43–54.[Medline]
[Order article via Infotrieve]
26.
Jarpe MA, Blalock JE. Complementary peptides:
applications of the molecular recognition theory to peptide and protein
purification and design. In: Basava C, Anantharamaiah GM, eds.
Peptides: Design, Synthesis, and Biological Activity.
Boston, Mass: Birkhauser; 1994:165–179.
27.
Martin SF, Feisk SW, Austin RE, Eaton HL. NMR study of
the possible interactions in solution of angiotensin II
with a peptide encoded by angiotensin II complementary RNA.
Proc Natl Acad Sci U S A. 1989;86:9767–9769.
28.
Zhou N, Moore GJ, Vogel HJ. Proton NMR studies of
angiotensin II and its analogues in aqueous solution.
J Protein Chem. 1991;10:333–343.[Medline]
[Order article via Infotrieve]
29.
DeGasparo M, Whitebread S, Einsle K, Heusser C. Are the
antibodies to a peptide complementary to angiotensin II
useful to isolate the angiotensin II receptor?
Biochem J. 1989;261:310–311.[Medline]
[Order article via Infotrieve]
30.
Guillemette G, Boulay G, Gagnon S, Bosse R, Escher E.
The peptide encoded by angiotensin II complementary RNA
does not interfere with angiotensin II action.
Biochem J. 1989;261:309–311.[Medline]
[Order article via Infotrieve]
31.
Wong PC, Timmermanns PB. Nonpeptide
angiotensin II receptor antagonists:
insurmountable angiotensin II antagonism of EXP3892 is
reversed by the surmountable antagonist DuP 753.
J Pharmacol Exp Ther. 1991; 258:49–57.
32.
Wong PC, Hart SD, Zaspel AM, Chiu AT, Ardecky RJ, Smith
RD, Timmermans PB. Functional studies of nonpeptide
angiotensin II receptor subtype-specific ligands: DuP 753
(AII-1) and PD123177 (AII-2). J Pharmacol Exp Ther. 1990;255:584–592.
33.
Wong PC, Bernard R, Timmermans PB. Effect of blocking
angiotensin II receptor subtype on rat sympathetic nerve
function. Hypertension. 1992;19:663–667.
34.
Herlihy JT, Murphy RA. Length-tension relationship of
smooth muscle of the hog carotid artery. Circ Res. 1973;33:275–283.
35.
Dillon PF, Murphy RA. Tonic force maintenance
with reduced shortening velocity in arterial smooth muscle.
Am J Physiol. 1982;242:C102–C108.
36.
Dillon PF, Murphy RA. High force development and
crossbridge attachment in smooth muscle from swine carotid arteries.
Circ Res. 1982;50:799–804.
37.
Dickenson CJ, Lawrence JR. A slowly developing pressor
response to small concentrations of angiotensin: its
bearing on the pathogenesis of chronic renal hypertension.
Lancet. 1063;1:1354–1356.
38.
Brown AJ, Casals-Stenzel J, Goffortd S, Lever AF,
Morton JJ. Comparison of fast and slow pressor effects of
angiotensin II in the conscious rat. Am J
Physiol. 1981;241:H381–H388.
39.
Melaragno MG, Fink GD. Enhanced slow pressor effect of
angiotensin II in two-kidney, one clip rats.
Hypertension. 1995;25:288–293.
40.
Dillon PF, Aksoy MO, Driska SP, Murphy RA. Myosin
phosphorylation and the cross-bridge cycle in
arterial smooth muscle. Science. 1981;211:495–497.
41.
Haeberle JR, Trybus KM, Hemric ME, Warshaw DM. The
effects of smooth muscle caldesmon on actin filament motility.
J Biol Chem. 1992;267:23001–23006.
42.
Lauritzen M. Cerebral blood flow in migraine and
cortical spreading depression. Acta Neurol Scand Suppl. 1987;113:1–40.[Medline]
[Order article via Infotrieve]
43.
Eberle AN, Huber M. Antisense peptides of ACTH and MSH:
tools for receptor isolation? In: Mol JNM, van der Krol AR, eds.
Antisense Nucleic Acids and Proteins. New York, NY: Marcel
Dekker; 1991:187–208.
44.
Goldstein A, Brutlag DL. Is there a relationship
between DNA sequences encoding peptide ligands and their receptors?
Proc Natl Acad Sci U S A. 1989;86:42–45.
45.
Jurzak M, Pavo I, Fahrenholz F. Lack of interaction of
vasopressin with its antisense peptides: a functional and immunological
study. J Recept Res. 1993;13:881–902.[Medline]
[Order article via Infotrieve]
46.
Masu Y, Nayakama K, Tamaki H, Harada Y, Kuno M,
Nakanishi S. cDNA cloning of bovine substance-K receptor through oocyte
expression system. Nature. 1987;329:836–838.[Medline]
[Order article via Infotrieve]
47.
Najem ES, Corigliano-Murphy A, Ferretii JA.
Conformational behavior of fragments of adrenocorticotropin and their
antisense peptides determined by NMR spectroscopy and CD
spectropolarimetry. FEBS Lett. 1989;250:405–410.[Medline]
[Order article via Infotrieve]
48.
Rasmussen UB, Hesch RD. On antisense peptides: the
parathyroid hormone as an experimental example and a critical
theoretical view. Biochem Biophys Res Commun. 1987;149:930–938.[Medline]
[Order article via Infotrieve]
49.
Johnson HM, Torres BA. A novel arginine
vasopressin-binding peptide that blocks arginine vasopressin modulation
of immune functions. Biochem Biophys Res Commun. 1988;141:2420–2423.
© 1998 American Heart Association, Inc.
Scientific Contributions
Augmentation of Aortic Ring Contractions by Angiotensin II Antisense Peptide
Abstract
Top
Abstract
Introduction
Methods
Results
Conclusions
References
Abstract—Previous biochemical
experiments have revealed two antisense peptide antagonists
to human angiotensin II (Ang II), one encoded in the cDNA
in the antiparallel reading, the other in the parallel reading. Neither
peptide's ability to produce physiological
antagonism has been demonstrated previously. Both peptides were tested
for their ability to antagonize Ang II–induced contractions on rabbit
aorta smooth muscle. Neither peptide had any direct contractile
activity. The antiparallel Ang II peptide had
physiological antagonism to Ang II contractions at
a lower sensitivity than reported in biochemical studies, and its
antagonist activity was partially blocked by Ang II
antiserum, suggesting that it is not an antipeptide but an Ang II
homologue. The parallel Ang II antipeptide also required high
concentrations for physiological inhibition. Its
contractile inhibition was not affected by Ang II antiserum and
diminished the Ang II contraction at high micromolar concentrations,
findings consistent with physicochemical data showing that it
is an Ang II complement. The concentration of either peptide required
to produce an antagonistic
physiological effect was too high to predict any
pharmacological usefulness. The parallel antipeptide, however,
significantly increased the force of muscle contractions at high
nanomolar concentrations, thus displaying a unique dual
augmentation/antagonist activity. This antipeptide seems to
have highly sequence-specific activity because other similar parallel
antipeptides had no activity. The parallel antipeptide augmentation
mimics the shift in the Ang II dose-response curve produced in
hypertension studies of the slow pressor effect of Ang II and may be
useful in deducing the currently unknown cause of the slow pressor
effect. It may also have some uses in migraine studies.
Key Words: antisense elements • peptides • angiotensin II • muscle, smooth, vascular
Introduction
Top
Abstract
Introduction
Methods
Results
Conclusions
References
These experiments
were undertaken to test the mechanisms of action and
physiological utility of Ang II antisense peptides.
Antisense peptides are the logical analogues of antisense
oligonucleotides. Just as the noncoding strand of DNA
can be used either directly to generate an antisense DNA sequence or
indirectly to generate antisense mRNA that can bind to and
inactivate the coding strand of DNA or its respective mRNA,
some investigators believe that it is possible to use the coding strand
of DNA or its respective mRNA to generate antisense peptides that are
assumed to bind directly to and inactivate the
sense-generated peptide. Thus, four different antisense approaches
exist. All have been applied to Ang II regulation. Antisense DNA
sequences have been genetically inserted into cells to regulate Ang II
receptor expression.1 Antisense RNA has been
exogenously introduced to limit angiotensinogen
expression.2 3 Assuming that antisense DNA
encodes a protein complementary to Ang II, Ruiz-Opazo et
al4 have used the antisense approach to isolate a
novel dual receptor for Ang II and vasopressin. Finally, investigators
have synthesized antisense peptide sequences based on two different
reading frames of Ang II mRNA (parallel and antiparallel; see later
section) resulting in Ang II antagonist
activity.5 6 7 8 9 10 11 12 13 This study focuses on Ang II
antisense peptide activity.14 15 
3'). The resulting antisense peptide
for human Ang II has the sequence
NH2-GLU-GLY-VAL-TYR-VAL-HIS-PRO-VAL-COOH. This sequence shall be
referred to as "Blalock peptide," or "Bl peptide" for short.
The second approach to antisense design was initiated both
theoretically and experimentally by
Root-Bernstein.21 22 23 24 It also uses the (-) strand
DNA sequence but reads it in the parallel or backward reading frame
(3'5'). The resulting antisense peptide sequence for human Ang II is
NH2-LEU-ALA-HIS-MET-TYR-VAL-GLY-LYS-COOH. In previous
publications,8 9 this peptide was referred to as
RB1 peptide. An inverted version of this
peptide—NH2-LYS-GLY-VAL-TYR-MET-HIS-ALA-LEU-COOH, referred to as
RB6—has significantly greater activity than
RB1.8 9 Both approaches are often used, sometimes
interchangeably, by those generating so-called antisense or
"complementary" peptides (reviewed in References 14, 25, and 2614 25 26 ).
Among the unresolved issues of the field are whether these two
approaches are interchangeable and whether the resulting peptides
actually represent "complements" or antipeptides that bind
directly to their target peptides. This assumption has been seriously
questioned. Despite claims of low nanomolar
activity,6 several groups independently have
demonstrated, using nuclear magnetic resonance
spectroscopy,13 27 28 soft ionization
electrospray mass spectrometry,12 and binding of
radiolabeled Ang II to immobilized
peptides,13 that there is no detectable binding
between Bl peptide and Ang II even at millimolar concentrations.
Moreover, DeGasparo et al29 and Guillemette et
al30 have directly challenged the finding that
the form of Bl peptide generated from rat mRNA has anti–Ang II
activity, whereas Soffer et al,5 Moore et
al,8 Weist et al,9 and
Jackson et al11 have concluded that Bl peptide
acts not as an Ang II antipeptide but as an Ang II receptor
antagonist. Moreover, Bl peptide is more than 80% similar
to Ang II (Table 1
). This sequence
similarity would tend to indicate that Bl peptide is not complementary
to Ang II but acts by binding to the receptor as a simple
sequence-variant antagonist.
View this table:
[in a new window]
Table 1. Amino Acid Sequences of RB, Bl, and Ang II
Peptides1
Methods
Top
Abstract
Introduction
Methods
Results
Conclusions
References
Solutions
PSS contained the following (in millimolar concentrations): NaCl
116; KCl 5.4; NaHCO3 19;
NaH2PO4 1.1;
CaCl2 2.5; MgSO4 1.2; EDTA
0.01; and glucose 11. PSS was aerated with 95%
O2/5% CO2 to maintain pH
7.4 and warmed to 37°C before addition to tissue baths. Isosmolar
high K+-PSS was made by reducing the NaCl
concentration to 46 mmol/L and increasing KCl to 75.4 mmol/L.
Ang II and Ang II antipeptides (Bl human configuration) were obtained
from BaChem Bioscience. Ang II antipeptide (RB6 configuration) and
variants (RB3, RB4, RB5, RB7) were manufactured at the Michigan State
University Peptide Synthesis Facility (Department of Biochemistry).
Additional variants lacking the glycine residue (see sequences in Table 1
) were synthesized by Daniel Holsworth at Houghten Pharmaceuticals,
Inc (San Diego, Calif). Rabbit anti-Ang II antiserum was obtained from
Peninsula Laboratories. All pharmaceuticals were refrigerated until
serially diluted in PSS on the day of the experiment.
All rabbits used were kept in university-approved facilities
before experimental use. All proper procedures were followed. Adult New
Zealand White rabbits of either sex were relaxed with 55 mg/kg
ketamine administered intramuscularly. After 15 minutes, the
rabbits were anesthetized with 50 mg/kg Nembutal administered
intraperitoneally. When the rabbits were
unresponsive to toe pinch, the abdomen was opened and the abdominal
aorta exposed. The aorta was teased from the vena cava and clamped at
both the rostral and caudal ends. The aorta was removed using surgical
scissors and placed in 4°C PSS. The aortic clamps were removed to
induce death.
Results
Top
Abstract
Introduction
Methods
Results
Conclusions
References
Fig 1 shows the dose-response curves
for control contractions of Ang II using individual 4-minute
contractions or cumulative contractions without relaxation between the
different doses. In these experiments, only at 1 nmol/L did the
individual contraction show a significantly higher relative force than
the cumulative force. In either case, 10 nmol/L Ang II produced a
maximum Ang II contraction. For 29 rings, 10 nmol/L Ang II
produced 0.85±0.06 g/mg force. Ang II produced 0.62±0.03 (n=8) of the
K+ contractions. This force is 
50% of the
maximum force that vascular smooth muscle can generate using
hypercalcemic K+ or histamine
solutions.36
View larger version (14K):
[in a new window]
Figure 1. Cumulative ( 
) and individual (
)
dose-response curves for Ang II (AII)–stimulated contractions of
rabbit aortic rings. The force symbols are mean±SE (n=4) and are
relative to the highest force for each method. *Significantly different
measurement (P<.02) between the two methods. shows the relative force of Ang II
cumulative dose-response curves in control solutions or in the presence
of 10 µmol/L Bl or 10 µmol/L RB6 peptides. As expected,
the Bl peptide caused a rightward shift of the dose-response curve,
with the relative force at 3, 10, and 30 nmol/L Ang II being
significantly lower in the presence of Bl peptide. Unexpectedly, Ang II
contractions in the presence of RB6 peptide were shifted to the left,
with significantly higher relative force generated at 1 and 3 nmol/L
Ang II. At these concentrations, it can make this tissue more sensitive
to Ang II.
View larger version (16K):
[in a new window]
Figure 2. Cumulative dose-response curves of rabbit aortic
rings to Ang II (AII) alone ( ) or in the presence of either 10
µmol/L RB6 peptide () or 10 µmol/L Bl peptide (
). The
force symbols are mean±SE (n=4) and are relative to the highest force
generated by each condition. *Significantly different measurement
(P<.02) from the control (C) at a given Ang II
concentration. shows the same data as Fig 2, but
here the force is measured relative to the tissue weight. In the case
of the RB6 peptide–generated leftward shift of the Ang II curve, this
presentation demonstrated that the increased relative
contractile strength seen in Fig 2 is not due to a reduction in
the maximum force generated by Ang II in the presence of RB6
peptide. The tissue weight–normalized force at 1 and 3 nmol/L Ang II
is higher in the presence of 10 µmol/L RB6 peptide. The Bl
peptide still inhibits the normalized force at 3, 10, and 30 nmol/L Ang
II.
View larger version (16K):
[in a new window]
Figure 3. Cumulative dose-response curves of rabbit aortic
rings to Ang II (AII) alone ( ) or in the presence of either 10
µmol/L RB6 peptide () or 10 µmol/L Bl peptide (). The
force symbols are mean±SE (n=4) and are normalized to the weight of
the tissue (grams of force per milligram of tissue). *Significantly
different measurement (P<.02) from the control (C) at a
given Ang II concentration., different
concentrations of RB6 peptide were added to 1 nmol/L contractions of
Ang II. There was a significant increase in Ang II force at every RB6
peptide concentration from 0.1 µmol/L to 10 µmol/L.
Additions of RB6 peptide alone over this range did not produce any
contractions at any concentration. The reversal of the curve at 1
µmol/L RB6 peptide indicates two competing processes, one augmenting
Ang II contractions competing with another that inhibits its
contraction.
View larger version (13K):
[in a new window]
Figure 4. RB6 peptide–induced variations in individual
contractile force of rabbit aortic rings stimulated by 1 nmol/L Ang II
(AII). The force symbols are mean±SE (n=6) and are relative to force
produced by 1 nmol/L Ang II. *Significantly different measurement
(P<.02) from the 1 nmol/L Ang II–generated
force. shows the inhibition of individual
10 nmol/L Ang II contractions by increasing concentrations of Bl
peptide. For each concentration, the peptide was added to the solution
before it was prewarmed before addition to the tissue bath.
Half-maximal inhibition occurs at approximately 2 µmol/L Bl
peptide. Addition of Bl peptide alone from 0.1 µmol/L to 10
µmol/L in half-log increments did not produce any measurable force at
any concentration.
View larger version (12K):
[in a new window]
Figure 5. Inhibition of 10 nmol/L Ang II (AII) contractions
by Bl peptide. The force symbols are mean±SE and are relative to the
force generated by 10 nmol/L Ang II alone. All are n=4 except for
-6.57 and -5.5.8 . Addition of 100 µmol/L RB6
peptide to 10 nmol/L Ang II solutions produced 0.49±0.05 (n=4) of the
Ang II-only force. No variant of RB6 produced a similar reduction in
the force generated by Ang II contractions (Table 2). Because of the uncertainty of the
mechanism(s) of the antipeptide inhibition, Ang II contractions were
made in the presence of Ang II antiserum, Ang II antipeptide, or both.
Using the Fig 1 individual dose-response curve to approximate the
decrease in Ang II using antiserum and/or RB6 peptide in individual
contractions, the combination of antiserum and RB6 reduces the Ang II
force by the expected amount if both antiserum and RB6 bind directly to
Ang II. The variation from the predicted force is 0.16 log units or a
factor of 1.45. The combination force is significantly less than with
the RB6 alone. The antiserum+RB6 contractions were compared with Ang
II-alone contractions, which followed. Potential tissue damage by RB6
was measured by comparing K+ contractions at the
beginning and end of the experiment. The postcontrol terminal
K+ contractions after 10 µmol/L RB6 were
1.03±0.04 (n=4) of the initial K+ contractions.
Terminal K+ contractions after 30 µmol/L
RB6 were 1.01±0.04 (n=4) of the initial K+
contractions. Neither pair was significantly different. The
augmentation of Ang II contractions by RB6 occurred at 10 µmol/L
RB6 and less (Fig 5), making damage to the tissue by RB6 unlikely.
View larger version (28K):
[in a new window]
Figure 6. Inhibition of 10 nmol/L Ang II (AII) contractions
of rabbit aortic rings by 0.07 mg/mL Ang II antiserum (AS) alone or in
combination with either 100 µmol/L RB6 peptide or 3
µmol/L Bl peptide. The measurements are mean±SE (n=4) and are
relative to 10 nmol/L Ang II contractions on the same rings.
*Significantly different force (P<.02) from the
RB6-alone inhibition. Antiserum+Bl is not different from Bl alone.
These results are striking. Ang II antiserum antagonizes Ang II
activity by direct binding to it. Blalock has asserted that Bl peptide
also antagonizes Ang II activity by binding to it. The effect of Ang II
antiserum plus Bl peptide on Ang II should therefore be additive. It is
not. The observed result is 0.59 log units less than is predicted from
the individual effects of the Ang II antiserum and Bl peptide. We
conclude that the Ang II antiserum must recognize Bl peptide as an
antigen, interfering with its activity as well as the activity of Ang
II. Bl peptide must therefore be an Ang II homologue
antagonist rather than an Ang II antipeptide. RB6, however,
behaves like an antipeptide. The effects of RB6 plus Ang II antiserum
on Ang II activity are additive and differ from the value predicted
from their independent activities by only 0.16 log units. Thus RB6 is
not recognized significantly by Ang II antiserum as an antigen and is
not behaving as an Ang II homologue antagonist.
View this table:
[in a new window]
Table 2. Relative Force of RB Peptide–Ang II Contractions
Versus Ang II Contractions ). The combined Ang
II inhibition of antiserum and Bl not only does not produce the
expected further decrease in Ang II force, but an Ang II force that is
not significantly different than the Bl peptide alone, and the mean is
higher. The predicted mean change is in the wrong direction and varies
by 0.59 log units or a factor of 38.9. This result is not
consistent with Bl peptide as well as antiserum binding Ang II,
but it is consistent with both Ang II and Bl binding directly
to antiserum. were all
inactive when tested at concentrations 10 000 times higher than the
Ang II available to the muscle tissue, as were a number of these
sequences lacking the glycine residue in the sequence (data not shown).
While it is possible that one of these peptides was tested just at the
cusp between its antagonistic and augmenting activities, it
is extremely unlikely that this is the case for all of the variant
peptides; even if this were the case, the antagonist
activity of such a peptide would be in the millimolar range and thus of
no significance. These data are consistent with RB6 activity
being highly sequence specific.
Conclusions
Top
Abstract
Introduction
Methods
Results
Conclusions
References
The physiological results of these experiments
indicate that Blalock peptide and some other antisense peptides of Ang
II have no clinical usefulness in inhibiting Ang II contractions. The
concentrations required are too high. On the other hand, an unexpected
nanomolar augmentation effect of both investigative and clinical
potential was discovered for RB6 peptide. ), whereas Bl peptide at the
same concentrations decreased the force of such contractions. RB6
peptide, in other words, has an augmenting effect on Ang II at low
concentrations and an antagonistic effect at higher
concentrations. This is, as far as we are aware, the first report of a
compound that has dual augmentation/antagonist effects on
smooth muscle and the first demonstration of a tissue activation effect
of an antisense peptide. We note, however, that a similar force
increase in smooth muscle contraction, known as the slow pressor
effect, occurs naturally as a result of chronic low-dose Ang II
exposure.37 38 39 The mechanism by which this slow
pressor effect is induced is unknown. RB6 peptide may provide clues to
this mechanism and a novel and much faster way to explore the
phenomenon. . Several possibilities exist to
explain the augmentation results. RB6 alone did not induce
contractions. This makes it unlikely that the increase in force was due
to either the opening of calcium channels or the release of
intracellular calcium stores by RB6. A direct effect on the
complex smooth muscle contractile mechanism, either the latch
mechanism40 or a thin filament
process,41 has not been demonstrated for any
hydrophilic agent, and it is unlikely that RB6 has such an effect. It
is unlikely that RB6 causes upregulation of receptor affinity or number
because the effect is transient on a time scale of a few minutes.
Therefore, RB6 most likely has its augmentation effect by facilitating
the effectiveness of Ang II activation, resulting in an increase in
intracellular calcium and thus increased force. Among the possibilities
that further research must distinguish are (1) RB6 peptide may bind to
Ang II, placing it in a preferred conformation for binding to its
receptor; (2) RB6 peptide may work allosterically at the receptor
itself to facilitate Ang II activity; (3) RB6 peptide may prevent
cellular sequestration or degradation of the Ang II receptor; and (4)
RB6 peptide may poorly activate a synergistic receptor system
in smooth muscle (a possibility made particularly interesting due to
the unexplained nature of the slow pressor effect). In each of these
cases, it is assumed that as the concentration of RB6 peptide
increases, its antagonistic activity to Ang II overwhelms
that of its contractile augmentation activity. The direct effect of
losartan31 32 33 on the Ang II receptor
indicates the receptor's sensitivity to functional conformational
change. The augmentation of the response by such a mechanism
(possibility 2 above) increases the scope of the potential manipulation
of this receptor. RB6 peptide may therefore open up new drug
development opportunities. and 2). RB7, which is the inverse of the RB6
sequence, was previously shown to have some anti-Ang II activity in
physicochemical tests and an Ang II–receptor
assay13 but had no measurable activity on smooth
muscle. Adding amino acids to the N and C termini of RB6 destroyed its
activity (RB5) and had no effect on the lack of activity displayed by
RB7 (RB3 and RB4). We conclude that both sequence and end effects are
critical components of the activity we are reporting here. We also note
that sequences lacking the glycine residue in the sequence also
destroyed peptide activity (data not shown). Thus, not only is the
activity displayed by RB6 unique, it is also extremely sensitive to any
alterations in the peptide. ). Such low sensitivity in vascular smooth
muscle contractions obviates its usefulness. Also, in contrast to the 1
order of magnitude shift produced by Bl peptide (Fig 1),
losartan shifts the Ang II dose-response curve of the rabbit
aorta by 2.5 orders of magnitude.31 32 ). The degree of
muscle contraction shows that, if anything, more Ang II was
functionally available. This result is only possible if the Ang II
antiserum bound up a significant proportion of Bl peptide, proving that
Bl peptide and Ang II are antigenically similar. In light of the fact
that Ang II antiserum can block Bl peptide activity, that Bl peptide is
sequentially very similar to Ang II, and that DeGasparo et
al29 have found that antibodies against Bl
peptide do not bind to the Ang II receptor, it is likely that antibody
to Bl peptide binds directly to Ang II because of molecular mimicry
between Ang II and Bl peptide. 5x10-6 mol/L (Reference 1313 ) is within the
right range. We calculate 80x10-6 mol/L for
the inhibition constant from the muscle contraction data. The
augmentation effect will alter the apparent inhibition constant
measurement because a higher concentration will be needed to overcome
the augmentation effect. Adding Ang II antiserum to RB6 peptide in the
presence of Ang II resulted in an additive decrease in Ang II activity,
as would be expected if each acted directly on Ang II without
interaction between the antiserum and RB6 peptide (Fig 6). Thus, our
results are consistent with physicochemical data indicating
that RB6 peptide is a true Ang II complement or antipeptide, not a
receptor antagonist,12 13 but not at
a physiologically useful concentration.
Selected Abbreviations and Acronyms
Ang II
=
angiotensin II
Bl
=
Blalock design
PSS
=
physiological salt solution
RB
=
Root-Bernstein design
Acknowledgments
This research was supported by US Public Health Service grant
42268 to Dr Dillon.
Footnotes
Reprint requests to Patrick F. Dillon, Department of Physiology, 108 Giltner Hall, Michigan State University, East Lansing, MI 44824.
References
Top
Abstract
Introduction
Methods
Results
Conclusions
References
1.
Phillips MI. Antisense inhibition and
adeno-associated viral vector delivery for reducing hypertension.
Hypertension. 1997;29:177–187.
This article has been cited by other articles:
 |
|
 |
|
  P. F. Dillon, R. S. Root-Bernstein, and C. M. Lieder Ascorbate enhancement of H1 histamine receptor sensitivity coincides with ascorbate oxidation inhibition by histamine receptors Am J Physiol Cell Physiol, November 1, 2006; 291(5): C977 - C984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. F. Dillon, R. S. Root-Bernstein, and C. M. Lieder Antioxidant-independent ascorbate enhancement of catecholamine-induced contractions of vascular smooth muscle Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2353 - H2360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Gomez, J. Miranda-Rios, E. Rudino-Pinera, D. I. Oltean, S. S. Gill, A. Bravo, and M. Soberon Hydropathic Complementarity Determines Interaction of Epitope 869HITDTNNK876 in Manduca sexta Bt-R1 Receptor with Loop 2 of Domain II of Bacillus thuringiensis Cry1A Toxins J. Biol. Chem., August 9, 2002; 277(33): 30137 - 30143. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||