(Hypertension. 1999;34:525-530.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Alternative Angiotensin II Formation in Rat Arteries Occurs Only at Very High Concentrations of Angiotensin I
From the Third Department of Internal Medicine, Osaka Medical College, Takatsuki City, Osaka, Japan.
Correspondence to Hikaru Nishimura, MD, Third Department of Internal Medicine, Osaka Medical College, 2-7 Daigakumachi, Takatsuki City, Osaka 569-8686, Japan. E-mail in3023{at}poh.osaka-med.ac.jp
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Abstract |
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Abstract—Contrary to previous reports, recent enzymatic assays showed the predominance of chymase-like activity in rat arteries. We determined the existence and significance of such alternative pathways in rat carotid arteries by measuring contraction of arterial rings in organ baths and blood pressure in conscious rats. Hamster aorta served as a positive control for chymase. Temocapril (30 µmol/L) inhibited the contractions to angiotensin (Ang) I (10-9 to 10-5 mol/L) except at high concentrations of Ang I (>10-7 mol/L). Addition of chymostatin (100 µmol/L) to temocapril exerted a synergistic inhibitory effect. Hamster aorta gave similar results, except that temocapril was 30-fold less effective than in rat arteries. [Pro11,D-Ala12]Ang I (10-8 to 10-5 mol/L), a chymase-specific substrate, provoked similar responses in rat and hamster arteries; chymostatin, but not temocapril, attenuated the responses. CV 11974 (30 µmol/L), an Ang II type 1 receptor antagonist, abolished the responses to both peptides. In conscious rats, Ang I (0.03 to 30 µg/kg) and [Pro11,D-Ala12]Ang I (7 to 700 µg/kg) produced similar pressor responses. Not only CV 11974 (1 mg/kg) but also temocapril (2 mg/kg) abolished Ang I–induced responses in vivo. CV 11974, but not temocapril, inhibited responses to [Pro11,D-Ala12]Ang I. Our results showed the presence of the alternative pathway in rat arteries, but it did not play a major role. Arteries with the opposing characteristics of chymase responded equally to [Pro11,D-Ala12]Ang I. These findings suggest that biochemical and [Pro11,D-Ala12]Ang I–derived results may not reflect the functional significance of chymase.
Key Words: angiotensin-converting enzyme • chymase • aorta • carotid arteries • hamster
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Introduction |
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Species differences exist in local angiotensin (Ang) II–forming pathways in the cardiovascular system mainly due to different characteristics of chymase.1 2 3 Human and hamster chymases effectively cleave Ang I to form Ang II,4 5 whereas rat chymase is Ang II–degrading.6 It is therefore generally believed that rat arteries do not have any alternative pathway other than an angiotensin-converting enzyme (ACE)-mediated pathway.3 This notion is supported by many reports indicating the equal effectiveness of ACE inhibitors and Ang II subtype 1 (AT1) receptor antagonists in rat models of cardiovascular disease. These inhibitors similarly lowered blood pressure and prevented cardiovascular remodeling in spontaneously hypertensive rats7 and TGR(mREN2)27 transgenic rats.8
Recently, however, some investigators reported the presence of chymase-like activity in the homogenates of rat heart and aorta.1 2 In particular, Akasu et al2 even showed that chymase-like enzyme, rather than ACE, is the major Ang II–forming enzyme in the rat cardiovascular system in vitro. Despite such elegant biochemical observations, however, there is little physiological evidence supporting the notion that chymase-like enzyme plays such a significant role in rat vasculature. The unavailability of a chymase-specific inhibitor has hampered the clarification of pathophysiological roles of chymase. To circumvent this problem, a chymase-specific substrate, [Pro11,D-Ala12]Ang I, was developed.9 10 This synthetic peptide has been used to show chymase-dependent Ang II formation in various species such as humans,9 11 baboons,12 hamsters,13 marmosets,14 and dogs,15 but not in rats.
The aims of this study were to determine functionally (1) whether the alternative Ang II–forming pathway exists in rat arteries and (2) whether it plays as significant a role as suggested by biochemical data. We compared the contractile responses to Ang I and [Pro11,D-Ala12]Ang I with and without ACE inhibitors (captopril and temocapril), an AT1 receptor antagonist (CV 11974), or a serine protease inhibitor (chymostatin) in rat and hamster arteries. On the basis of the different characteristics of hamster and rat chymases,4 6 we used hamster aorta as a positive control for chymase-dependent Ang II formation.13 Our study demonstrates the presence of the alternative pathway in rat arteries. However, contrary to the biochemical observations, functionally the non-ACE pathway does not play a major role in rat vasculature either in vitro or in vivo.
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Methods |
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Animals
Male Syrian hamsters (n=14, 155±4 g) and Wistar-Kyoto/Izm rats (n=40, 397±5 g) were obtained from Japan SLC (Shizuoka, Japan) and the Disease Model Cooperative Research Association (Kyoto, Japan), respectively. They were kept in a quiet room at constant temperature (20°C to 22°C) and under a 12:12-hour light-dark cycle. The experiments were performed in accordance with the institutional guidelines of the Osaka Medical College for the use of experimental animals.
Contraction Studies in Rat and Hamster Arteries
Under chloral hydrate anesthesia (350 mg/kg IP),
animals were killed by exsanguination. The thoracic aorta of hamsters
and the carotid arteries of rats were rapidly removed and cut into
2-mm-long rings under a microscope. We did not use rat aorta because of
weak and variable responses and marked tachyphylaxis to Ang II,
which precluded a construction of cumulative concentration-response
curves. Instead, we compared rat carotid arteries with hamster aorta
because both are conductance vessels with a similar size and similar
dose-dependent responses to Ang II. Four rings with intact
endothelium were mounted simultaneously in
a Mulvany myograph (model 610 mol/L, JP Trading) filled with
physiological salt solution (5 mL) of the following
composition (mmol/L): NaCl 119.0, NaHCO3 25, KCl
4.7, CaCl2 · 2H2O
2.5, MgSO4 · 7H2O
1.2, glucose 5.5, and
KH2PO4 1.2. The solution in
the baths was constantly aerated with 95% O2 and
5% CO2 and kept at 37°C (pH 7.4). Contractile
force, measured with isometric transducers (DSC-6, JP Trading), was
stored and analyzed by a computerized system using MacLab
software (AD Instruments Ltd). After 45 minutes of equilibration with a
resting tension of 1.5 g for rat carotid arteries and 2 g for
hamster aorta, the rings were primed by exposure to 50 mmol/L KCl
(2 times) with intervening washings. Then the ring segments were
incubated with either vehicle, temocapril (30 µmol/L), captopril
(100 µmol/L), CV 11974 (30 µmol/L), or chymostatin
(100 µmol/L) for 30 minutes. After the tension was readjusted to
the respective resting values, we added Ang I
(10-9 to 10-5 mol/L) or
[Pro11,D-Ala12]Ang
I (10-8 to 10-5 mol/L) in
a cumulative fashion. The concentrations of these substrates were
selected to achieve similar contractile responses.13 14
Ring preparations that did not receive any antagonist(s)
but only vehicle set up in parallel served as controls. We added
100 µmol/L
NG-nitro-L-arginine
methyl ester (L-NAME) and 10 µmol/L indomethacin
in all baths to avoid a possible difference in basal release of nitric
oxide in the arteries and to exclude a possible effect of ACE
inhibitors on the kinin-prostaglandin
system.3 13 16
In a separate set of rings, we determined the time to the onset of response and the time to reach the peak of tension by incubating the rings with either Ang I (10-6 mol/L) or [Pro11,D-Ala12]Ang I (10-5 mol/L).
Blood Pressure Measurement in Conscious Rats
Under anesthesia with 4% chloral hydrate (350
mg/kg), catheters (PE 50) were inserted into the right carotid artery
and jugular vein of rats and were exteriorized in the interscapular
area. The catheters were filled with heparinized saline (10 IU/mL), and
the rats were allowed to recover overnight. The carotid catheter was
connected to a micromanometer (PC350, Millar
Instruments) to record blood pressure continuously with the
computerized system using MacLab software. After a 30-minute
stabilization period, Ang I (0.03 to 30 µg/kg) or
[Pro11,D-Ala12]Ang
I (7 to 700 µg/kg) was administered via the jugular catheter to
achieve comparable pressor responses.13 14 On a separate
day, after a 30-minute incubation with temocapril (2 mg/kg) or CV 11974
(1 mg/kg), the dose-dependent responses to the peptides were compared.
Blood pressure measurement was not done in hamsters because it has
already been reported with the same protocol.13
Pharmacological Agents
Chymostatin, phenylephrine, L-NAME,
indomethacin, and Ang I were purchased from Sigma
Chemical Co.
[Pro11,D-Ala12]Ang
I was synthesized at the Peptide Institute. CV 11974 was a gift of
Takeda Chemical Industries (Osaka, Japan). Temocapril and captopril
were supplied by Sankyo Pharmaceutical. Chymostatin was first dissolved
in dimethyl sulfoxide and then diluted in the solution in baths so that
the final dimethyl sulfoxide concentration was 0.1%. This
concentration of dimethyl sulfoxide was added to each bath to exclude
any effect on the contractile responses. All drugs were diluted in the
physiological salt solution and expressed as final
molar concentrations in the organ baths.
Statistics
The contractile responses of the isolated
arterial rings were expressed as percentage of contraction
induced by 10 µmol/L phenylephrine. The negative log
molar value (pD2) of the concentration at
50% of the maximum contraction (EDmax) was
calculated from the fits of each separate curve. Statistical evaluation
was done by Student's t test for paired and unpaired
observations. In the isolated vessel study, n denotes the number of
experiments, and in the experiments in vivo, n equals the number of
animals studied. Values are expressed as mean±SEM. A value of
P<0.05 was considered statistically significant.
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Responses to Ang I and [Pro11,D-Ala12]Ang I in the Carotid Arteries of Rats
Figure 1A shows dose-dependent contractile responses to Ang I in rat carotid arteries (n=16) in the presence and absence of various inhibitors. Temocapril completely suppressed the contractions except at very high concentrations of Ang I (>10-7 mol/L), shifting the pD2 from 7.34±0.11 to 6.12±0.08 (P<0.001, n=12). Ang I concentration needed for the onset of contraction was shifted from 10 nmol/L to 3 µmol/L in the presence of temocapril, indicating that a 300-fold higher dose of Ang I was necessary to yield the first response. In contrast, chymostatin had no effect on the Ang I–induced contraction (Figure 1A, n=7), indicating the predominance of the ACE pathway in rat arteries. However, when chymostatin was combined with temocapril, there was a synergistic inhibitory effect (pD2 6.12±0.10 to 5.86±0.09, P<0.01, n=11), suggesting the presence of a non-ACE pathway, but only at high Ang I concentrations. Whereas the EDmax to Ang I was not affected with temocapril or the combination of temocapril and chymostatin, CV 11974 completely abolished the responses (Figure 1A, n=8).
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Unexpectedly, [Pro11,D-Ala12]Ang I produced dose-dependent responses comparable to those found with Ang I in rat arteries (EDmax 64±8% versus 68±7% with Ang I, P=0.76, n=16, Figure 1B). In contrast to Ang I, temocapril did not affect the contractile responses elicited by [Pro11,D-Ala12]Ang I (Figure 1B, n=7); however, chymostatin shifted the dose-response curve to the right (pD2 6.25±0.10 to 5.89±0.09, P<0.01, n=10). These findings indicate that the responses to [Pro11,D-Ala12]Ang I were mediated by chymostatin-sensitive, non-ACE enzyme(s). Unlike Ang I, there was no synergistic inhibitory effect from combining chymostatin and temocapril (pD2 5.88±0.08 versus 5.89±0.09 with chymostatin alone, P=0.84, n=7), further confirming the absence of ACE involvement in the [Pro11,D-Ala12]Ang I–induced responses. CV 11974 completely inhibited contractile responses to the synthetic peptide (Figure 1B, n=8), implying that the responses to [Pro11,D-Ala12]Ang I were AT1 receptor–mediated.
Responses to Ang I and
[Pro11,D-Ala12]Ang I in the Aorta
of Hamsters
To compare the functional contribution of different
characteristics of chymases to vascular Ang II formation, the same
protocol was repeated in hamster aorta. Temocapril significantly
inhibited the contractions to Ang I, shifting pD2
from 7.63±0.12 to 6.38±0.08 (P<0.001) without affecting
the EDmax (Figure 2A, n=12). Compared with rat arteries,
however, temocapril was 30-fold less potent in hamster arteries,
because a 10-fold higher concentration of Ang I (from 30 to 300 nmol/L)
was needed to elicit the first response (Figure 2A), whereas a
300-fold higher Ang I dose was required in rat arteries (Figure 1A). Chymostatin had no effect on Ang I–induced contraction
(Figure 2A, n=7), suggesting that even in hamster arteries, the
alternative pathway is not a main pathway. The addition of chymostatin
to temocapril shifted the dose-response curve to the right
(pD2 6.38±0.10 to 5.92±0.09,
P<0.05, n=11). The synergistic inhibitory
effect was similar in hamster and rat arteries, requiring 3 times
higher doses of Ang I to elicit the first response in each vessel. CV
11974 significantly suppressed the responses to Ang I
(P<0.001, Figure 2A, n=8).
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[Pro11,D-Ala12]Ang I induced dose-dependent responses similar to those induced by Ang I (EDmax 77±10% versus 78±11% with Ang I, P=0.85, Figure 2B, n=16). In contrast to Ang I, the inhibitory effects of various inhibitors on the responses to [Pro11,D-Ala12]Ang I were similar in hamster and rat arteries. As shown in Figure 2B, chymostatin shifted pD2 from 6.58±0.09 to 6.04±0.08, P<0.05, without affecting the EDmax (n=11). Although temocapril did not affect the responses (n=7), CV 11974 inhibited them significantly (P<0.001, Figure 2B, n=8). As with rat arteries, the addition of temocapril to chymostatin exerted no synergistic effect (pD2 6.05±0.09 versus 6.04±0.08 with chymostatin alone, P=0.82, n=7). Compared with Ang I, [Pro11,D-Ala12]Ang I was 12-fold and 11-fold less potent in rat and hamster arteries, respectively, in terms of the pD2 values (rat versus hamster arteries, P=0.74); [Pro11,D-Ala12]Ang I produced EDmax similar to that of Ang I in each blood vessel. These findings indicate that the sensitivity and EDmax to [Pro11,D-Ala12]Ang I relative to those of Ang I were similar in rat and hamster vessels despite different characteristics of each chymase.
To rule out the possibility that the inhibitory effects on Ang I–induced contraction we obtained are specific for temocapril, we also evaluated the effect of captopril (100 µmol/L) in rat and hamster arteries (n=8 each). We found no significant differences in the inhibitory effects of these ACE inhibitors in our experimental settings (captopril data not shown).
Time to the Onset of Response and to Reach the Peak of
Tension
In another set of rat and hamster arteries (n=10 each), we
compared the time course of contractile responses to each peptide. Ang
I (10-6 mol/L) and
[Pro11,D-Ala12]Ang
I (10-5 mol/L) induced the same maximum
contractile responses in rats (66±4% versus 67±3%) and hamsters
(78±1% versus 79±1%). However, the onset of contractile response
was significantly delayed with
[Pro11,D-Ala12]Ang
I compared with Ang I in the vessels of rats (67±6 versus 39±4
seconds, P<0.01) and hamsters (57±5 versus 34±4 seconds,
P<0.01). The time from the onset to the peak response was
also longer with
[Pro11,D-Ala12]Ang
I than with Ang I (rats, 267±19 versus 216±13 seconds,
P<0.05; hamsters, 308±20 versus 255±9 seconds,
P<0.05).
Pressor Responses to Ang I and
[Pro11,D-Ala12]Ang I in
Conscious Rats
To determine the contribution of the alternative pathway observed
in the isolated rat arteries to the in vivo Ang II formation, we
compared pressor responses to both peptides and the
inhibitory effects of temocapril and CV 11974 in conscious
rats (n=28). Baseline mean arterial pressure was
100±4 mm Hg, and heart rate was 312±11 bpm. Neither temocapril
nor CV 11974 affected baseline blood pressure or heart rate (data not
shown). Intravenous injections of Ang I or
[Pro11,D-Ala12]Ang
I produced similar dose-dependent pressor responses (Figure 3A and 3B, n=14 each), confirming the
results obtained in in vitro experiments. In contrast to the findings
in the isolated arteries, however, temocapril completely suppressed the
Ang I–induced responses even at high concentrations of the peptide, as
did CV 11974 (Figure 3A, n=7 each), suggesting that the non-ACE
pathway found in vitro does not play a role in vivo. As found in the
isolated arteries, CV 11974, but not temocapril, abolished the
responses to
[Pro11,D-Ala12]Ang
I (Figure 3B, n=7 each), indicating that the pressor responses
to the synthetic peptide were mediated by enzyme(s) other than ACE.
Chymostatin could not be used in vivo because dimethyl sulfoxide, in
which chymostatin was dissolved, is toxic to animals.
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Discussion |
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Recent enzymatic data showed the predominance of non-ACE pathway in rat arteries,2 whereas previous functional data denied even the existence of such a pathway in rat vasculature.3 The present study clarified these discrepancies by addressing the questions of (1) whether the alternative pathway exists in rat arteries and (2) whether such a non-ACE pathway has physiological significance. Our study demonstrated the existence of an ACE-independent Ang II–forming pathway in rat carotid arteries, as evidenced by a greater inhibitory effect of CV 11974 and the combination of chymostatin and temocapril compared with temocapril alone on Ang I–induced contraction. The vasoconstrictive responses to [Pro11,D-Ala12]Ang I, which were not affected by temocapril, further confirmed the presence of the alternative pathway in rat arteries.
In contrast to the present results, Okunishi et al3 failed to show any additive inhibitory effect of combining captopril and chymostatin on responses to Ang I in rat arteries, thus denying the presence of the alternative pathway in rats. The discrepancy is probably because the concentration of Ang I used in their study was too low (10-7 mol/L). At this concentration, we also found that captopril or temocapril almost completely suppressed the Ang I–induced contraction in rat arteries. Consistent with our data, the synergistic inhibitory effect of captopril and chymostatin was also reported in the perfused rat hindlimb.17 Thus, the predominance of the ACE or non-ACE pathway appears to be highly dependent on the concentrations of Ang I used. Most investigators used only an extremely high concentration of Ang I (10-6 mol/L) to show the importance of chymase-dependent Ang II formation in human11 and dog14 arteries and human detrusor muscle.18 However, if one uses only such an unphysiologically high concentration of Ang I, even in rat arteries the alternative pathway predominates over the ACE pathway (Figure 1A).
The inhibitory effect of ACE inhibitors in our study was mainly due to the blockade of the renin-angiotensin system, because we used L-NAME and indomethacin to exclude a possible effect of ACE inhibitors on the kinin-prostaglandin system. In rat arteries, temocapril completely suppressed the responses to submaximal concentrations of Ang I, so that as much as a 300-fold higher dose of Ang I was necessary to yield a first contractile response to Ang I in the presence of temocapril. This is in contrast to human16 and hamster arteries, which required 3-fold and 10-fold higher Ang I concentrations, respectively, to elicit the first response. These findings indicate that in rat arteries, the ACE pathway plays a greater role than in human vessels. In further support of this view, chymostatin had no effect on Ang I–induced contraction in rat arteries, a finding in contrast to human arteries.3 11 16 Moreover, the non-ACE pathway in rat vessels was detected only when the concentrations of Ang I were extremely high. However, it is unlikely that the blood vessels are exposed to such high concentrations of Ang I in vivo, because plasma Ang I levels are relatively low.19 20 This can explain why there was no difference in the inhibitory effects of temocapril and CV 11974 on Ang I–induced pressor responses in our conscious rats. This interpretation is further supported by the equal effectiveness of ACE inhibitors and AT1 receptor antagonists in hypertensive rats.7 8 In TGR(mREN2)27 transgenic rats, a hypertension model with the increased tissue renin-angiotensin system, quinapril and losartan equally prevented cardiac and vascular hypertrophy.8 These findings suggest that even at the tissue level, the alternative pathway does not play a significant role in rats. However, we cannot exclude the presence of another substrate or endogenous inhibitor for rat chymase. Further studies are needed to clarify this issue.
The synthetic substrate for chymase, [Pro11,D-Ala12]Ang I, does not interact with Ang II receptors but is supposedly converted by chymase to Ang II, inducing AT1 receptor–mediated vasoconstriction.9 13 Despite opposing characteristics of their chymases (Ang II–forming versus degrading),4 6 rat and hamster arteries responded similarly to [Pro11,D-Ala12]Ang I. Moreover, the pressor responses to [Pro11,D-Ala12]Ang I in our conscious rats are also comparable to those reported in conscious hamsters,13 baboons,12 dogs,15 and marmosets,14 raising doubt that [Pro11,D-Ala12]Ang I is not chymase-specific. This concern is further strengthened by the delay in the onset and the time to peak response with [Pro11,D-Ala12]Ang I compared with Ang I in rat and hamster vessels. The delay is probably due to a slower conversion of [Pro11,D-Ala12]Ang I to Ang II by non-ACE enzyme(s). This finding may be related to differences in the distribution of ACE and chymase.21 22 This is unlikely, however, because the present method allowed equal accessibility of the substrates intraluminally and extraluminally. Because the conversion velocity of Ang I to Ang II is in this order: chymase>ACE>cathepsin G,23 the delay may be due to the contribution of enzyme(s) other than chymase, such as cathepsin G, to the conversion of [Pro11,D-Ala12]Ang I. In support of this speculation, the responses to [Pro11,D-Ala12]Ang I were attenuated by chymostatin, which inhibits not only chymase but also cathepsin G.5 Taken together, the results obtained with [Pro11,D-Ala12]Ang I may overestimate the physiological importance of chymase-dependent Ang II formation, as suggested by others.24 Although several investigators claimed the vasoconstrictor responses to [Pro11,D-Ala12]Ang I as being chymase-dependent,9 11 12 13 14 15 our results in rats suggest that responses to [Pro11,D-Ala12]Ang I simply mean the presence of the ACE-independent pathway. The functional significance of the chymase-dependent pathway should also be determined with several concentrations of Ang I. To elucidate this issue, however, one should develop a chymase-specific inhibitor.
There are discrepancies in biochemical and functional evidence regarding the predominance of ACE versus chymase in various tissues. Although biochemical data showed the predominance of chymase-like activity in dog heart,24 human detrusor muscle,18 and rat and hamster aorta,2 13 the functional results showed the predominance of ACE pathway in each tissue.13 18 24 Thus, the biochemical data may overestimate the chymase-like activity, as suggested by Balcells et al.1 Several reasons can be postulated to explain a possible overestimation. First, in the biochemical studies, extremely high concentrations of Ang I were used (in the range of 10-4 mol/L),1 2 13 18 which is not physiological.2 Second, chymase is normally stored in the secretory granules of mast cells and must be actively secreted into the interstitium to exert its action.22 However, in the process of tissue homogenization, large quantities of intracellular Ang II–forming enzymes, including chymase, would gain free access to a high concentration of Ang I. Other interesting biochemical and functional discrepancies are that (1) even though chymase-like activity predominated equally over ACE activity in the homogenates of human and rat arteries,2 the present and previous functional observations3 16 revealed a marked difference in the Ang II–forming pathways in rat and human vessels and (2) although biochemically, chymase-like enzyme-dependent Ang II formation was severalfold higher in hamster than in human arteries,2 functional studies by us and others13 16 gave results opposite to the biochemical data. Together, these findings suggest that the biochemical results do not apply to the in vivo system.25 We emphasize a need for a combined functional and biochemical approach.24 Recently, Hollenberg et al25 clearly showed the contribution of ACE-independent pathways in humans in vivo by using various classes of agents that block several enzymes in the renin-angiotensin system.
In summary, our in vitro study showed the presence of the alternative pathway mediated partly by chymostatin-sensitive enzyme(s) in rat arteries. However, contrary to the biochemical data, its functional contribution to Ang II formation in rat vasculature appears to be negligible, as substantiated by our in vivo results. Despite the different characteristics of their chymases, rat and hamster arteries responded similarly to [Pro11,D-Ala12]Ang I. These data suggest that caution should be exercised in extrapolating the results obtained from tissue homogenates and [Pro11,D-Ala12]Ang I to the in vivo importance of chymase.
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Acknowledgments |
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We are grateful for the excellent technical and secretarial assistance of Mariko Fujimura.
Received February 25, 1999; first decision March 24, 1999; accepted May 20, 1999.
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References |
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