Effects of Tonin, an Angiotensin II-Forming Enzyme, on
Vascular Smooth Muscle in the Normal Rabbit
R A U L GARCIA, M.D.,
ERNESTO L. SCHIFFRIN, M.D.,
AND JACQUES GENEST,
GAETAN THIBAULT, P H . D . ,
M.D.
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SUMMARY Tonin is an enzyme of the serine protease family present in different rat tissues which releases
angiotensin II (AH) directly from angiotensinogen and the tetradecapeptide renin substrate and from angiotensin I (AI). Tonin potentiates the effect of norepinephrine (NE) in the rat mesenteric artery preparation and
in the aortic strips from normal and hypertensire rats. In rabbit aortic and mesenteric artery strips tonin potentiates the effect of NE, almost doubling its response. A similar effect was observed on the KG and All-induced
contraction. This tonin-induced potentiation is reversible and long-lasting, persisting for 1 to 2 hours after being added into the tissue bath. In 75% of the vascular strips assayed, tonin elicited a contraction with a short
latency period and with a maximum tension ranging from a few milligrams to over 1 g. To clarify the
mechanisms of tonin effect on vascular smooth muscle, a variety of agents have been used. Neither indomethacin, saralasin, nor a- or /S-adrenergic blockers changed the direct contraction or the potentiation induced
to NE. Db-cAMP and ttaeophylllne blocked the potentiation to the response to NE. A C»l+-free medium,
La'*, and verapamil produced a 75% inhibition of the direct tonin-induced contraction. Papaverine,
isoproterenol, and theophylline relaxed the same contraction. Enzymatic inactivation of tonin blocked completely the direct contraction but not the potentiation to NE.
These experiments suggest that the vasoactive effect of tonin may be mediated by the release of intracellular-bound calcium, an effect dependent on a proteolytic effect of tonin, and by increasing the cellular
permeability to calcium, which is not of a proteolytic effect. It is suggested that tonin remains attached to the
vascular strips by mechanisms as yet not clarified. (Hypertension 3 (supp I): 1-101—1-106, 1981)
KEY WORDS • tonin • vascular smooth muscle • vasoconstriction mechanisms •
vascular relaxation • calcium antagonists • norepinephrine • arterial reactivity
T
ONIN is an enzyme of the serine proteases
family that is present in different rat tissues and releases angiotensin II (All) from
angiotensin I (AI) and directly from a natural substrate present in plasma, or from the synthetic tetradecapeptide renin substrate. 1 ' J It has been purified to
homogeneity3 and recently crystallized.4
It has been demonstrated6 that tonin infusion into
the rat mesenteric artery preparation potentiates the
vasoconstrictor response to norepinephrine (NE). As
this effect was similar to that produced by All and
was not abolished by saralasin, the possibility that All
was generated at a site inaccessible to the antagonist
was suggested. However, a direct vasoactive effect of
tonin itself could not be ruled out.
We have previously presented evidence that tonin
may be involved in the pathogenesis of one-kidney one
clip hypertension in the rat" and rabbit.7 More
recently," we have shown that tonin also potentiates
the contraction induced by NE in aortic strips from
normotensive and hypertensive rats. This effect was
more pronounced in the one-kidney one clip hypertensive animals and was not abolished by saralasin,
which is consistent with our previous results in the
mesenteric preparation. The mechanism by which
tonin potentiates the effect of NE in the vascular
smooth muscle is by no means clear. The fact that this
effect is not inhibited by saralasin casts some doubts
on the in situ generation of All by tonin, as previously
assumed. 8 The present experiments have been
designed to clarify this point.
From the Clinical Research Institute of Montreal, Montreal,
Quebec, Canada.
Supported by a group grant given by the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension of the Clinical Research Institute of Montreal.
Address for reprints: Raul Garcia, M.D., Clinical Research
Institute of Montreal, 110 Pine Avc West, Montreal, Quebec,
Canada, H2W 1R7.
Materials and Methods
Male New Zealand rabbits weighing 1.8 to 2.0 kg
were fed Purina rabbit chow and allowed free access to
tap water. The day of each experiment the animals
1-101
1-102
PATHOPHYSIOLOGY OF HYPERTENSION
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were anesthetized with sodium pentobarbital (Nembutal, 30 mg/kg, i.v.). The thoracic aorta and mesenteric artery were rapidly removed and helically cut as
described by Furchgott and Bhadrakom.' Each vascular strip of approximately 2 mm width and 20 to 25
mm length for the mesenteric artery, and 3 mm width
and 30 to 35 mm length for the aorta, was suspended
individually in a 20 ml tissue bath containing Krebs
solution at 37°C, which was continuously oxygenated
with a mixture of 95% O2 and 5% CO, (pH 7.4). The
strips were mounted between a fixed base and a forcedisplacement transducer (GrasS'FT-03C). The contractions were recorded on a Grass Model 5 or Model
7 polygraph.
A tension of 1000 to 1500 mg was applied to each
mesenteric strip and 2500 to 3000 mg to each aortic
strip, and the strips were allowed to equilibrate for 2 '
hours before the experimental procedures began. The
bathing fluid was removed and the strip tension adjusted to the starting value at 15-minute intervals.
The composition of the solution used in this study
was (mmole/liter): NaCl 119, KC1 4.7, KH,PO 4 1.8,
MgSO 4 - 7H 2 O 1.17, CaCl, • 6H,O 2.5, NaHCO,
25.0, and dextrose 5.5.
Fresh solutions of NE (L-norepinephrine bitartrate,
Sigma Chemicals) were prepared immediately before
use. Each arterial strip was exposed to NE, and a
dose-response curve was obtained. Once the contraction reached a stable plateau, the tissue was washed
with fresh Krebs until the tension returned to the baseline and a new dose was assayed. The interval between
doses was at least 15 minutes.
When a maximal response was attained, an ED M
value (concentration of the agonist required to
produce 50% of the maximal response) was estimated.
The contractile response to an ED M dose was studied
before and 5 minutes after the addition of tonin to the
bath to a final concentration of 1.7 X 10"7 mole/liter.
The tonin used was purified from rat submaxillary
glands.'
In some experiments, dose-response curves to All
(Hypertensin, Ciba) and potassium chloride were obtained, and the effect of tonin on the response to an
ED M dose of the agonist studied.
To investigate the mechanisms of the tonin effects
on vascular smooth muscle, the following pharmacological agents were used: indomethacin
(1.4 X 10"' to 1.4 X 10"4 mole/liter, Sigma Chemicals); phenoxyenzamine (10~5 mole/liter, Smith, Kline
and French, I.A.C.); propranolol (10"* mole/ liter,
DL-propranolol hydrochloride, Sigma Chemicals);
isoproterenol (10° mole/liter, DL-isoproterenol
hydrochloride, Sigma Chemicals); theophylline (10~J
mole/liter, Sigma Chemicals); dibutyryl cyclic AMP
(Db-cAMP) (10-3 mole/liter, Sigma Chemicals);
papaverine (10~4 mole/liter, papaverine hydrochloride, Sigma Chemicals); Sar1, Ala'-angiotensin II
(10~* to 1.5 X 10 5 mole/liter, Peninsula Laboratories,
Inc.); verapamil (10* mole/liter, isoptin hydrochloride, Knoll AG-Ludwigshafen); and lanthanum
chloride (2 X 10"4 to 4 X 10"s mole/liter, Sigma
Chemicals).
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MAY-JUNE,
1981
Enzymatically inactive tonin was prepared by incubating 1 mg of tonin with 10 s M diisopropyl fluorophosphate (DFP) for 24 hours at room temperature
and 48 hours at 6°C. The DFP was removed after incubation by dialyzing the mixture for 24 hours at 6°C.
An enzymatic inhibition of 95% was attained, as
measured by His-Leu released from AI and a fluorometric method.2 Statistical analysis was performed by
the Student's paired t test.
Results
As seen in figure 1 and table 1, tonin potentiates the
response to NE in the aorta and mesenteric artery
strips. A similar effect was observed on the potassium
chloride- and All-induced contractions, increasing the
response from 692 ± 74 to 1193 ± 108 mg (n = 6,
p < 0.001) in the former and from 521 ± 61 to
851 ± 188 mg (n = 6, p < 0.001) in the latter. This
tonin-induced potentiation, though reversible, is longlasting, even after repeated washings of the strips,
returning gradually to basal levels in 1 to 2 hours after
being added into the bath.
In contrast to what has previously been described in
the rat, 8 in 75% of the rabbit aortic and mesenteric
strips tonin elicited by itself a contraction (fig. 2),
Im/n
NE
Imin
f
TONIN
NE
FIGURE 1. Effect of tonin on the response to NE of the
rabbit aortic (upper panel) and mesenteric (lower panel)
strips.
TABLE 1. Potentiation by Tonin of the Contractile Response to an EDfio Dose of Norepinephrine by the Rabbit
Aorta and Mesenteric Artery Strips
Contraction Contraction
force
force
before
after
tonin
tonin
Increase
Strip
n
(mg)
(mg)
(%)
712 ±96*
Aorta
19
386 ± 5 3
191 ± 7
Mesenteric
8
197 ± 2 2
409 ±43*
216 ± 18
artery
*p < 0.001. Mean ± SEM.
TONIN AND VASCULAR SMOOTH MUSCLE/Garcia et al.
Imin
FIGURE 2. Direct contraction induced by tonin in two rabbit aortic strips.
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which appears immediately, with a slow phasic component and a maximum contraction ranging from a
few milligrams to over 1 g, which returns to the
baseline 15 to 30 minutes after washing the strips. No
correlation was found between the contraction
produced by tonin and the potentiation induced to
various agents, as potentiation was present even in the
absence of a tonin-induced contraction.
Of those pharmacological agents that ave been
used, neither indomethacin, saralasin, nor a- or /3adrenergic blockers have changed the direct vasoconstriction or the potentiation induced by tonin.
The Db-cAMP at concentrations that do not interfere with NE prevents the potentiation induced by
tonin to NE, reducing the response from 180% ± 6%
in its absence to 96% ± 9% in its presence (n = 6,
p < 0.01) (fig. 3). A late potentiation appears once the
Db-cAMP has been washed away, without adding
tonin for a second time. This late potentiation is not
seen if tonin was not added previously and is reversed
by a new addition of Db-cAMP. Similar results were
found with theophylline (not shown).
Isoproterenol, a /3-adrenergic agonist, papaverine,
and theophylline relax the tonin-induced contraction
(figs. 4 and 5). In contrast, dopamine, in a phenoxybenzamine-treated strip, does not produce any change.
The effect of tonin is partially inhibited in a Ca I + free solution (fig. 5). In the upper two channels, it can
be seen that this inhibition is reversed when the strips
are washed with a normal Ca 1+ -Krebs. In the lower
two channels, it is shown that La a+ blocks this reversion, which appears once La3+ has been washed away.
This enhanced contraction is not apparent in control
Imin
NE
NE
•i
Db-cAMP TONIN i
- *•• •
FIGURE 3.
-_±L
Db-cAMP
WASH-OUT / \
A
|
«
Effect of Db-cAMP on tonin-induced potentiation of NE in the rabbit aortic strips.
Imin
t
DOPAMINE (10 - * M )
PAPAVERINE(IO"*M;
tmin
4
TOWN
I
1-103
ISOPftO-'M)
i
3FIGURE 4. Effect of dopamine, papaverine, and isoproterenol on the tonin-induced contraction
in the rabbit aortic strips.
PATHOPHYSIOLOGY OF HYPERTENSION
1-104
I mm
2
Co *FREE
-KRESS
2
3
'Co *
-KRESS
THEOPHY.(K)- M)
SUPP
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HYPERTENSION, VOL
3, No 3, MAY-JUNE, 1981
The DFP treatment abolishes completely the contractile effect of tonin on the aortic strips; however,
the potentiation induced to NE was only reduced to
74% ± 6% of the maximum effect (fig. 8).
Discussion
Ca2>FREE
-KRESS
'Co2*
-KRESS
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FIGURE 5. Tracings obtained from simultaneous experiments on rabbit aortic strips before and after the addition ofCat+ to the tissue bath in the absence and presence of
La**. Upper two channels, show the effect of theophylline.
strips in which no tonin has been added. These results
are quantified in figure 6. Verapamil, another calcium
antagonist, partially inhibits the tonin-induced contraction. This inhibition is similar to that produced by
La 3+ and a Ca 2+ -free medium (fig. 6).
The effect of different La 3+ concentrations on tonininduced potentiation of the response of the aortic strip
to N E is shown in figure 7 (three upper channels). If
they are compared with the lower channel in which
no La 3+ has been added, an inhibition is observed
that is more pronounced in the phasic phase of the
contraction.
600
Co2' FREE-KBES5
Lo 3 *
VERAMMB.
9.500
E
MEAN!SCM
*p<aooi
* * p<aoi
300
50
200
s wo
(n=1IJ
(n=8)
(n = 6)
6. Effect ofCa*+-free Krebs solution and calcium
antagonists on the tonin-induced contraction in the rabbit
aortic strip. Left panel: Reduction of the contraction in absolute values. Right panel: The percent of the maximum
contraction in the same group.
FIGURE
As tonin, an angiotensin II-producing enzyme,
potentiates the effect of NE in the rat mesenteric
artery preparation and this effect was not blocked by
saralasin, the possibility of All formation at a site inaccessible to the antagonist was suggested.4 Later, a
similar effect was found in rat aortic strips" which was
also not abolished by saralasin.
In the present study on rabbit vascular strips, we
have demonstrated that tonin not only potentiates the
effect of NE-, KC1-, and All-induced contractions,
but elicits by itself a contraction that was not present
in our previous experiments in the rat. In a Ca2+-free
medium, this tonin-induced contraction is reduced to
27% of the maximum contraction, an inhibition that
was reversed after the preparation was washed with a
normal Krebs solution. As this effect is not seen if
tonin has not been added into the bath, it suggests that
tonin increases the permeability of the cell membrane,
allowing Ca ! + to enter the muscular cell from the extracellular space and triggering the contraction.
In smooth muscle, La 3+ is considered to compete
with Ca 2+ for extracellular negative sites* and for ion
channels, thus interfering with the influx of calcium
ions through drug-operated ion channels.11
The addition of La 3+ to the tissue bath with a normal Ca 2+ concentration also partially blocks the contraction produced by tonin (fig. 6), further suggesting
that this contraction could at least be partially induced
by an influx of Ca a+ . Furthermore, when calcium is
restored to a Ca2+-free solution (fig. 5), La 3+ abolishes
the restorative effect of Ca a+ on tonin contraction.
Verapamil, another calcium antagonist,12 also
blocked the tonin-induced contraction to the same extent as Ca !+ -free medium or La s+ (fig. 6).
Db-cAMP, which probably lowers cytoplasmatic
Ca I+ by increasing its sequestration by the sarcoplasmic reticulum, 13 blocked the potentiation
produced by tonin of the NE-induced contraction, an
effect that is reversed once the Db-cAMP is washed
from the preparation. La"+ also blocked this potentiation of NE (fig. 7), suggesting that this effect of tonin
is also calcium-mediated. Probably the same
mechanism could be involved in the potentiation of the
KG- and All-induced contractions. The same results
as with Db-cAMP were found with theophylline, a
well-known phosphodiesterase inhibitor.14
The fact that tonin is still capable of producing a
contraction in a Ca 1+ free-medium or in the presence
of calcium antagonists strongly suggests that the increased permeability of the cell membrane to Ca I + is
not the only mechanism involved.
The finding that propranolol or phenoxybenzamine
has no effect on the tonin-induced contraction suggests
that neither a- nor j8-adrenergic receptors are im-
TONIN AND VASCULAR SMOOTH MUSCLE/Garcia et al.
1-105
Imin
LA 3 *(2»IO"' 1 M)
NE
TONIN
I
LA 3 *(2»IO" 3 M)
TONIN
1
LA 3 *(4»l0' 3 )
TONIN
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TONIN
t
FIGURE 7. Tracings obtained from simultaneous experiments on rabbit aortic strips. The effects of La'+
on the potentiation induced by tonin in the response to NE is shown.
plicated. Prostaglandin release has been involved in
the action of catecholamines15 and A l l " on smooth
muscle. However, since indomethacin has no effect on
direct contractions from tonin or on tonin potentiation of the contraction produced by NE, this
possibility seems unlikely. Another explanation
previously suggested that the All formation in situ
can be fairly ruled out, at least from our present
results in rabbit smooth muscle, since: 1) the effect of
tonin is not inhibited by saralasin; and 2) in the experiments in which DFP-treated tonin has been used,
DIRECT
VASOC0NSTRICTI0N
POTENTIATION TO THE
NE-INDUCED CONTRACTION
%IOO
PC0.05
O
UJ
T
t
UJ
S 50
<
Z
UJ
o
TONIN TONINOFP
TONIN TONINDFP
FIGURE 8. Effect of enzymatically inactive tonin on the
rabbit aortic strip expressed as percent of the maximum
effect produced by active tonin. Left panel: Effect of DFP
treatment on the direct effect contraction induced by tonin.
Right panel: Effect of DFP treatment on the potentiation by
tonin of the contraction produced by NE.
the potentiation of NE is only slightly depressed even
when tonin is 95% enzymatically inactive.
Contractions induced in smooth muscle by substances such as epinephrine are distinctly biphasic.17
The phasic or fast component utilizes a bound source
of intracellular calcium, and the tonic or slow component is more dependent on extracellular calcium."
Since the contraction produced by tonin is only partially inhibited by a Ca2+-free medium or by calcium
antagonists, it seems logical to conclude that both the
tonin-induced potentiation of NE and its direct effect
may be mediated by two mechanisms: 1) the dislodging of internally bound calcium, which is not dependent on external calcium concentration; and 2) an increase in the permeability of the cell membrane, which
allows calcium to enter the cell from the extracellular
space. The first of these mechanisms is completely
blocked by the treatment of tonin with DFP, suggesting a proteolytic effect on the cellular membrane,
which is reversible, however. The second mechanism
does not seem to be of a proteolytic nature, as it is
only slightly inhibited by DFP treatment (fig. 8).
Relaxation of tonin-induced contraction by /Sadrenergic stimulation with isoproterenol and by
theophylline could be the result of a decrease of intracellular calcium concentration has been demonstrated by Mueller and Van Breeman. 1 '
The finding that the described tonin effects on
vascular smooth muscle are persistent after repeatedly
washing the strips, or after the removal of a variety of
inhibitors, suggests that tonin may remain attached to
the vascular tissue in a still not precise manner.
These results may partially explain the mechanisms
by which tonin could be involved in the pathogenesis
of experimental hypertension, as suggested.*"*
1-106
PATHOPHYSIOLOGY OF HYPERTENSION
Acknowledgments
The authors thank Louise Theroux and Suzanne Diebold for
technical assistance and Vivianne Jodoin for secretarial help.
References
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17. Healy JK, Bohr DF: Angiotensin II responses of rabbit aortic
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rabbit.
R Garcia, E L Schiffrin, G Thibault and J Genest
Hypertension. 1981;3:I101
doi: 10.1161/01.HYP.3.3_Pt_2.I101
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