From the Departments of Internal Medicine I (L.M. de L., A.M.B.B.,
M.A.D.H.S.) and Pharmacology (L.M. de L., A.H.J.D., P.R.S.), Erasmus
University Rotterdam, The Netherlands.
Correspondence to A.H. Jan Danser, PhD, Department of Pharmacology, Room EE 1418b, Erasmus University, Dr Molewaterplein 50, 3015 GE Rotterdam, Netherlands.
Local Ang II production in the heart is often invoked to
explain the results of heart failure trials evaluating the effects of
drugs that block angiotensin
formation.5 6 Knowledge of cardiac Ang I and II
production, however, is still fragmentary and little is known
about its regulation and physiological
significance.3
In a previous study we used a modified Langendorff rat heart
preparation to investigate the production of Ang I in cardiac
tissue.7 This isolated heart preparation enabled
us to collect IST separately from the CE. During perfusion with renin
and angiotensinogen, Ang I appeared in the transudate. This
Ang I was produced in cardiac tissue and was not derived from Ang I in
the perfusate. There was no indication that the heart was
capable of producing Ang I independent of arterially
delivered renin and angiotensinogen. Observations in
nephrectomized pigs also indicated that the cardiac tissue levels of
Ang I and II are determined by kidney-derived
renin.4
Here we report on a study in which we used the same modified
Langendorff rat heart preparation to investigate the production
of Ang II. The presence of Ang II in tissue depends on
arterial delivery of this peptide and on its local
generation. Ang II may be formed from arterially delivered
Ang I or from in situ synthesized Ang I. Extracellular Ang II is known
to be taken up into cells by AT1-receptor
endocytosis.8 9 10 11 The adrenals, the kidneys, and
the heart accumulate Ang II from the circulation by an
AT1-receptor–mediated process, probably
endocytosis.12
The questions addressed by the present study are: In which tissue
compartments is Ang II localized in the heart, where is it produced,
and how are its levels in cardiac tissue influenced by
AT1-receptor–mediated cellular uptake?
Preparation of the Modified Langendorff Heart
Male Wistar rats (Harlan, Zeist, The Netherlands; 280 to 400 g)
were anesthetized with pentobarbital (60 mg/kg IP) and
heparinized (5000 U/kg IV). The hearts (1.0 to 1.4 g) were rapidly
excised, cooled in ice-cold Tyrode's buffer (125 mmol/L NaCl,
4.7 mmol/L KCl, 1.4 mmol/L CaCl2,
20 mmol/L NaHCO3, 0.4 mmol/L
NaH2PO4, 1.0 mmol/L
MgCl2, 10 mmol/L D-glucose, pH 7.4) until
contractions stopped, and prepared for Langendorff perfusion.
Carbogen-gassed (95% O2/5%
CO2) Tyrode's buffer at 37°C was continuously
perfused immediately after cannulation of the aorta at a constant
perfusion pressure of 80 mm Hg. Subsequently the
pulmonary artery was cannulated and the caval and
pulmonary veins were carefully ligated. After the ligation
procedure, which took 30 to 45 minutes, the hearts were allowed to
stabilize for 30 minutes.
With this modified Langendorff heart preparation it is possible
to collect separately CE and IST.7 13 CE, ejected
by the right ventricle, was collected through the cannulated
pulmonary artery. IST, which drips from the heart, was
collected at the apex. IST flow was 0.03 to 0.16 mL/min, corresponding
with 0.7% to 2% of the coronary flow. An IST flow >2% of
the coronary flow, which occurred in one out of four to five
heart preparations, was considered to be an indication of leakage, for
example, from veins that were not properly
ligated.7 Hearts with such high IST flow were
therefore not used. IST is derived from the ISF, and the Ang I and II
concentrations in IST are considered to be
representative for the Ang I and II concentrations in
ISF.7
Perfusions With RAS Components and Collection of CE and
IST
CE and IST were collected during and after the infusions. One-minute (4
to 8 mL), 4-minute (16 to 40 mL), or 10-minute samples (40 to 80 mL) of
CE were collected into BSA-coated 10- or 50-mL polystyrene tubes.
Individual drops of IST (
The Eppendorf cups and polystyrene tubes were kept on ice during the
perfusions so that the samples were rapidly cooled during their
collection and remained cold (0° to 4°C) during the experiment.
After the experiment was finished, the samples for Ang I and II
measurements were frozen at -80°C.
Perfusion With Ang II to Study the Localization of
Arterially Delivered Ang II
In a separate series of experiments, the steady-state levels of Ang II
in CE, IST, and cardiac tissue were measured during perfusions with Ang
II in the presence of the AT1-receptor
antagonist losartan (10-6
mol/L) in the perfusion fluid. Coronary flow in these
experiments was 4 to 11 mL/min.
Perfusion With Ang I to Study the Localization of
Arterially Delivered Ang I and Locally Generated
Ang II
In a separate series of experiments, the steady-state levels of Ang I
and II in CE, IST, and cardiac tissue were measured during perfusions
with Ang I in the presence of the AT1-receptor
antagonist losartan (10-6
mol/L) in the perfusion fluid. Coronary flow in these
experiments was 4 to 9 mL/min.
Perfusions With Renin Combined With Angiotensinogen to
Study the Localization of Locally Generated Ang I and II
Samples of CE and IST collected from 31 to 40 minutes and from 46 to 55
minutes were used to determine the steady-state levels of Ang I and II.
Collections from 41 to 45 and from 56 to 60 minutes had been used for
other measurements, reported in our previous
study.7 The frozen hearts were used to measure
the steady-state tissue levels of Ang I and II.
Measurements of Ang I and II
The Ang I and II concentration of CE and IST samples obtained during
infusion with renin combined with angiotensinogen, as well
as the cardiac tissue concentrations of Ang I and II both during Ang I
and II perfusion and during combined renin and
angiotensinogen perfusion, were measured by
radioimmunoassay after SepPak extraction and reversed-phase HPLC
separation.4 14 15 To measure tissue Ang I and
II, the frozen hearts were minced and homogenized (1:10,
wt/vol) in an iced solution of 0.1 mol/L HCl/80% ethanol.
Homogenates were centrifuged at 20 000g
for 25 minutes at 4°C. Ethanol in the supernatant was evaporated
under constant air flow. The remainder of the supernatant was diluted
in 20 mL 1% ortho-phosphoric acid and centrifuged again at
20 000g. The supernatant was diluted with 1%
ortho-phosphoric acid 1:1 (vol/vol). The tissue homogenate
supernatants and CE and IST samples were concentrated over SepPak
columns (SepPak C18, Waters), and the concentrated extracts were
subjected to HPLC followed by radioimmunoassay.
125I-labeled Ang I had been added as an internal
standard to the CE and IST samples before SepPak extraction and to
tissue before its homogenization. Recovery was
better than 70%, and the Ang I and II results were corrected for
incomplete recovery. The lowest level of Ang I that could be measured
with the Ang I radioimmunoassay after HPLC separation was 2.0 fmol/g in
cardiac tissue, 0.05 fmol/mL in CE, and 2.5 fmol/mL in IST. The lowest
level of Ang II that could be measured with the Ang II radioimmunoassay
after HPLC separation was 1.0 fmol/g in cardiac tissue, 0.05 fmol/mL in
CE, and 1.5 fmol/mL in IST.
Calculations
in which [AngIST] is the steady-state
Ang I or II concentration in IST and [AngCE] is
the steady-state Ang I or II concentration in CE. ISF volume is the
cardiac interstitial fluid volume (0.23 mL/g), determined
previously as the difference between the distribution volume of
51Cr-EDTA and
125I-HSA,7 and IVF volume
is the intravascular fluid volume (0.38 mL/g), determined previously as
the distribution volume of
125I-HSA.7 In this
calculation, it is assumed that [AngCE] is
representative for the angiotensin
concentration in most of the intravascular fluid compartment
(coronary venules and veins and right ventricle).
Statistical Analysis
After discontinuation of the Ang II perfusion, Ang II disappeared from
IST, in a monophasic way, with a t1/2 of
1.4±0.3 minutes (mean±SD, n=5).
The cardiac tissue level of Ang II (per gram of tissue), shortly after
the Ang II perfusion had been switched off, was
During perfusion with Ang II in the presence of the
AT1-receptor antagonist
losartan (n=5), the steady-state Ang II levels in CE and IST
were not significantly different from the levels during Ang II
perfusion in the control situation (Fig 2
Localization of Arterially Delivered Ang I and Locally
Generated Ang II During Perfusion With Ang I
The steady-state Ang II level in IST was higher than in CE, whereas the
steady-state Ang I level in IST was lower than in CE. This, together
with the finding that during perfusion with Ang II the Ang II level in
IST was also lower than in CE (see above), indicates that part of the
Ang II present in IST during Ang I perfusion was not derived from
Ang II in the perfusate.
The cardiac tissue level of Ang I (per gram of tissue), shortly after
the Ang I perfusion had been switched off, was
During perfusion with Ang I in the presence of the
AT1-receptor antagonist
losartan (n=5), the steady-state Ang I and II levels in CE and
IST were not significantly different from those during Ang I perfusion
in the control situation (Fig 3
Localization of Locally Generated Ang I and II During Combined
Renin/Angiotensinogen Perfusion
The cardiac tissue level of Ang I, 18.2 (11.4 to 22.8) fmol/g, shortly
after the combined renin/angiotensinogen perfusion had been
switched off, was close to the level predicted on the basis of the
presence of Ang I in the cardiac extracellular fluid compartments (see
"Methods"),
The cardiac tissue level of Ang II, 4.8 (2.8 to 6.4) fmol/g, was higher
than the level predicted on the basis of the presence of Ang II in the
cardiac extracellular fluid compartments,
During combined renin/angiotensinogen perfusion in the
presence of the AT1-receptor
antagonist losartan (n=3), the Ang I and II levels
measured in CE, IST, and cardiac tissue were similar to the levels
measured in the control experiments without losartan. Thus
blockade of the AT1-receptor–mediated uptake of
extracellular Ang II in these experiments did not decrease tissue Ang
II to an undetectably low level as it did in the Ang I and II perfusion
experiments.
As expected, part of the Ang I present in the perfusate
during the combined renin/angiotensinogen perfusions and
formed by the reaction of arterially delivered renin with
arterially delivered angiotensinogen was
converted to Ang II and ejected by the CE. Ang I to II conversion by
the coronary vascular bed has been reported by
others.16 17 More interesting is our finding that
during these combined renin/angiotensinogen perfusions, the
concentration of Ang II in the IST was several times higher than in the
CE, whereas during perfusions with Ang II, the Ang II concentration in
the IST was lower than in the CE. These results indicate that a
substantial part of Ang II in the ISF was not derived from the Ang II
that was present in the perfusate.
At the time we finished this study, a report was published by
Dell'Italia et al18 on measurements of Ang II in
the ISF space of the dog heart by using microdialysis probes. The Ang
II level in the ISF was higher than in blood plasma and did not change
after systemic infusion of Ang I. These results indicate
compartmentalization of Ang II in the heart between the
interstitial and intravascular fluid compartments. Our
results are in agreement with this conclusion and suggest that Ang II
in the ISF is formed outside the intravascular fluid compartment.
In the combined renin/angiotensinogen perfusion
experiments, the Ang II level we measured in cardiac tissue (expressed
per gram of tissue) was higher than in CE (expressed per milliliter of
fluid). It was in fact as high as in the IST. This is an indication
that the locally produced Ang II is not restricted to the extracellular
fluid and that a large part of this Ang II might be located in the
cells, because the intravascular and interstitial fluid
compartments comprise
AT1-receptor–mediated binding of Ang II to
cardiac cells is also indicated by our results obtained during the Ang
I or II perfusions, which showed that the cardiac tissue concentration
of Ang II during these perfusions fell to an undetectably low level
when the specific AT1-receptor
antagonist losartan was added to the perfusion
fluid. Losartan, however, had no significant effect on the
cardiac tissue concentration of locally formed Ang II during the
combined renin/angiotensinogen perfusions. This is in
accordance with observations in rats, which showed an increase and not
a decrease in cardiac tissue Ang II after 8 days of treatment of the
animals with losartan, an increase that is likely to be related
to the stimulated renin release from the kidney after this
drug.19 The apparent discrepancy with respect to
the observed effects of losartan, between the results of the
Ang I and II perfusions on the one hand and the combined
renin/angiotensinogen perfusion on the other hand, raises
the possibility that during the combined
renin/angiotensinogen perfusion, local Ang II
production in the heart is taking place in a compartment that
is not reached by losartan, that is, within the cells.
The finding that the measured levels of Ang II in cardiac tissue in the
Ang I and II perfusion experiments were lower than calculated on the
basis of their presence in the extracellular fluid may be explained as
follows. Most of the extracellular fluid in the Langendorff heart
preparation is localized in the intravascular compartment, that is, the
coronary vascular bed and the right
ventricle.7 The half-life of Ang I and II is
In the combined renin/angiotensinogen perfusion experiments
these losses of extracellular Ang II might have been overcome, at least
partly, by ongoing formation of this peptide during the time that
elapsed until the tissue was frozen. The tissue levels of Ang II we
measured in these combined renin/angiotensinogen perfusion
experiments are therefore probably more close to the true levels at the
time of perfusion than the tissue levels we measured in the Ang I and
II perfusion experiments. Moreover, the Ang II concentration in the
extracellular fluid was several orders of magnitude higher during the
Ang I and II perfusions than during combined
renin/angiotensinogen perfusion. Because of these higher
levels of extracellular Ang II a smaller proportion of it will be bound
to the cardiac cell AT1-receptors and taken up in
the cells, where it is protected against the rapid degradation to which
extracellular Ang II is exposed.11 12 20 Finally,
if it is true that Ang II is produced within the cells during combined
renin/angiotensinogen perfusion, this intracellular Ang II
will similarly be protected against this rapid degradation.
The results of the renin/angiotensinogen perfusion
experiments indicate that in contrast with Ang II, most of the locally
produced Ang I in the heart is restricted to the cardiac extracellular
fluid compartments. Likely sites of cardiac Ang I production
are the ISF and a site closer to the blood compartment, possibly
vascular surface-bound renin (Fig 7
Received August 19, 1997;
first decision September 11, 1997;
accepted December 15, 1997.
© 1998 American Heart Association, Inc.
Scientific Contributions
Localization and Production of Angiotensin II in the Isolated Perfused Rat Heart
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—We used a modification of
the isolated perfused rat heart, in which coronary effluent and
interstitial transudate were separately collected, to
investigate the localization and production of
angiotensin II (Ang II) in the heart. During combined renin
(0.7 to 1.5 pmol Ang I/mL per minute) and angiotensinogen
(6 to 12 pmol/mL) perfusion (4 to 8 mL/min) for 60 minutes (n=3), the
steady-state levels of Ang II in interstitial transudate in
two consecutive 10-minute periods were 4.3±1.5 and 3.6±1.5 fmol/mL
compared with 1.1±0.4 and 1.1±0.6 fmol/mL in coronary
effluent (mean±half range). During perfusion with Ang II (n=5),
steady-state Ang II in interstitial transudate was 32±19%
of arterial Ang II compared with 65±16% in
coronary effluent (mean±SD, P<.02). During
perfusion with Ang I (n=5), Ang II in interstitial
transudate was 5.1±0.6% of arterial Ang I compared with
2.2±0.3% in coronary effluent (P<.05). The
tissue concentration of Ang II in the combined
renin/angiotensinogen perfusions (per gram) was as high as
the concentration in interstitial transudate (per
milliliter). Addition of losartan (10-6 mol/L) to
the renin/angiotensinogen perfusion (n=3) had no
significant effect on the tissue level of Ang II, whereas
losartan in the perfusions with Ang I (n=5) or Ang II (n=5)
decreased tissue Ang II to undetectably low levels. The results
indicate that the heart is capable of producing Ang II and that this
can lead to higher levels in tissue than in blood plasma. Cardiac Ang
II does not appear to be restricted to the extracellular fluid. This is
in part due to AT1-receptor–mediated cellular uptake of
extracellular Ang II, but our results also raise the possibility of
intracellular Ang II production.
Key Words: angiotensin II • heart • receptors, angiotensin
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Angiotensin II is the
primary mediator of the physiological actions of
the RAS and has an important function in cardiovascular
homeostasis. Ang II in the circulation is produced by the conversion of
Ang I by ACE of the vascular endothelium. Ang I is
generated in the circulation by the action of renin from the kidney on
its substrate, angiotensinogen, produced by the liver. The
various RAS components have all been identified in
tissues,1 2 3 4 which suggests the existence of a
locally acting RAS in the tissues, apart from the RAS in the
circulation.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Chemicals and Reagents
[Ile5]-Ang-(1–10) decapeptide (Ang I)
and [Ile5]-Ang-(1–8) octapeptide (Ang II) were
obtained from Bachem. BSA was from Sigma, 1,10-phenanthroline was from
Merck, and sodium pentobarbital was obtained from Apharma. The
AT1-receptor antagonist
losartan was a kind gift of Dr R.D. Smith, Du Pont Merck,
Wilmington, Del. The renin inhibitor remikiren was a kind
gift of Dr P. van Brummelen, Hoffmann-La Roche, Basel, Switzerland.
Renin was prepared from porcine kidneys as described
before.7 Angiotensinogen was prepared
from plasma of nephrectomized pigs.7 All other
reagents were of standard laboratory grade.
All experiments were performed under the regulation of the
Animal Care Committee of the Erasmus University, Rotterdam, The
Netherlands, in accordance with the "Guiding principles in the care
and use of animals" as approved by the American
Physiological Society.
The Langendorff hearts were perfused with Tyrode's buffer
through the cannulated aorta. After a 30-minute stabilization period,
RAS components were infused through a T-connection into the cannulated
aorta. 
50 µL each minute) and 4-minute (200
µL) or 9- to 10-minute (500 µL) samples of IST were collected
into BSA-coated 1.5-mL Eppendorf cups. The Eppendorf cups and
polystyrene tubes contained a mixture of inhibitors, 5, 10,
or 25 µL in the Eppendorf cups (for the individual drops of IST and
the 4-minute and 10-minute IST samples, respectively), and 250, 2500,
and 5000 µL in the polystyrene tubes (for the 1-minute, 4-minute, and
10-minute CE samples), to prevent the ongoing formation of Ang I, the
conversion of Ang I to Ang II, and the degradation of Ang I and II. The
mixture consisted of 0.2 mmol/L of the renin inhibitor
remikiren, 125 mmol/L disodium EDTA, and 25 mmol/L
1,10-phenanthroline.7 Remikiren is an
inhibitor of human renin (IC50
7x10-10 mol/L). It also inhibits porcine renin
(IC50 5x10-8
mol/L).4
Langendorff hearts were perfused for 15 minutes with Ang II. Ang
II diluted with Tyrode's buffer to a concentration of 400 pmol/mL was
infused into the perfusion system at a rate of 0.1 mL/min. Ang II
caused a transient fall in coronary flow that stabilized in 2
to 3 minutes at 4 to 9 mL/min. After the Ang II infusion had been
switched off, the heart was either quickly removed from the perfusion
apparatus and transferred into liquid nitrogen or subjected
to a 10-minute washout period. The tissue was frozen within 45 seconds
after the Ang II infusion had been stopped. One-minute samples of CE
and individual drops of IST were collected during the infusion and
washout periods in order to study the uptake and washout kinetics of
Ang II. A steady state was reached within 5 minutes. Samples of CE and
IST that had been collected from 7 to 10 minutes and from 11 to 14
minutes were used to determine the steady-state levels of Ang II. The
frozen hearts that were not subjected to a washout period were used to
measure the steady-state tissue levels of Ang II.
Langendorff hearts were perfused for 15 minutes with Ang I. Ang
I diluted with Tyrode's buffer to a concentration of 400 pmol/mL was
infused into the perfusion system at a rate of 0.1 mL/min.
Coronary flow in these experiments was 5 to 10 mL/min. The
uptake and washout kinetics of Ang I are known from our previous
study.7 After the Ang I infusion had been
switched off, the heart was removed from the perfusion
apparatus and immediately transferred into liquid nitrogen.
Samples of CE and IST collected from 7 to 10 minutes and from 11 to 14
minutes were used to determine the steady-state levels of Ang I and II.
The frozen hearts were used to measure the steady-state tissue levels
of Ang I and II.
Langendorff hearts were perfused for 60 minutes with renin and
angiotensinogen. Porcine renin diluted with Tyrode's
buffer to a concentration of 60 pmol Ang I/min per milliliter and
undiluted porcine angiotensinogen (500 pmol/mL) were
infused into the perfusion system both at a rate of 0.1 mL/min.
Coronary flow was 4 to 8 mL/min. The final concentrations of
renin and angiotensinogen in the perfusion fluid ranged
from 0.7 to 1.5 pmol Ang I/mL per minute and from 6 to 12 pmol/mL,
respectively. These experiments were performed in the presence or
absence of the AT1-receptor
antagonist losartan (10-6
mol/L) in the perfusion fluid. The renin and
angiotensinogen solutions were kept at 4°C until they
reached the aorta, in order to prevent angiotensin
generation outside the heart.7 After the
perfusion had been switched off, the hearts were frozen in liquid
nitrogen.
All angiotensin measurements were performed by a
technician who did not know which experiments had been carried out with
losartan and which without this drug. The Ang I and II
concentrations in CE and IST samples, collected during Ang I and II
perfusions, were measured directly with sensitive
radioimmunoassays.14 15 Measurements were made in
50 µL of undiluted CE and in 50 µL IST diluted with 25 µL of 0.25
mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl. The lowest
measurable Ang I concentration was 15 fmol/mL in CE and 40 fmol/mL in
IST. The lowest measurable Ang II concentration was 10 fmol/mL in CE
and 25 fmol/mL in IST.
To determine in which tissue compartments Ang I and II are
localized in the heart, the measured tissue levels of Ang I and II were
compared with the levels predicted on the basis of the presence of
these peptides in the cardiac extracellular fluid compartments. If the
measured tissue levels are higher than predicted,
angiotensin is not restricted to the extracellular fluid
compartments. If the measured tissue levels are lower than predicted,
angiotensin has disappeared from the extracellular fluid
compartments during the period between the moment the heart has been
removed from the perfusion equipment and the moment the tissue is
transferred into liquid nitrogen. The predicted tissue level (fmol/g)
was calculated as follows: Predicted tissue Ang
concentration =[AngIST]xISF
volume+[AngCE]xIVF volume
The Ang I and II levels in CE, IST, and cardiac tissue during
Ang I or II perfusion are expressed as a percentage of the
arterial levels of Ang I (Ang I perfusion) or Ang II (Ang
II perfusion). Ang I and II levels in CE, IST, and cardiac tissue
during renin combined with angiotensinogen perfusion are
given as absolute values. Differences between the experimental groups
were evaluated for statistical significance by Student's unpaired
t test. Differences within groups were evaluated for
statistical significance by Student's paired t test.
Bonferroni's correction was applied to adjust for multiple
comparisons. Differences were assigned to be significant for values of
P<.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Localization of Arterially Delivered Ang II During
Perfusion With Ang II
During perfusion with Ang II (n=10), the steady-state Ang II level
in CE was 65% of the arterial level (Fig 1
and Fig 2). Thus 35% of the
arterially delivered Ang II was removed by the heart during
a single passage of the perfusate. The intravascular fluid
volume of the Langendorff heart was measured in our previous study of
this model and was found to be 0.38 mL/g. Heart weight was 1.0 to
1.4 g, thus the intravascular fluid volume per heart was 0.38 to
0.53 mL. The perfusate flow was 4 to 9 mL/min; therefore the
perfusate transit time of the isolated heart preparation was
0.04 to 0.13 minutes. It can be concluded, therefore, that 35% of the
arterially delivered Ang II was removed in 0.04 to 0.13
minutes, which corresponds with a t1/2 of
0.06 to 0.21 minute. A steady-state level in IST was reached within 5
minutes. It was 35% of the arterial level (39% in the
experiments in which also the Ang II washout was measured, Fig 1; 32%
in the experiments in which also the tissue Ang II concentration was
measured, Fig 2). The steady-state Ang II concentration in IST was
significantly lower than in CE.
View larger version (18K):
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Figure 1. Ang II concentration in IST ( 
) and CE (
)
during and after perfusion with Ang II (n=5). Data (mean±SEM) are
presented as a percentage of the arterial Ang II
concentration.
View larger version (20K):
[in a new window]
Figure 2. Steady-state Ang II concentration in CE and IST
during perfusion with Ang II for 15 minutes and the Ang II
concentration in cardiac tissue shortly after the perfusion. Perfusions
were carried out in the absence (n=5) or presence (n=5) of
losartan (10-6 mol/L). The mean of the levels of
the 7- to 10-minute and 11- to 14-minute perfusion periods was taken as
the steady-state concentration. Data (mean±SD) are presented
as a percentage of the arterial Ang II concentration. Ang
II in IST was lower than in CE (P<.02) and higher than
in cardiac tissue (P<.05) both with and without
losartan. n.d. indicates not detectable. 5% of the
arterial level (per milliliter of perfusate) (Fig 2
). This is lower than predicted on the basis of the presence of Ang II
the cardiac extracellular fluid compartments (see "Methods"). The
predicted tissue concentration of Ang II would be 30% to 35% of the
arterial concentration. This discrepancy suggests that
extracellular Ang II was rapidly degraded in the short period
(maximally 45 seconds) between the moment the Ang II infusion had been
stopped and the moment the tissue was transferred into liquid
nitrogen. ). Ang II, which was above the
detection limit in all cardiac tissue samples in the control Ang II
perfusion experiments, could not be detected in the tissue samples in
the Ang II/losartan perfusion experiments. This is likely to be
due to blockade of the AT1-receptor–mediated
cellular uptake of extracellular Ang II.
During perfusion with Ang I (n=5), the steady-state Ang I level in
CE was 48% of the arterial level (Fig 3). Thus 52% of arterially
delivered Ang I was removed by the heart during a single passage of
perfusate. Part of the Ang I removal was caused by conversion
to Ang II (Fig 4).
View larger version (16K):
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Figure 3. Steady-state Ang I concentration in CE and IST
during perfusion with Ang I for 15 minutes and the Ang I concentration
in cardiac tissue shortly after the perfusion. Perfusions were carried
out in the absence (n=5) or presence (n=5) of losartan
(10-6 mol/L). The mean of the levels of the 7- to
10-minute and 11- to 14-minute perfusion periods was taken as the
steady-state concentration. Data (mean±SD) are presented as a
percentage of the arterial Ang I concentration. Ang I in
IST was lower than in CE (P<.02) and higher than in
cardiac tissue (P<.02) both with and without
losartan.
View larger version (20K):
[in a new window]
Figure 4. Steady-state Ang II concentration in CE and IST
during perfusion with Ang I for 15 minutes and the Ang II concentration
in cardiac tissue shortly after the perfusion. Perfusions were carried
out in the absence (n=5) or presence (n=5) of losartan
(10-6 mol/L). The mean of the levels of the 7- to
10-minute and 11- to 14-minute perfusion periods was taken as the
steady-state concentration. Data (mean±SD) are presented as a
percentage of the arterial Ang I concentration. Ang II in
IST was higher than in CE (P<.05) and cardiac tissue
(P<.05) both with and without losartan. n.d.
indicates not detectable. 5% of the
arterial level (per milliliter of perfusate) (Fig 3). This is lower than predicted on the basis of the presence of Ang I
in the cardiac extracellular fluid compartments (see "Methods").
The predicted tissue concentration of Ang I would be 22% of the
arterial concentration. This discrepancy suggests that
extracellular Ang I, like extracellular Ang II, was rapidly degraded in
the short period between the moment the Ang I infusion had been stopped
and the moment the tissue was transferred into liquid nitrogen. and Fig 4). Ang II, which was above the
detection limit in all cardiac tissue samples in the control Ang I
perfusion experiments, could not be detected in the tissue samples in
the Ang I/losartan perfusion experiments, which is probably due
to blockade of the AT1-receptor–mediated
cellular uptake of extracellular Ang II.
Results of the combined renin/angiotensinogen
perfusions are shown in Fig 5 and Fig 6. During combined
renin/angiotensinogen perfusion in the absence of
losartan (n=3), the Ang I levels in IST in the 31- to 40-minute
and 46- to 55-minute perfusion periods were 4.2 (1.5 to 8.2) and 3.2
(0.9 to 6.5) times higher than in CE, respectively (mean and range).
The Ang II levels in IST in these perfusion periods were 4.8 (2.7 to
6.8) and 3.8 (2.2 to 5.7) times the levels in CE.
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Figure 5. Steady-state Ang I concentration in CE and IST
during combined renin/angiotensinogen perfusion for 60
minutes and the Ang I concentration in cardiac tissue shortly after the
perfusion. Perfusions were carried out in the absence (n=3) or presence
(n=3) of losartan (10-6 mol/L). Data are
presented as mean and half range.
View larger version (26K):
[in a new window]
Figure 6. Steady-state Ang II concentration in CE and IST
during combined renin/angiotensinogen perfusion for 60
minutes and the Ang II concentration in cardiac tissue shortly after
the perfusion. Perfusions were carried out in the absence (n=3) or
presence (n=3) of losartan (10-6 mol/L). Data are
presented as mean and half range. 20 fmol/g. 1.5 fmol/g. This suggests
that the presence of the locally formed Ang II was not limited to the
intravascular and interstitial fluid.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In a previous series of experiments we studied the
production of Ang I by the modified rat Langendorff heart, in
which the ISF transudate could be collected separately from the
CE.7 Our present study uses the same isolated
heart model and focuses on the production of Ang II. 38% and 23% of tissue weight,
respectively.7 Cellular binding and uptake
through AT1-type Ang II receptors is known to
occur.8 9 10 11 125I-Ang II
infused into the left cardiac ventricle of intact pigs is accumulated
in cardiac tissue by an AT1-receptor–dependent
process, and the kinetics of Ang II accumulation and disappearance are
compatible with receptor-mediated
endocytosis.12 1
minute in the ISF compartment.7 In the
intravascular compartment it is even shorter, 0.1 to 0.2 minute. It
is therefore possible that the short period between the moment the
perfusions had been stopped and the tissue had been transferred into
liquid nitrogen was long enough for the endothelial
peptidases to cause substantial breakdown of intravascular Ang I and
II. ).7 The question
of where in the tissue the Ang II is formed is incompletely answered by
the present study. Part of it is formed at the vascular
endothelial and endocardial
surfaces.16 21 22 23 Our observations in the
present study on the effects of blockade of
AT1-receptor–mediated uptake of Ang II, which
did not show a change in the tissue level of locally produced Ang II as
opposed to a decrease in the tissue level of arterially
administered Ang II, raise the interesting possibility of intracellular
Ang II formation (Fig 7). There is experimental evidence that neonatal
rat cardiac myocytes are capable of binding and internalizing renin and
prorenin through a mannose 6-phosphate receptor and that prorenin is
activated in the cells.24 Internalized
Ang II has a long half-life, and there is growing evidence that
intracellular Ang II can serve important
functions.25 26 27
AT1-receptor–mediated uptake of Ang II and the
evidence that the formation and degradation of Ang I and II in cardiac
tissue are highly compartmentalized has implications for the effects of
drugs that interfere with Ang II receptor binding
(AT1-receptor antagonists) or reduce
the production of Ang II (renin inhibitors, ACE
inhibitors). These drugs may have different effects on the
Ang II levels in different cardiac tissue compartments.
View larger version (34K):
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Figure 7. Proposed scheme of Ang I and
II production in the heart: Intravascular and
interstitial fluid compartments as well as blood vessel
wall and cardiac cells (endothelial cells, myocytes,
fibroblasts, macrophages) are depicted. Circulating renin and
angiotensinogen (Aog) both enter the
interstitial fluid compartment. Renin may also bind to the
vascular wall and cardiac cells.7 24 ACE is present on
endothelial cells and possibly also on cardiac cells.
Ang I and II are metabolized by peptidases while passing through the
vascular wall. Ang I and II in the interstitial fluid are
mainly generated outside the vascular fluid compartment. Tissue
Ang I and II generation may occur not only in the
interstitial fluid or on the cell surface but also within
cells, for instance after renin uptake by the cells.24
Binding of Ang II to the AT1-receptor is followed by
internalization of the AT1-receptor–Ang II complex.
Selected Abbreviations and Acronyms
ACE
=
angiotensin-converting enzyme
Ang I, II
=
angiotensin I, II
AT1
=
angiotensin II type 1 receptor
BSA
=
bovine serum albumin
CE
=
coronary effluent
HPLC
=
high-performance liquid chromatography
HSA
=
human serum albumin
ISF
=
interstitial fluid
IST
=
interstitial transudate
RAS
=
renin-angiotensin system
Acknowledgments
This study was supported by the Netherlands Heart Foundation,
research grant 96.019.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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