(Hypertension. 1997;29:1240-1251.)
© 1997 American Heart Association, Inc.
Articles |
Renin-Angiotensin System Components in the Interstitial Fluid of the Isolated Perfused Rat Heart
Local Production of Angiotensin I
From the Departments of Internal Medicine (L.M. de L., J.P. van K., M.A.D.H.S.) and Pharmacology (L.M. de L., A.H.J.D., R.G.S., P.R.S.), Erasmus University Rotterdam (Netherlands).
Correspondence to A.H. Jan Danser, PhD, Department of Pharmacology, Rm EE1418b, Erasmus University, Dr. Molewaterplein 50, 3015 GE Rotterdam, Netherlands.
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Abstract We used a modification of the isolated perfused rat heart, in which coronary effluent and interstitial transudate were separately collected, to investigate the uptake and clearance of exogenous renin, angiotensinogen, and angiotensin I (Ang I) as well as the cardiac production of Ang I. The levels of these compounds in interstitial transudate were considered to be representative of the levels in the cardiac interstitial fluid. During perfusion with renin or angiotensinogen, the steady-state levels (mean±SD) in interstitial transudate were 64±34% (P<.05 for difference from the arterial level, n=8) and 108±42% (n=6) of the arterial level, respectively; the levels in coronary effluent were not significantly different from those in interstitial transudate. Ang I was not detectable in interstitial transudate during perfusion with Tyrode's buffer or angiotensinogen. It was very low in interstitial transudate during perfusion with renin and rose to much higher levels during combined renin and angiotensinogen perfusion. The total production rate of Ang I present in interstitial fluid could be largely explained by the renin-angiotensinogen reaction in the fluid phase of the interstitial compartment. In contrast, the total production rate of Ang I present in coronary effluent and the net ejection rate of Ang I via coronary effluent were, respectively, 4.6±2.2 and 2.8±1.3 (P<.01 and P<.05 for difference from 1.0, n=6) times higher than could be explained by Ang I formation in the fluid phase of the intravascular compartment. Ang I from the interstitial fluid contributed little to the Ang I in the intravascular fluid and vice versa. These data reveal two tissue sites of Ang I production, ie, the interstitial fluid and a site closer to the blood compartment, possibly vascular surface-bound renin. There was no evidence that the release of locally produced Ang I into coronary effluent and interstitial transudate occurred independently of blood-derived renin or angiotensinogen.
Key Words: renin • angiotensinogen • angiotensin • heart • extracellular space
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A local RAS in the heart may contribute to the pathogenesis of congestive heart failure, cardiac hypertrophy and remodeling, and reperfusion arrhythmias.1 2 3 4 The RAS components—renin, angiotensinogen, ACE, and Ang I and II, which are all present in circulating blood—have also been identified in cardiac tissue.5 6 In addition, the tissue concentrations of Ang I and II are too high to be explained simply by passive diffusion out of the blood and distribution into the ISF.6
Perfusion of the Langendorff isolated rat heart with Ang I leads to the appearance of Ang II in the coronary effluent,7 8 and perfusion with renin leads to the appearance of both Ang I and II.7 Part of the Ang I in the coronary venous blood appears to originate from local production at cardiac tissue sites,9 but it is not known how much of this local production depends on renin that is synthesized by the heart and how much on renin from the kidney. It has been reported that in pigs, the release of locally produced Ang I into the coronary circulation is directly proportional to the level of renin activity in circulating plasma9 and that the cardiac tissue levels of renin, Ang I, and Ang II are undetectably low 30 hours after bilateral nephrectomy, when plasma renin activity was practically zero.6 In the rat, the cardiac tissue levels of Ang I and II were also lowered by nephrectomy, although small quantities of Ang II are still detectable in both cardiac tissue and blood.5 These observations indicate that the presence of Ang I and II in cardiac tissue depends at least in part on kidney-derived renin.
Little is known about the cardiac uptake of blood-derived RAS components. The sites of cardiac Ang I and II production are also unknown. A unique model to address these issues is a modified version of the Langen-dorff heart,10 11 12 which enables the investigator to separately collect the CE and IST derived from the ISF compartment. We report here on the use of this model to study the transport and distribution of blood-derived renin and angiotensinogen and to investigate the local intracardiac production of Ang I.
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Chemicals
[Ile5]Ang-(1-10) decapeptide (Ang I) was obtained from Bachem, 51Cr-EDTA and 125I-labeled HSA from Amersham, bovine serum albumin from Sigma Chemical Co, 1,10-phenanthroline from Merck, and sodium pentobarbital from Apharma. The angiotensin type 1 receptor antagonist losartan was a kind gift of Dr R.D. Smith, DuPont Merck, Wilmington, Del. The renin inhibitor remikiren was a kind gift of Dr P. van Brummelen, Hoffmann–La Roche, Basel, Switzerland. All other reagents were of standard laboratory grade.
Preparation of Renin and Angiotensinogen
Renin was prepared from rat or porcine kidneys. Both rat renin
and porcine renin were used to perfuse the rat Langendorff hearts. Most
of these perfusions were carried out with porcine renin because of the
limited availability of sufficient quantities of rat renin.
Angiotensinogen was prepared from plasma of nephrectomized rats, pigs, or sheep. Rat angiotensinogen was used as a substrate for rat renin measurements, and porcine or sheep angiotensinogen was used for porcine renin measurements. Under the conditions of our experiments, sheep angiotensinogen yielded higher quantities of Ang I than porcine angiotensinogen when incubated with porcine renin. The angiotensinogens were also used to perfuse the Langendorff heart. Most of these perfusions were carried out with porcine or sheep angiotensinogen because of the limited availability of sufficient quantities of rat angiotensinogen.
For renin preparation, kidney tissue was homogenized (1:1, wt/vol) with a polytron (PT10/35, Kinematica) in 0.01 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl. The homogenate was dialyzed for 48 hours at 4°C against 0.05 mol/L glycine buffer, pH 3.3, containing 0.001 mol/L disodium EDTA and 0.095 mol/L NaCl.6 This was followed by dialysis for 24 hours against 0.1 mol/L phosphate buffer, pH 7.4, containing 0.001 mol/L disodium EDTA and 0.075 mol/L NaCl. The content of the dialysis bag was then collected, and denatured protein was removed by centrifugation at 20 000g for 20 minutes at 4°C. The supernatant ("semipurified renin") was stored at -80°C. The renin concentration was 125 pmol Ang I/min per milliliter in the rat renin preparation and 600 pmol Ang I/min per milliliter in the porcine renin preparation, as assessed by incubation with rat and porcine angiotensinogen, respectively.6
Angiotensinogen was prepared as described before.13 The semipurified preparations of rat, porcine, and sheep angiotensinogen were stored at -80°C. The angiotensinogen concentrations in these preparations were 2500, 500, and 300 pmol/mL, respectively.
Preparation of Modified Langendorff Heart
All experiments were performed under the regulations of the
Animal Care Committee of the Erasmus University, Rotterdam,
Netherlands, in accordance with the "Guiding Principles in the Care
and Use of Animals" approved by the American
Physiological Society.
Male Wistar rats (Harlan, Zeist, 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, and 10 mmol/L D-glucose, pH 7.4) until contractions stopped, and prepared for Langendorff perfusion. Continuously carbogen-gassed (95% O2/5% CO2) Tyrode's buffer at 37°C was perfused immediately after cannulation of the aorta at a constant perfusion pressure of 80 mm Hg. Coronary flow was between 4 and 8 mL/min. 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 stabilized for 30 minutes.
With this modified Langendorff heart preparation, it is possible to collect separately CE and IST.10 11 12 CE, ejected by the right ventricle, was collected via the cannulated pulmonary artery. Dead space of the pulmonary cannula was 0.1 mL. IST, which keeps dripping 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 greater than 2% of the coronary flow was considered to be an indication of leakage, eg, from veins that were not properly ligated. Hearts with such a high IST flow were therefore not used.
Checking for Leakage of Perfusate Into the IST
The hearts were perfused with red blood cells to check for
leakage of perfusate into the IST. The blood cells were
isolated from heparinized (30 U/mL) rat blood and washed two times with
carbogen-gassed Tyrode's buffer containing heparin (30 U/mL). The
cells were diluted in this buffer to a concentration of
5x109 cells/mL and infused via a
T-connection into the cannulated aorta with a
Harvard 22 pump at a speed of 0.1 mL/min for either 10 or 20 minutes.
The cells were counted in CE and IST samples collected from the moment
the infusion was started to the end of infusion.
Measurements of Intravascular Fluid and ISF Volumes
The hearts were perfused with 125I-HSA or
51Cr-EDTA. The radiolabeled markers were diluted to a
concentration of 0.2 µCi/mL with Tyrode's buffer and infused via a
T-connection into the cannulated aorta at a
speed of 0.1 mL/min. 125I-HSA was infused for 1 minute and
51Cr-EDTA for 10 minutes.
One-minute CE samples and individual IST drops (approximately 50 µL) were collected during the infusion period, and the hearts were removed immediately after the perfusion had been switched off. Radioactivity levels (counts per minute) of CE, IST, and the whole heart were measured with a Minaxi 5000 multiple channel gamma-counter (Packard Instruments). Intravascular and extracellular fluid volumes (milliliters per gram heart wet weight) were considered to be equal to the distribution volumes of 125I-HSA and 51Cr-EDTA, respectively, in the heart and were calculated as the ratio between the radioactivity of the heart (counts per minute per gram) and the radioactivity of CE (counts per minute per milliliter). The intravascular compartment contains the fluid present in the coronary vascular bed and right ventricle. The ISF volume (milliliters per gram) was calculated by subtracting the intravascular fluid volume from the extracellular fluid volume.
Perfusions With RAS Components and Collection of CE and
IST
The Langendorff hearts were perfused with Tyrode's buffer via
the cannulated aorta. The buffer contained the angiotensin
type 1 receptor antagonist losartan in a
concentration of 10-6 mol/L. This
concentration is sufficient to prevent Ang II–mediated
vasoconstriction.14 After a 30-minute stabilization
period, the RAS components were infused via a
T-connection into the cannulated aorta at a
speed of 0.1 mL/min.
CE and IST were collected during and after the infusions. One-minute (4- to 8-mL) or 4- to 5-minute (16- to 40-mL) samples of CE were collected into bovine serum albumin–coated 10- or 50-mL polystyrene tubes, and 1-minute (approximately 50-µL) or 9- to 10-minute (approximately 450- to 500-µL) samples of IST were collected into bovine serum albumin–coated 1.5-mL Eppendorf cups. The Eppendorf cups and polystyrene tubes used to collect samples for Ang I measurement contained a mixture of inhibitors—5 or 25 µL in the Eppendorf cups (for the 1-minute and 9- to 10-minute IST samples) and 250 or 2500 µL (for the 1-minute and 9- to 10-minute CE samples) in the polystyrene tubes—to prevent ex vivo formation of Ang I, conversion of Ang I to Ang II, and degradation of Ang I. 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.6 Remikiren is an inhibitor of human renin (IC50, 7x10-10 mol/L). It also inhibits porcine renin (IC50, 5x10-8 mol/L).6 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 measurement were frozen at -80°C. Samples for the measurement of renin and angiotensinogen were frozen at -20°C.
Control Perfusion With Tyrode's Buffer to Study Release of
Endogenous Renin, Angiotensinogen, and Ang
I
Langendorff hearts were perfused with Tyrode's buffer for 40
minutes. The 30-minute stabilization period after heart preparation
(see above) was omitted here because we assumed that after such a long
period, the endogenous RAS components would have been
washed away from the CE and IST. Nine-minute collections of CE and IST
were used for measurement of Ang I. Each 9-minute collection was
followed by a 1-minute collection for measurement of renin and
angiotensinogen.
Perfusion With Renin, Angiotensinogen, or Ang I to
Study Uptake and Washout of Exogenous RAS Components
Langendorff hearts were perfused with porcine renin, diluted 1:8
with Tyrode's buffer; with undiluted porcine
angiotensinogen; or with Ang I, diluted to a concentration
of 400 pmol/mL with Tyrode's buffer. The RAS components were infused
into the perfusion system for 60 minutes (renin and
angiotensinogen) or 15 minutes (Ang I). After the infusion
had been switched off, the hearts were either rapidly frozen in liquid
nitrogen or subjected to a 10-minute washout period.
One-minute samples of CE and individual drops of IST were collected during the infusion and washout periods for measurement of renin, angiotensinogen, or Ang I. The frozen hearts were used for measurement of the steady-state tissue levels of these RAS components. Hearts frozen after 60 minutes of perfusion with Tyrode's buffer served as controls.
Perfusion With Renin or Angiotensinogen or Renin
Combined With Angiotensinogen to Study Cardiac
Production of Ang I
Langendorff hearts were perfused with rat renin or
angiotensinogen to study Ang I production from
endogenous (rat) angiotensinogen and renin,
respectively. Other Langendorff hearts were perfused with porcine or
sheep angiotensinogen to investigate whether these hearts
were capable of producing Ang I in the absence of exogenous renin,
possibly by the action of a reninlike enzyme in the heart. Renin,
diluted 1:4 with Tyrode's buffer, or undiluted
angiotensinogen was infused into the perfusion system for
40 minutes, followed by a 10-minute washout. Nine-minute collections of
CE and IST were used for measurement of Ang I. Each 9-minute collection
was followed by a 1-minute collection for measurement of renin or
angiotensinogen.
Finally, a number of Langendorff hearts were perfused with porcine renin combined with porcine or sheep angiotensinogen to study the cardiac production of Ang I from both exogenous renin and exogenous angiotensinogen. Renin, diluted 1:4 with Tyrode's buffer, and undiluted angiotensinogen were infused into the perfusion system for 60 minutes, followed by a 30-minute washout. The renin and angiotensinogen solutions were kept at 0° to 4°C with ice until they reached the cannulated aorta. Ten-minute collections of CE and IST were used for measurement of Ang I. Each 10-minute collection period was followed by a 5-minute collection for measurement of renin and angiotensinogen.
Biochemical Measurements
Renin
The concentration of renin in CE, IST, or cardiac tissue
homogenate was determined by measuring the rate of Ang I
generation during incubation, at pH 7.4 and 37°C, with known amounts
of angiotensinogen in the presence of a mixture of ACE,
angiotensinase, and serine protease
inhibitors.6 Renin concentration was defined
as the maximal Ang I generation rate (Vmax) at
saturating concentrations of angiotensinogen.
For measurement of renin in cardiac tissue, the hearts were rapidly frozen in liquid nitrogen. The frozen hearts were then minced and homogenized (1:3, wt/vol) in 0.01 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl, with a polytron (PT10/35). Homogenate used for the measurement of renin was treated as follows: One milliliter of homogenate was dialyzed for 48 hours at 4°C against 0.05 mol/L glycine buffer, pH 3.3, containing 0.095 mol/L NaCl.6 15 This was followed by dialysis at 4°C for 24 hours against 0.1 mol/L phosphate buffer, pH 7.4, containing 0.075 mol/L NaCl. The content of the dialysis bags was then collected; denatured protein was removed by centrifugation at 20 000g for 20 minutes at 4°C; and volume was adjusted to 1 mL with phosphate buffer. Experiments in which 0.1 mL rat or porcine renin was added to 1 g frozen tissue before homogenization showed that the recovery of renin was better than 90%.
In the experiments in which the hearts were perfused with Tyrode's buffer (control perfusion) or with rat renin, the renin concentration was determined by incubation with rat angiotensinogen. In the experiments in which the hearts were perfused with porcine renin, the renin concentration was determined with the use of porcine angiotensinogen. In the experiments in which the hearts were perfused with porcine renin combined with porcine angiotensinogen or sheep angiotensinogen, the renin concentration was determined by using porcine or sheep angiotensinogen, respectively.
The incubation mixture of the renin assay consisted of (1) 100 µL undiluted CE or 100 µL IST diluted 1:3 in 0.01 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl, or 100 µL acid-pretreated tissue homogenate; (2) 100 µL of 0.01 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl; (3) 200 µL angiotensinogen; and (4) 14 µL of an inhibitor mixture containing phenylmethylsulfonyl fluoride (0.07 mol/L), disodium EDTA (0.14 mol/L), 8-hydroxyquinoline sulfate (0.10 mol/L), and aprotinin (2000 kallikrein inhibiting units per milliliter).
Incubation time was 1 or 2 hours, and Ang I generation was linear during this period. Ang I was measured with a sensitive radioimmunoassay.16 Vmax was calculated according to the equation Vmax=Vx(Km+[S])/[S], in which V is the measured Ang I generation rate, Km is the Michaelis-Menten constant, and [S] is the angiotensinogen concentration in the incubate. Km was determined by 5-, 10-, and 20-minute incubations of renin with serial dilutions of angiotensinogen and by constructing Lineweaver-Burk plots of the measured Ang I generation rates. Km for the reaction of rat renin with rat angiotensinogen was 2400 pmol/mL, which agrees with the values reported in the literature.17 18 Km was 420 pmol/mL for the reaction of porcine renin with porcine angiotensinogen and 110 pmol/mL for the reaction of porcine renin with sheep angiotensinogen.
The lowest level that could be measured for rat renin (incubated with rat angiotensinogen) was approximately 10 fmol Ang I/min per milliliter in CE, 40 fmol/min per milliliter in IST, and 40 fmol Ang I/min per gram in cardiac tissue. For porcine renin (incubated with either porcine or sheep angiotensinogen), it was approximately 5 fmol Ang I/min per milliliter in CE, 25 fmol Ang I/min per milliliter in IST, and 25 fmol Ang I/min per gram in cardiac tissue.
Angiotensinogen
The concentration of angiotensinogen in CE, IST, or
cardiac tissue homogenate was measured as the maximum
quantity of Ang I generated during incubation, at pH 7.4 and 37°C,
with porcine kidney renin in the presence of a mixture of ACE,
angiotensinase, and serine protease
inhibitors.6 For measurement of
angiotensinogen in cardiac tissue, the frozen hearts were
rapidly minced and homogenized as described above under
"Renin," but the dialysis step used for renin measurement was
omitted here. Experiments in which 0.1 mL rat, porcine, or sheep
angiotensinogen was added to 1 g frozen tissue before
homogenization showed that the recovery of
angiotensinogen was better than 90%.
The incubation mixture of the angiotensinogen assay consisted of (1) 100 µL undiluted CE, 100 µL IST diluted 1:3 in 0.01 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl, or 100 µL tissue homogenate; (2) 150 µL porcine renin diluted 1:50 in 0.01 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl; and (3) 14 µL inhibitor mixture (see above). Incubation time was 1 hour, and the conditions of the assay were chosen in such a way that Ang I formation was completed within 1 hour.
The lowest levels of angiotensinogen that could be measured were 0.1 pmol/mL in CE, 0.4 pmol/mL in IST, and 0.4 pmol/g in cardiac tissue.
Ang I
The Ang I concentration of CE and IST, collected
during Ang I perfusion, was measured directly with a sensitive Ang I
radioimmunoassay.16 Measurements were made in 50 µL
undiluted CE and 50 µL IST diluted 1:1.5 in 0.25 mol/L
phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl. Recovery of
Ang I added to CE or IST was better than 95%. The lowest levels of
Ang I that could be measured with the direct
radioimmunoassay were 15 fmol/mL in CE and 40 fmol/mL
in IST.
The Ang I concentration of CE, IST, or cardiac tissue during perfusion with Tyrode's buffer, renin, angiotensinogen, or renin combined with angiotensinogen was measured by radioimmunoassay after SepPak extraction and reversed-phase high-performance liquid chromatographic (HPLC) separation.6 9 16 For measurement of Ang I, 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 of 1% ortho-phosphoric acid and centrifuged again at 20 000g. The supernatant was diluted with 1% ortho-phosphoric acid (1:1, vol/vol). CE, IST, or tissue homogenate supernatants were concentrated over the SepPak columns (C18, Waters), and the concentrated extracts were subjected to HPLC followed by radioimmunoassay. 125I-labeled Ang I had been added to the samples before SepPak extraction (CE and IST samples) or before cardiac tissue homogenization (tissue homogenate samples) as an internal standard. Recovery was better than 70%, and the Ang I results were corrected for incomplete recovery. The lowest levels of Ang I that could be measured with the Ang I radioimmunoassay after HPLC separation were 0.05 fmol/mL in CE, 2.5 fmol/mL in IST, and 2.0 fmol/g in cardiac tissue.
Calculations
In our calculations, IST that is dripping from the heart
is distinguished from the ISF that is present in cardiac tissue. A
distinction is also made between exogenous arterially
delivered Ang I and endogenous Ang I formed in the
Langendorffpreparation.
The production of endogenous Ang I present in
ISF (femtomoles per minute) was calculated as follows:
 | (1) |
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The clearance of Ang IISF was calculated according to the
equation
 | (2) |
The production rate of Ang IISF (femtomoles per
minute), predicted on the basis of the
renin-angiotensinogen reaction in the fluid phase of the
interstitial compartment, was derived from the following
equation:
 | (3) |
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 | (4) |
Ang IISF production calculated according to
Equation 1
is referred to as "measured" Ang IISF
production, because the independent variables in this
equation were measured experimentally. Ang IISF
production predicted by Equation 3 is referred to as
"predicted" Ang IISF production.
The production rate of endogenous Ang
ICE (femtomoles per minute) was calculated as follows:
 | (5) |
The net ejection rate of endogenous Ang I (femtomoles per
minute) via CE was calculated as follows:
 | (6) |
 | (7) |
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 | (8) |
Ang ICE production calculated according to Equation 5 is referred to as "measured" Ang ICE
production because the independent variables in this
equation were measured experimentally. Ang ICE
production predicted by Equation 7 is referred to as
"predicted" Ang ICE production.
Our analysis is based on the following assumptions: (1) The disappearance of exogenous Ang I from IST and from the cardiac ISF follows first-order kinetics, and the half-life of exogenous Ang I is not different between the two fluid compartments; (2) the steady-state concentrations of exogenous Ang I in IST and ISF are not different; and (3) these assumptions also apply to endogenous Ang I.
In assumptions 1 and 2 above, the ISF is considered to represent a single compartment and the only source of IST. This is supported by kinetic studies of the transport of inert low- and high-molecular-weight substances from the perfusion fluid into IST19 and by studies of the release of cardiac enzymes and metabolites into IST11 12 19 20 as well as by studies of cardiac glucose uptake, in which glucose levels in CE and IST were compared in the same type of Langendorff model as we used here.10 For assumptions 1 and 2 to be valid, it is important to exclude leakage of perfusate directly into IST caused by damage to the coronary vessels or inadequate ligation of the veins. Perfusions with red blood cells showed that such leakage only marginally contributed to the formation of IST (see "Results").
Assumption 3 implies that in the ISF the half-life of endogenous Ang I is the same as the half-life of exogenous Ang I. The half-life of Ang I in ISF is determined by its metabolism by peptidases, by its back-diffusion into the intravascular compartment, and by its loss via IST. It seems logical to assume that in the ISF these mechanisms act on endogenous Ang I in the same way as on exogenous Ang I.
Is the half-life of endogenous Ang I in the IST drop
also the same as the half-life of endogenous Ang I in the
ISF? As described in "Results," the half-life of exogenous Ang I
in IST in the collection tube was much longer than the half-life in the
IST drop while it was still on the cardiac surface. There may be little
back-diffusion of Ang I from the IST drop into the ISF, and Ang I in
the IST drop may be less exposed to peptidases than Ang I in the ISF.
This would result in a longer half-life in the IST drop on the cardiac
surface than in the ISF in cardiac tissue, so that the level of
endogenous Ang I in the IST might be higher than the level
of endogenous Ang I in the ISF. Equation 1 would then lead
to an overestimation of the true production of the Ang I
present in the ISF. However, the Ang I production
calculated according to Equation 1 was close to the value predicted by
Equation 3 (see "Results"), and since Equation 3 gives the lowest
possible value, the level of endogenous Ang I does not
appear to be higher in IST than in the ISF.
Statistical Analysis
Data are expressed as mean±SD except when indicated otherwise.
In the uptake and washout experiments, the concentrations in CE, IST,
and cardiac tissue are expressed as a percentage of the
arterial concentration. In the Ang I generation
experiments, the concentrations in CE and IST are given as absolute
values. Intraindividual differences were evaluated for statistical
significance by Student's paired t test. Differences were
considered to be significant for values of P<.05.
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Leakage of Perfusate Into the IST
During 10- and 20-minute perfusions with red blood cells, the cell counts in IST were approximately 1% of the counts in simultaneously collected CE (Table 1
).
Leakage of perfusate into IST may occur when the
coronary vessels are damaged or after the veins have been
improperly ligated. Our results show that such leakage only marginally
contributed to the formation of IST.
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Intravascular and ISF Volumes
125I-HSA, when infused into the Langendorff rat heart,
entered the IST and reached levels comparable to those in CE after
approximately 10 minutes. After 1-minute perfusion with
125I-HSA, the level in IST was approximately 10% of the
level in CE. We used the 125I-HSA level in
CE collected during the first minute of infusion and the level in
cardiac tissue after 1 minute of infusion to calculate the
intravascular fluid volume, which consists of the fluid in the
coronary vascular bed and the right ventricle. It was 38%
(mean, n=4) of the cardiac wet weight (Table 2), which
is in agreement with previous studies in isolated
hearts.21
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51Cr-EDTA, when infused into the perfusion system,
reached levels in IST comparable to those in CE within 5 minutes of
infusion. No further increase was observed when the infusion was
prolonged. We used 51Cr-EDTA levels in CE and cardiac
tissue after 10 minutes of infusion to calculate the extracellular
fluid volume. It was 61% (mean, n=6) of the cardiac wet weight (Table 2). Since 38% of the cardiac weight consisted of intravascular fluid,
the ISF volume is estimated to be 23% of the cardiac wet weight.
Release of Endogenous Renin,
Angiotensinogen, and Ang I
During 40 minutes of perfusion with Tyrode's buffer immediately
after preparation of the Langendorff hearts (n=4), renin and Ang I were
below the detection limit of the assay in both CE and IST.
Angiotensinogen was undetectable in CE but not in IST.
Angiotensinogen in IST was 4±1 pmol/mL after 10 minutes of
perfusion with Tyrode's buffer and decreased to just above the
detection limit (0.4 pmol/mL) after 40 minutes.
Uptake and Washout of Exogenous Renin, Angiotensinogen,
and Ang I
Renin
During perfusion with porcine renin (n=8), the steady-state renin
level in CE was 78% of the arterial level (mean value,
P<.05 for difference from the arterial level;
see Table 3). Thus, some of the arterially
delivered renin was removed from the perfusate by the heart. A
steady-state level in IST was reached within 20 minutes (Fig 1), and it was not significantly different from that in
CE (Table 3, Fig 1). After discontinuation of the renin perfusion,
renin disappeared from CE in a biphasic pattern (Fig 2).
The rapid first phase had a t1/2 of 0.42±0.03 minutes, and
the slow second phase had a t1/2 of 3.3±0.8 minutes. Renin
disappeared from IST in a monophasic way, with a t1/2 of
3.9±1.4 minutes.
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) and IST (
) during perfusion of the modified Langendorff
heart with these RAS components. Values (means and SEM) are expressed
as percentage of the arterial level.
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The cardiac tissue level of renin (per gram tissue), immediately after
the renin perfusion had been switched off, was 56±9% (n=5) of the
arterial level (per milliliter perfusate). Since
the renin level in CE was 78% of the arterial level and
not different from the level in IST, and since in a different series of
experiments the cardiac extracellular space was found to be 0.61 mL/g
heart wt (mean value, n=6; see Table 2), the cardiac tissue level of
renin is expected to be 0.61x78=48% (mean) of the
arterial level, assuming that renin in the heart is located
in the extracellular fluid compartment and that 1 g tissue
corresponds with a volume of 1 mL.6 22 This calculated
value is close to the renin level that was actually measured in cardiac
tissue (56% of the arterial level). Thus, the cardiac
tissue level of renin is consistent with its location in the
extracellular fluid at a concentration similar to that of CE. Renin was
not detectable in control hearts perfused with Tyrode's buffer
(n=4).
Angiotensinogen
During perfusion with porcine angiotensinogen (n=6),
the steady-state angiotensinogen level in CE was not
significantly different from the arterial level (Table 3).
Thus, in contrast with renin, removal of angiotensinogen
from the perfusate by the heart could not be demonstrated.
Angiotensinogen did reach the IST, and in IST a
steady-state level comparable to that in CE was reached after
approximately 30 minutes (Fig 1). After discontinuation of the
angiotensinogen perfusion, angiotensinogen
disappeared rapidly (t1/2, 0.54±0.32 minutes) from CE in a
monophasic way (Fig 2), and there was no evidence for a slow second
phase, as there was during renin washout. Angiotensinogen
disappeared from IST, also in a monophasic way, with a t1/2
of 2.9±1.0 minutes.
The cardiac tissue level of angiotensinogen (per gram
tissue), immediately after the angiotensinogen perfusion
had been switched off, was 53±18% (n=5) of the arterial
level (per milliliter perfusate). Since the
angiotensinogen levels in CE and IST were not different
from the arterial level, and since in a different series of
experiments the cardiac extracellular fluid space was found to be 0.61
mL/g (mean, n=6; see Table 2), the cardiac tissue level of
angiotensinogen is expected to be 0.61x100=61% (mean) of
the arterial level, assuming that
angiotensinogen is located in the extracellular fluid
compartment and that 1 g tissue corresponds with a volume of 1
mL.6 22 This calculated value is similar to the
angiotensinogen level that was actually measured in cardiac
tissue (53% of the arterial level). Thus, as with renin,
the cardiac tissue level of angiotensinogen is
consistent with its location in the extracellular fluid at a
concentration similar to that of CE. Angiotensinogen was
not detectable in control hearts perfused with Tyrode's buffer
(n=4).
Ang I
During perfusion with Ang I (n=5), the steady-state Ang I level in
CE was 60% of the arterial level (mean, P<.01
for difference from the arterial level; see Table 3). Thus,
40% of the arterially delivered Ang I was removed from the
perfusate by the heart. A steady-state level of Ang I in IST
was reached within 5 minutes (Fig 1). It was 17% of the
arterial level, which was significantly lower than the
level in CE (P<.01). After discontinuation of the Ang I
perfusion, Ang I disappeared rapidly from CE in a monophasic way (Fig 2), with a t1/2 less than 0.5 minute. Ang I disappeared
from IST, also in a monophasic way, with a t1/2 of 0.9±0.6
minute. Ang I added to samples of CE and IST after they had been
collected from control hearts perfused with Tyrode's buffer (n=2) had
a half-life longer than 40 minutes at 37°C (in the absence of the
mixture of ACE and angiotensinase inhibitors
routinely used during CE and IST collection). Thus, the rapid
disappearance of Ang I from IST observed in the Ang I perfusion
experiments was not caused by the presence of peptidases in IST but by
the rapid removal of Ang I from the cardiac ISF.
The cardiac tissue level of Ang I (per gram tissue), immediately
after the Ang I perfusion had been switched off, was less than 5%
(n=3) of the arterial level (per milliliter
perfusate). The Ang I levels in CE and IST were 60% and 17%
of the arterial level, respectively, whereas in a different
series of experiments, the intravascular and ISF spaces were 0.38 and
0.23 mL/g, respectively (see Table 2). Thus, the cardiac tissue level
of Ang I is expected to be (0.38x60)+(0.23x17)=27% of the
arterial level, assuming that Ang I is located in the
extracellular fluid compartment and that 1 g tissue corresponds
with a volume of 1 mL.6 22 The difference from the
measured result might be related to the rapid degradation of Ang I in
the vascular compartment. It is possible that the short period between
the moment the Ang I perfusion was stopped and the moment the tissue
was transferred into liquid nitrogen was long enough for the
endothelial peptidases to cause a loss of most of the
Ang I. Ang I was not detectable in control hearts perfused with
Tyrode's buffer (n=3).
Cardiac Production of Ang I
Results obtained during perfusion with rat renin (n=4), in the
absence of exogenous angiotensinogen, are shown in Fig 3. Ang I was not detectable in CE and IST samples
collected before renin was infused into the perfusion system. In
samples collected during renin perfusion, Ang I remained undetectable
in CE, whereas in IST Ang I rose to levels above the detection limit of
the assay. After an initial increase, Ang I in IST decreased, despite
the continuous perfusion with renin. This may be due to the washout of
endogenous (rat) angiotensinogen during the
course of the experiment.
|
Ang I was not detectable in CE and IST samples collected during perfusion with rat, porcine, or sheep angiotensinogen (n=3). Thus, there was no evidence for cardiac Ang I production by endogenous (rat) renin or reninlike enzymes (cathepsins).
Ang I was easily detectable in samples of CE and IST collected during
perfusion with porcine renin combined with porcine or sheep
angiotensinogen (Fig 4 and Tables 4 and 5). During combined renin and
angiotensinogen perfusion, the level of Ang I in CE had
reached its steady-state maximum in the 30- to 40-minute and 45- to
55-minute samples but not in the 0- to 10-minute and 15- to 25-minute
samples. This contrasts with the levels of renin and
angiotensinogen, which had reached their steady-state
maximum in CE within less than 5 minutes (see Fig 1). The slow increase
of Ang I in CE, as compared with the rapid increase of renin and
angiotensinogen, is an indication that the
renin-angiotensinogen reaction in the fluid phase of the
intravascular compartment was not the only source of Ang I in CE. Also
in IST, Ang I had reached its steady-state maximum in the 30- to
40-minute and 45- to 55-minute samples but not in the 0- to 10-minute
and 15- to 25-minute samples; this corresponds well with the time
required for renin and angiotensinogen to reach their
steady-state maximum in IST (see Fig 1).
|
|
|
Figs 5 and 6 give the measured Ang
IISF and Ang ICE production rates as
derived from Equations 1, and 5 (see "Calculations" above) for the
30- to 40-minute and 45- to 55-minute samples. The measured
production rates are compared with the production rates
predicted by Equations 3, and 7, which are based on the assumption that
the renin-angiotensinogen reaction in the intravascular and
interstitial compartments is occurring in the fluid phase
only. The renin and angiotensinogen levels that were
entered into these calculations are shown in Tables 4 and 5.
|
|
As shown in Figs 5 and 6, the production of Ang
IISF could be accounted for by the
renin-angiotensinogen reaction in the fluid phase of the
interstitial compartment. The production of Ang
ICE, however, was too high to be accounted for by the
renin-angiotensinogen reaction in the fluid phase of the
intravascular compartment. In the porcine renin/porcine
angiotensinogen perfusion experiments, the measured Ang
ICE production rate was 4.6±2.2 times (average of
the two samples collected at 30 to 40 minutes and 45 to 55 minutes of
perfusion, P<.01 for difference from 1.0; n=6) the rate
predicted on the basis of the renin-angiotensinogen
reaction in the fluid phase. In the porcine renin/sheep
angiotensinogen perfusion experiments, the discrepancy was
even greater, the measured production rate being 7.1 (4.1 to
10.1) times (mean value and range, n=3) the predicted rate.
The measured production rate of Ang ICE may be
somewhat higher than the true production rate because the
measured production rate accounts for the extraction of
arterially delivered Ang I, whereas during combined renin
and angiotensinogen perfusion, Ang I is formed in the
perfusate during its passage through the coronary
vascular bed. Fig 7, therefore, gives the net ejection
rate of Ang I via CE, as derived from Equation 6, which is less than
the total production of Ang ICE. It can be seen
that even with this underestimation of the true Ang ICE
production rate, results were higher than predicted on the
basis of the renin-angiotensinogen reaction in the fluid
phase. The measured net ejection rate of Ang I was 2.8±1.3 times
(P<.05 for difference from 1.0, n=6) the predicted rate in
the porcine renin/porcine angiotensinogen perfusion
experiments, and 4.3 (2.5 to 6.1) times (mean value and range, n=3) the
predicted rate in the porcine renin/sheep angiotensinogen
experiments. Therefore, it appears that part of the Ang I in CE is
produced at tissue sites. The Ang IISF production
rate was much lower than the Ang ICE production
rate. The contribution of Ang I from the ISF to the Ang I level in CE
was therefore minimal.
|
|
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study of the cardiac uptake and production of RAS components made use of a modified Langendorff heart model that was perfused with an albumin-free buffer solution under normoxic conditions. In this model, the CE is ejected by the right ventricle via the cannulated pulmonary artery, and a small amount (0.7% to 2%) of the infusion fluid entering the coronary arteries passes through the vascular wall, seeps through the heart tissue, and reaches the epicardial surface. This transudate fluid, which is referred to in this article as IST, keeps dripping from the apex, and we assumed the levels of renin, angiotensinogen, and Ang I in this fluid to be representative of the levels in the cardiac ISF.
There is indeed strong evidence to support this assumption. The protein concentration in IST is much higher than in the CE.10 Creatine kinase, lactate dehydrogenase, and malate dehydrogenase activity levels are about 100 times higher in IST than CE.19 Glucose is taken up by the heart primarily from the ISF, whereas lactate is primarily released into this fluid rather than into CE, and this is reflected by the glucose and lactate concentrations in IST and CE.10 The levels of renin and angiotensinogen that were reached in IST during infusions of these RAS components into the perfusion system were similar to the levels in CE. Using a Langendorff heart preparation somewhat different from ours, Wienen and Kammermeier19 found that during perfusion with dextran T-70 (molecular weight [MW] 70 kD), the dextran level in IST was equal to that in CE; the same observation was made for albumin (MW 70 kD) during perfusion with 0.01% bovine serum albumin. In the experimental setup Wienen and Kammermeier used,19 IST was collected by slight suction under a latex cap over the ventricles, thereby minimizing the risk of evaporation. In view of the agreement between the results obtained by these authors and the results obtained in our experiments, it is safe to conclude that evaporation had a minimal effect on the measured concentrations of RAS components in IST. The half-life of Ang I in IST in the collection tube was much longer than the half-life in the IST drop while it was still on the cardiac surface. Therefore, the rapid washout of Ang I from the IST, while it was still on the cardiac surface (as observed after the discontinuation of Ang I perfusion), reflects the rapid disappearance of Ang I from the ISF in cardiac tissue. All of these observations, taken together, support the view that the composition of IST indeed reflects the composition of the cardiac ISF. The levels of exogenous arterially delivered Ang I are probably similar in the two fluid compartments, as are the levels of endogenous Ang I formed in the Langendorff preparation. This is indicated by our finding that the Ang I level measured in IST in the combined renin and angiotensinogen perfusion experiments was not different from the level predicted on the basis of renin-angiotensinogen reaction in the fluid phase of the interstitial compartment.
In our renin and angiotensinogen perfusion experiments, the release of renin (MW 48 kD) and angiotensinogen (MW 65 kD) into the IST was slow compared with the release of Ang I into the IST during Ang I perfusion. This is probably related to the much smaller molecular size of Ang I (MW 1.297 kD). Similar observations have been published with respect to dextran T-70 (MW 70 kD) and disulfin blue (MW 0.566 kD).19 It should be noted that in order to obtain sufficient amounts of IST for Ang I measurement, we did not add serum or serum albumin to the perfusion fluid. Albumin, however, reduces not only the movement of water and low-molecular-weight solutes from the perfusate into the IST but also the transport of other proteins, including albumin itself.23 Our results may therefore quantitatively differ from the situation in vivo.
Our measurements of renin and angiotensinogen in IST and CE, together with measurements of the tissue levels during perfusion with these RAS components, indicate that most of the renin and angiotensinogen was localized in the extracellular fluid compartment. Some of the infused renin may bind to the cell surface or may have been taken up by the cells. Cardiac membrane fractions contain renin,6 and binding of renin to vascular membranes has been reported.24 Cellular uptake of renin followed by intracellular proteolytic destruction may explain why, during perfusion with renin, the renin level in CE was lower than the arterial level.
In our perfusion experiments, approximately 40% of the infused Ang I was removed from the perfusate by the heart. This is in accordance with studies in intact pigs which demonstrated that in the coronary vascular bed, 45% to 50% of the arterially delivered Ang I was removed from the circulation,25 most likely by peptidases on the endothelial surface.
An important aspect of the present study is the evidence it provides for local Ang I formation by the heart, outside the perfusate compartment. According to our calculations, the level of Ang I in IST during combined renin and angiotensinogen perfusion can be explained by the renin-angiotensinogen reaction in the fluid phase of the interstitial compartment. In contrast, most of the Ang I in CE was not formed by the renin-angiotensinogen reaction in the fluid phase of the intravascular compartment. Ang I from the intravascular compartment contributed little to the Ang I level in IST, and Ang I from the cardiac ISF contributed little to the Ang I level in CE. Most of the Ang I in CE appears to be formed at tissue sites, and the direction of Ang I release from these sites seems to be toward the intravascular compartment rather than the interstitial compartment. We assume, therefore, that these sites are close to the luminal surface of the blood vessel wall and represent blood vessel wall–bound renin, as suggested by Swales and Thurston26 more than 20 years ago. The finding that the Ang I level in CE was not higher than in IST does not argue against this conclusion, because Ang I, after its release into the intravascular fluid, is rapidly washed away by the flow of perfusate. The present study extends earlier observation in intact pigs, which suggested that the Ang I concentration in coronary venous plasma was too high to be explained by the plasma renin activity.9 With the use of biochemical and immunohistochemical methods, renin has been demonstrated in the endothelial cells of normal human gastroepiploic arteries.27 In our experiments, renin disappeared from the coronary effluent in a biphasic manner after the renin perfusion had been stopped. The slow second phase of the disappearance curve may correspond with the endothelial compartment. Such a slow second phase was not observed in the disappearance curve of angiotensinogen.
Fig 8 presents a hypothetical scheme that is
compatible with our observations. It shows two sites of Ang I formation
outside the perfusion fluid, namely, the vascular surface and the ISF.
Some renin and also the peptidases involved in Ang I
metabolism are bound to the vascular
endothelial cells. Angiotensinogen and
Ang I are present in the fluid phase. Most of the Ang I
present in CE is produced by endothelium-bound
renin. Most of the Ang I present in the ISF compartment is produced
by renin present in the fluid phase of this compartment.
|
The local formation of Ang I at cardiac tissue sites depends on arterially delivered renin. In our perfusion experiments, Ang I was undetectable in CE and IST collected after 30 minutes of perfusion with Tyrode's buffer, before the infusion of renin into the perfusion system had been started. Also, CE collected from the classic Langendorff heart model did not contain Ang I, unless renin had been added to the perfusion fluid.7 Conclusive evidence that cardiac Ang I production depends on blood-derived renin comes from our studies of the effect of nephrectomy on the cardiac tissue levels of Ang I and II in pigs.6 Both peptides became undetectable in cardiac tissue after bilateral nephrectomy. In the present study, Ang I levels in IST and CE were much higher during combined renin and angiotensinogen perfusion than during perfusion with renin alone. It is therefore reasonable to conclude that Ang I production by the heart not only depends on arterially delivered renin but also on arterially delivered angiotensinogen.
The production of Ang I at cardiac tissue sites may, via conversion of Ang I to Ang II, lead to local concentrations of Ang II that are higher than can be obtained with arterially delivered Ang II. That the local formation of Ang I is of physiological importance is suggested by our recent observations in intact pigs on the effect of intracoronary administration of a specific renin inhibitor on cardiac contractility.28 The inhibitor reduced cardiac contractility, whereas the time course of this effect was not correlated with the effect of the inhibitor on the circulating levels of Ang I and II. In addition to its short-term effect on cardiac contractility, Ang II also has long-term effects; it promotes left ventricular hypertrophy and the remodeling that occurs after myocardial infarction.1 2 3 Ang I produced locally in the heart may, after its conversion to Ang II, participate in these processes. This is in keeping with the view that the long-term beneficial effects of ACE inhibitors in left ventricular hypertrophy and heart failure are determined not only by the decrease in circulating Ang II but also by a decrease in the conversion of locally formed Ang I.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This work was supported by the Netherlands Heart Foundation, Research Grant 91.121.
Received May 6, 1996; first decision May 27, 1996; accepted November 5, 1996.
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J. J. Saris, F. H. M. Derkx, J. M. J. Lamers, P. R. Saxena, M. A. D. H. Schalekamp, and A. H. J. Danser Cardiomyocytes Bind and Activate Native Human Prorenin : Role of Soluble Mannose 6-Phosphate Receptors Hypertension, February 1, 2001; 37(2): 710 - 715. [Abstract] [Full Text] [PDF]
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 G. PRESCOTT, D. W. SILVERSIDES, S. M. L. CHIU, and T. L. REUDELHUBER Contribution of circulating renin to local synthesis of angiotensin peptides in the heart Physiol Genomics, November 9, 2000; 4(1): 67 - 73. [Abstract] [Full Text] [PDF]
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W. C. De Mello and A. H. J. Danser Angiotensin II and the Heart : On the Intracrine Renin-Angiotensin System Hypertension, June 1, 2000; 35(6): 1183 - 1188. [Abstract] [Full Text] [PDF]
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S. Engeli, R. Negrel, and A. M. Sharma Physiology and Pathophysiology of the Adipose Tissue Renin-Angiotensin System Hypertension, June 1, 2000; 35(6): 1270 - 1277. [Abstract] [Full Text] [PDF]
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P. C. Almeida, V. Oliveira, J. R. Chagas, M. Meldal, M. A. Juliano, and L. Juliano Hydrolysis by Cathepsin B of Fluorescent Peptides Derived From Human Prorenin Hypertension, June 1, 2000; 35(6): 1278 - 1283. [Abstract] [Full Text] [PDF]
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 S. A. Katz, J. A. Opsahl, S. E. Wernsing, L. M. Forbis, J. Smith, and L. J. Heller Myocardial renin is neither necessary nor sufficient to initiate or maintain ventricular hypertrophy Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2000; 278(3): R578 - R586. [Abstract] [Full Text] [PDF]
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J. J. Saris, M. A. van Dijk, I. Kroon, M. A. D. H. Schalekamp, and A. H. J. Danser Functional Importance of Angiotensin-Converting Enzyme-Dependent In Situ Angiotensin II Generation in the Human Forearm Hypertension, March 1, 2000; 35(3): 764 - 768. [Abstract] [Full Text] [PDF]
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 A.H.J. Danser, J. J Saris, M. P Schuijt, and J. P van Kats Is there a local renin--angiotensin system in the heart? Cardiovasc Res, November 1, 1999; 44(2): 252 - 265. [Abstract] [Full Text] [PDF]
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K. C Wollert and H. Drexler The renin-angiotensin system and experimental heart failure Cardiovasc Res, September 1, 1999; 43(4): 838 - 849. [Abstract] [Full Text] [PDF]
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C. A.M van Kesteren, J. J Saris, D. H.W Dekkers, J. M.J Lamers, P. R Saxena, M. A.D.H Schalekamp, and A.H.J. Danser Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen: evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II Cardiovasc Res, July 1, 1999; 43(1): 148 - 156. [Abstract] [Full Text] [PDF]
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N. Sambandam, M. A. Abrahani, E. St. Pierre, O. Al-Atar, M. C. Cam, and B. Rodrigues Localization of Lipoprotein Lipase in the Diabetic Heart : Regulation by Acute Changes in Insulin Arterioscler Thromb Vasc Biol, June 1, 1999; 19(6): 1526 - 1534. [Abstract] [Full Text] [PDF]
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A. T. Hirsch, J. A. Opsahl, M. M. Lunzer, and S. A. Katz Active renin and angiotensinogen in cardiac interstitial fluid after myocardial infarction Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H1818 - H1826. [Abstract] [Full Text] [PDF]
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C.-C. Wei, Q. C. Meng, R. Palmer, G. R. Hageman, J. Durand, W. E. Bradley, D. M. Farrell, G. H. Hankes, S. Oparil, and L. J. Dell'Italia Evidence for Angiotensin-Converting Enzyme– and Chymase-Mediated Angiotensin II Formation in the Interstitial Fluid Space of the Dog Heart In Vivo Circulation, May 18, 1999; 99(19): 2583 - 2589. [Abstract] [Full Text] [PDF]
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 A. Virdis, L. Ghiadoni, M. Marzilli, E. Orsini, S. Favilla, P. Duranti, S. Taddei, P. Marraccini, and A. Salvetti Adenosine causes the release of active renin and angiotensin II in the coronary circulation of patients with essential hypertension J. Am. Coll. Cardiol., May 1, 1999; 33(6): 1677 - 1684. [Abstract] [Full Text] [PDF]
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D. N. Muller, K. F. Hilgers, S. Mathews, V. Breu, W. Fischli, R. Uhlmann, and F. C. Luft Effects of Human Prorenin in Rats Transgenic for Human Angiotensinogen Hypertension, January 1, 1999; 33(1): 312 - 317. [Abstract] [Full Text] [PDF]
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J. P. van Kats, A. H. J. Danser, J. R. van Meegen, L. M. A. Sassen, P. D. Verdouw, and M. A. D. H. Schalekamp Angiotensin Production by the Heart : A Quantitative Study in Pigs With the Use of Radiolabeled Angiotensin Infusions Circulation, July 7, 1998; 98(1): 73 - 81. [Abstract] [Full Text] [PDF]
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L. M. de Lannoy, A.H. J. Danser, A. M.B. Bouhuizen, P. R. Saxena, and M. A.D.H. Schalekamp Localization and Production of Angiotensin II in the Isolated Perfused Rat Heart Hypertension, May 1, 1998; 31(5): 1111 - 1117. [Abstract] [Full Text] [PDF]
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 D. N. Muller, W. Fischli, J.-P. Clozel, K. F. Hilgers, J. Bohlender, J. Menard, A. Busjahn, D. Ganten, and F. C. Luft Local Angiotensin II Generation in the Rat Heart : Role of Renin Uptake Circ. Res., January 23, 1998; 82(1): 13 - 20. [Abstract] [Full Text] [PDF]
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K. F. Hilgers, E. Bingener, C. Stumpf, D. N. Muller, R. E. Schmieder, and R. Veelken Angiotensinases Restrict Locally Generated Angiotensin II to the Blood Vessel Wall Hypertension, January 1, 1998; 31(1): 368 - 372. [Abstract] [Full Text] [PDF]
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C. A. M. van Kesteren, A. H. J. Danser, F. H. M. Derkx, D. H. W. Dekkers, J. M. J. Lamers, P. R. Saxena, and M. A. D. H. Schalekamp Mannose 6-Phosphate Receptor–Mediated Internalization and Activation of Prorenin by Cardiac Cells Hypertension, December 1, 1997; 30(6): 1389 - 1396. [Abstract] [Full Text]
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