Angiotensin II receptors and renin release in rat glomerular afferent arterioles. The purpose of recent studies was to investigate the expression of angiotensin II (Ang II) receptor sites in afferent arterioles freshly isolated from the rat kidney, and the role of Ang II on renin release by these vessels. The method of isolation and purification of renal microves-sels was based on iron oxide infusion into the kidneys and separation of the afferent arterioles from glomeruli and connective tissue with the aid of a magnetic field, successive passages through various sieves, and harvesting with collagenase. Ang II receptor characteristics were evaluated by radioligand binding studies using the non-peptide Ang II antagonists of AT1 (Dup-753 and -532) and AT2 (PD-123319 and CGP-42112) receptors. AT1 antagonists displaced up to 80% of the Ang II binding with high affinity (3 nM), whereas the remaining 20% showed low affinity for the Dup compounds and CGP-42112 (>10 µM), and intermediate affinity for PD-123319 (12 µM). These data suggest the existence of two Ang II receptor subtypes in the renal vasculature of the rat. In separate experiments, renin release by isolated afferent arterioles in vitro was 9 ng/hr/mg under control conditions. Ang II (0.1 µM) inhibited renin secretion by 20%, whereas the adenylyl cyclase activator forskolin (10 µM) stimulated renin secretion by 50%. In arterioles isolated from rats chronically treated with a converting enzyme inhibitor (perindoprilate) to reduce endogenous formation of Ang II, renin release increased 20-fold under control conditions in vitro and was further stimulated by forskolin. These results demonstrate that this preparation is a useful tool to study the functional role of Ang II and the control of renin release in the afferent arterioles

Ardaillou, Raymond

Arendshorst, William

Chatziantoniou, Christos

Dussaule, Jean-Claude

Angiotensin II receptors and renin release in rat glomerular afferent arterioles. The purpose of recent studies was to investigate the expression of angiotensin II (Ang II) receptor sites in afferent arterioles freshly isolated from the rat kidney, and the role of Ang II on renin release by these vessels. The method of isolation and purification of renal microves-sels was based on iron oxide infusion into the kidneys and separation of the afferent arterioles from glomeruli and connective tissue with the aid of a magnetic field, successive passages through various sieves, and harvesting with collagenase. Ang II receptor characteristics were evaluated by radioligand binding studies using the non-peptide Ang II antagonists of AT1 (Dup-753 and -532) and AT2 (PD-123319 and CGP-42112) receptors. AT1 antagonists displaced up to 80% of the Ang II binding with high affinity (3nM), whereas the remaining 20% showed low affinity for the Dup compounds and CGP-42112 (>10 µM), and intermediate affinity for PD-123319 (12 µM). These data suggest the existence of two Ang II receptor subtypes in the renal vasculature of the rat. In separate experiments, renin release by isolated afferent arterioles in vitro was 9 ng/hr/mg under control conditions. Ang II (0.1 µM) inhibited renin secretion by 20%, whereas the adenylyl cyclase activator forskolin (10 µM) stimulated renin secretion by 50%. In arterioles isolated from rats chronically treated with a converting enzyme inhibitor (perindoprilate) to reduce endogenous formation of Ang II, renin release increased 20-fold under control conditions in vitro and was further stimulated by forskolin. These results demonstrate that this preparation is a useful tool to study the functional role of Ang II and the control of renin release in the afferent arterioles

Arendshorst, William J.

Elsevier - Publisher Connector 

Kidney International, Vol. 46 (1994), pp. 1570—1573
Angiotensin II receptors and renin release in rat glomerular
afferent arterioles
CHRISTOS CRkTZIANT0NI0u, JEAN-CLAUDE DUSSAULE, WILLIAM J. ARENDSHORST,
and RAYMOND ARDAILLOU
INSER.M U.64, Tenon Hospita4 Paris, France, and Department of Physiology, University of North Carolina, Chapel Hi14 North Carolina, USA
Angiotensin H receptors and renin release in rat glomerular afferent
arterioles. The purpose of recent studies was to investigate the expression
of angiotensin II (Ang II) receptor sites in afferent arterioles freshly
isolated from the rat kidney, and the role of Ang II on renin release by
these vessels. The method of isolation and purification of renal microves-
sels was based on iron oxide infusion into the kidneys and separation of
the afferent arterioles from glomeruli and connective tissue with the aid of
a magnetic field, successive passages through various sieves, and harvest-
ing with collagenase. Ang II receptor characteristics were evaluated by
radioligand binding studies using the non-peptide Ang II antagonists of
AT1 (Dup-753 and -532) and AT2 (PD-123319 and CGP-42112) receptors.
AT1 antagonists displaced up to 80% of the Ang II binding with high
affinity (3 nM), whereas the remaining 20% showed low affinity for the Dup
compounds and CGP-42112 (>10 .tM), and intermediate affinity for
PD-123319 (12 gM), These data suggest the existence of two Ang II
receptor subtypes in the renal vasculature of the rat. In separate experi-
ments, renin release by isolated afferent arterioles in vitro was 9 ng/hr/mg
under control conditions. Ang 11(0.1 jiM) inhibited renin secretion by
20%, whereas the adenylyl cyclase activator forskolin (10 jiM) stimulated
renin secretion by 50%. In arterioles isolated from rats chronically treated
with a converting enzyme inhibitor (perindoprilate) to reduce endogenous
formation of Ang II, renin release increased 20-fold under control
conditions in vitro and was further stimulated by forskolin. These results
demonstrate that this preparation is a useful tool to study the functional
role of Mg II and the control of renin release in the afferent arterioles.
The renin-angiotensin system (RAS) plays a major role in the
renal function. Although several investigations attempted to
localize the exact sites of angiotensin II (Ang II) action on the
renal vasculature, controversy still remains on whether Ang II can
act on the preglomerular vessels [1]. Micropuncture and clearance
techniques and studies on microdissected vessels suggest that Ang
II constricts preferentially the efferent arteriole [2, 3] On the
other hand, data obtained from in vitro visualization models
(juxtaglomerular perfused nephron, hydronephrotic kidney) sup-
port the view that Ang II cnn act on afferent arterioles [4, 5].
Previous studies from our laboratories indirectly supported the
latter hypothesis. Ang II receptors have been identified on
isolated glomeruli and cultured mesangial cells, a structure that is
thought to share several common characteristics with preglomeru-
lar vascular smooth muscle cells [6, 7]. Furthermore, injection of
Ang II directly into the renal artery decreased renal blood flow by
© 1994 by the International Society of Nephrology
50 to 60% [8]. Renal vasoconstriction of this magnitude is difficult
to be achieved without participation of preglomerular vessels.
The purpose of the present study was to provide convincing
evidence demonstrating the existence of functional receptor sites
of Ang II in the preglomerular vasculature. For this reason we
developed a technique to isolate in vitro pure and viable prepa-
rations of preglomerular vessels from the rat kidney, and we
subsequently studied the regulation and expression of the renin-
angiotensin system in these vessels.
Methods
Isolation of afferent arterioles
The technique to isolate afferent arterioles from the rat kidney
was the same as previously described [9]. Briefly, a magnetized
iron oxide suspension (1% Fe3O4 in isotonic saline) was perfused
into the renal arteries of seven-week-old Wistar-Kyoto (receptor
binding studies) or Sprague Dawley (renin release studies) rats (6
rats per experiment) (Fig. 1A). Thereafter the kidneys were
removed, decapsulated, and the cortex was dissected from the
medulla. Cortical tissue was homogenized with a Polytron homog-
enizer, and the iron oxide-loaded tissues (renal vessels and
glomeruli) were removed from the crude homogenate with the aid
of a magnet (Fig. 1B). Afferent arterioles were separated from
larger vessels and glomeruli using repetitive passages through
needles and sieves of decreasing sizes (20 to 23 gauge and 150 to
90 jxm, respectively). The microvessels were recovered from the
top of the 125 jim sieve and harvested with collagenase (55 U/mI)
for 30 minutes at 37°C to remove the surrounding connective
tissue. Only vascular preparations containing >90% of afferent
arterioles were retained for subsequent experiments.
Receptor binding studies
Aliquots of microvessels (40 j.tg of protein) were incubated with
increasing concentrations of 3H-Ang II as previously described
[9]. Nonspecific binding was determined in the presence of an
excess of unlabeled Ang 11(10 jim). In competitive inhibition
studies, the AT1 receptor antagonists Dup-753 and Dup-532
(DuPont) and the AT2 receptor antagonists PD-123319 (Parke-
Davis) and CGP-42112 (Ciba-Geigy) were used to displace 3H-
Mg II. Analyses of the data using the LIGAND program gave
estimates of the maximum specific binding (Bmj and dissocation
constant (Kd).
1570
4/t*j -
-4
Longitudinal section of a kidney after
'perfusion. B. Representative example
rent arteriole attached to a
us. Note that iron oxide particles do
letely fill glomerular capillaries.
Renin release evaluation
Aliquots of microvessels (150 to 200 tg of protein) were
incubated at 37°C in a final volume of 1 ml DMEM/F12 (1:1)
medium containing 1 mcvi Ca2. Incubations lasted one and three
hours in the absence or presence of 10 .tM of forskolin or 0.1 iM
of Ang II. Renin release was measured by radioimmunoassay as
the amount of Ang I produced by incubation of supernatants with
an excess of rat renin substrate (plasma of binephrectomized rats)
Chatziantoniou et al: RAS in afferent arterioles 1571
1572 Chatziantoniou et al: RAS in afferent arterioles
Renin release was expressed in nanograms of Ang I gener-
ated per hour of incubation per milligram of protein of afferent
arterioles.
Angiotensin converting enzyme (ACE) inhibition
In separate experiments, rats were treated with a converting-
enzyme inhibitor (perindoprilate, 40 mg/kg/day) for five days to
eliminate endogenous Ang II. Isolation of afferent arterioles and
renin release assay were performed as described above.
Results
Receptor binding studies
The properties of Ang II receptor sites were characterized in
afferent arterioles from seven-week-old rats using equilibrium
radiolabeled techniques. Scatchard analysis revealed one class of
high affinity receptors (Kd = 1 flM) with a density of 200 fmollmg
of protein.
To identif' the Ang II subtype(s) expressed in these renal
vessels, the AT1 antagonists Dup-753 and Dup-532, and the AT2
antagonists PD-123319 and CGP-42112 were used to displace
unlabeled Ang II. The AT1 antagonists displaced almost com-
pletely the Ang II binding. Analysis of the displacement curve by
the LIGAND program indicated that a two-site model produced
a significantly better fit than the one-site model (P < 0.00 1). In the
two-site model, Dup-753 binds to one site with high affinity (3 nM),
whereas it displays 500-fold lower affinity for the other site (1.5
tkM; (Fig. 2A). The high-affinity site is the most abundant and
occupies 80% of the total Ang II receptor sites. The low-affinity
Ang II site occupies the remaining 20% of the total receptor sites.
The putative AT2 antagonist PD-123319 displaced up to 25% of
the Ang II binding, with a rather medium affinity (100 nM; Fig.
2B). The other AT2 antagonist, CGP-42112, showed very low
affinity to the Ang II receptor sites (> 10 tM).
To determine whether or not the high affinity site that Dup-753
bound to is distinct from the medium affinity site recognized by
PD-123319, competitive inhibition curves were performed by
adding a standard concentration of PD-123319 (1 jiM) to increas-
ing concentrations of Dup-753, and alternatively, by adding a
standard concentration of Dup-753 (10 nM) to increasing concen-
trations of PD-123319. As shown in Figure 2A, PD-123319
provided an additive displacement (15 to 20%) that was uniform
in all tested concentrations of Dup-753. Similarly, Dup-753 dis-
placed an additional 20 to 25% of the sites in all PD-123319 tested
concentrations (Fig. 2B).
Renin release studies
The viability of the afferent arterioles was checked by measure-
ments of creatine kinase activity (CKA) in the medium. No
changes in the release of CKA were observed between 0 and 6
hours after isolation of the vessels. On the contrary, CKA was
increased eightfold at 24 hours compared to 0 hours. For this
reason, renin release measurements were performed at 1 and 3
hours after isolation of afferent arterioles. During this period, the
rate of secretion of renin was increased significantly with time.
After 1 hour renin release in supernatants was 10 2 ng/hr/mg,
and after 3 hours it was 22 2 ng/hr/mg (Table 1).
To investigate whether renin release can be regulated in our
vascular preparation in vitro, we used forskolin and Ang II, two
agents that are known to stimulate and inhibit renin release,
Normal Perindoprilate
lhr 3hr lhr 3hr
Control 10 2 22 2 153 16 242 19
Forskolin 15 2 34 4 217 16 394 42
Ang II 8 1 15 2 125 10 160 14
Values are expressed in nglhr/mg and are means SE of 4 independent
experiments.
P < 0.05 vs. control
respectively. Addition of forskoljn (10 jiM) increased renin release
by 40% after 1 hour and 50% after 3 hours compared to control
values. In contrast, when exogenous Ang 11(0.1 jiM)was added,
renin release by afferent arterioles was decreased by 35% at 3
hours (Table 1).
To examine if in vivo regulation of the expression of the
A
120
100
80
60
40
20
0
10_il lO_lO 10-s 10-8 10 10-6 10 10
DUP 753, M
B
110
100
90
80
70
60
VC
0
.0
0)CI
C
0
.0
0)CI
Fig. 2. Displacement curves of 3H-Ang II binding to preglomerular vessels
by increasing concentrations of the AT2 antagonist Dup 753 in absence and
presence of the AT2 antagonist PD 123319 (1 jiM) (A) and inversely, by
increasing concentrations of PD 123319 in absence and presence of Dup 753
(10 nM) (B). Values are means SE for 4 experiments of each condition.
Table 1. Renin release from afferent arterioles freshly isolated from
normal and perindoprilate-treated rats
50
10_il 10-s 10-8 10-i 10-8 10-s 10-i
PD 123319,
Chatziantoniou et al: RAS in afferent arterioles 1573
renin-angiotensin system can induce changes in renin release that
can be detected in isolated afferent arterioles, a group of rats was
treated chronically with a converting enzyme inhibitor (perindo-
prilate) to reduce production of endogenous Ang IL Afferent
arterioles from rats treated with perindoprilate were found to
release renin at a rate 15-fold higher than control. Addition of
forskolin further increased renin release (50 and 70% above
control at 1 and 3 hr, respectively), whereas addition of exogenous
Ang II inhibited renin release (30% below control at 3 hr).
Discussion
In the present studies, we isolated afferent arterioles of high
purity, and in sufficient quantity to perform several biochemical
studies such as receptor characterization or responses to a variety
of stimuli. This vascular preparation is viable in vitro, and it was
used to study Ang II receptor sites in biochemical (binding
characteristics) and functional (renin release regulation) terms.
The use of nonpeptide antagonists of Ang II indicated the
existence of two subtypes of Ang II receptors in the afferent
arterioles of rats. One site appears to be a typical vascular AT1
subtype since it displays high affinity to the AT1 antagonists
Dup-753 and Dup-532 and very low affinity to the AT2 antagonists
PD-123319 and CGP-42112. This subtype is predominant and
occupies 80% of the total number of Ang II receptor sites. The
other receptor subtype displays intermediate affinity to the AT2
antagonist PD-123319 and low affinity to AT1 antagonists. How-
ever, it does not appear to be the AT2 subtype found in the
adrenal gland, because it shows low affinity to CGP-421 12. Several
recent studies using molecular biology techniques indicated the
expression of two AT1 subtypes (designated as ATia and AT1b) in
the rat renal kidney [11, 121. Of particular interest, use of
quantitative PCR suggested that the relative ratio of the AT1,,
versus ATIb subtype in the renal cortex was 4:1 [12].
The characterization of Ang II receptor sites in freshly isolated
afferent arterioles does not necessarily mean that these sites are
functional. To verify the adequacy of this vascular preparation for
functional studies, we investigated the responsiveness of the
afferent arterioles to agents that control the expression of the
renin-angiotensin system, such as Ang II and forskolin. In control
rats, renin release was increased with time, it was inhibited by
exogenous Ang II, and it was stimulated by forskolin. It would be
interesting to investigate whether the two Ang II receptor sub-
types found in our preparation influence renin release differently.
In rats chronically treated with an ACE inhibitor, baseline renin
release was increased 15-fold. Such a large increase in renin
release is in agreement with studies indicating that in response to
ACE inhibition vascular smooth muscle cells of the renal vascu-
lature from afferent arteriole up to interlobar and arcuate arteries
express and release renin to a rate several times higher than the
normal [131. Interestingly, in the perindoprilate-treated rats,
isolated afferent arterioles continued to be responsive to exoge-
nous stimuli since renin release was increased or decreased
following exposure to forskolin or Ang II, respectively (Table 1).
In conclusion, we have described a reproducible technique to
isolate from the rat kidney afferent arterioles of high purity and in
sufficient quantities to perform biochemical studies such as Ang II
receptor characterization and regulation. The metabolic respon-
siveness of our vascular preparation to mediators of Ang II
receptor expression and renin release suggests that this model is a
useful tool to study the autocrine and paracrine regulation of the
renin-angiotensin system at the cellular level.
Acknowledgments
This work was financially supported by the "Institut National de Ia Sante
et de Ia Recherche Medicale" and the "Faculté de Médecine St Antoine",
and by a grant from the National Heart, Lung, and Blood Institute
(HL-02334). Dr. C. Chatziantoniou is recipient of a European Commu-
nities fellowship (Human Capital and Mobility).
Reprint requests to Pr. Raymond Ardaillou, INSERM U.64, Tenon Hos-
pital, 4 rue de Ia Chine, 75020 Paris, France.
References
1. NAVAR LG: Physiological role of the intrarenal renin-angiotensin
system. Fed Proc 45:1411—1413, 1986
2. EDWARDS RM: Segmental effects of norepinephrine and angiotensin
II on isolated renal microvessels. Am J Physiol 244 (Renal Fluid
Electrol Physiol 13):F526—F534, 1983
3. I-L&u. JE: Regulation of glomerular filtration rate and sodium excre-
tion by angiotensin II. Fed Proc 45:1431—1437, 1986
4. CARMINES PK, MORRISON TK, NAVAR LG: Angiotensin II effects on
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5. LOUTZENHISER R, EPSiruN M, HAYASHI K, TAKENAKA T, FOR5TER H:
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6. SRAER J, SRAER JD, ARDAILLOU R, MIMOUNE 0: Evidence for renal
glomerular receptors for angiotensin II. Kidney mt 6:241—246, 1974
7. FOIDART J, SRAER J, DELARUE F, MAHIEU P, ARDAILLOU R: Evidence
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8. Cn.TzIAroNIou C, DANIELS FH, ARENDSHORST WI: Exaggerated
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9. CHATZIANToNIOU C, ARENDSHORST WJ: Angiotensin receptor sites in
renal vasculature of rats developing genetic hypertension. Am J
Physiol 265 (Renal Fluid Electrolye Physiol 34):F853—F862, 1993
10. MENARD J, C&n KJ: Measurement of renin activity concentration
and substrate in rat plasma by radioimmunoassay of angiotensin I.
Endocrinology 90:422—430, 1972
ii. YE QM, HEALY DP: Characterization of an angiotensin type-i
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12. BOUBY N, CoRvoL P, BANIuR L, LLORENS-CORTES C: Localization
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13. CASELLAS D, DUPONT M, KASKEL FJ, INAGAMI T, MOORE LC: Direct
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Angiotensin II receptors and renin release in rat glomerular afferent arterioles 

Carolina Digital Repository

Kidney International, Vol. 46 (1994), pp. 1570—1573Angiotensin II receptors and renin release in rat glomerularafferent arteriolesCHRISTOS CRkTZIANT0NI0u, JEAN-CLAUDE DUSSAULE, WILLIAM J. ARENDSHORST,and RAYMOND ARDAILLOUINSER.M U.64, Tenon Hospita4 Paris, France, and Department of Physiology, University of North Carolina, Chapel Hi14 North Carolina, USAAngiotensin H receptors and renin release in rat glomerular afferentarterioles. The purpose of recent studies was to investigate the expressionof angiotensin II (Ang II) receptor sites in afferent arterioles freshlyisolated from the rat kidney, and the role of Ang II on renin release bythese vessels. The method of isolation and purification of renal microves-sels was based on iron oxide infusion into the kidneys and separation ofthe afferent arterioles from glomeruli and connective tissue with the aid ofa magnetic field, successive passages through various sieves, and harvest-ing with collagenase. Ang II receptor characteristics were evaluated byradioligand binding studies using the non-peptide Ang II antagonists ofAT1 (Dup-753 and -532) and AT2 (PD-123319 and CGP-42112) receptors.AT1 antagonists displaced up to 80% of the Ang II binding with highaffinity (3 nM), whereas the remaining 20% showed low affinity for the Dupcompounds and CGP-42112 (>10 .tM), and intermediate affinity forPD-123319 (12 gM), These data suggest the existence of two Ang IIreceptor subtypes in the renal vasculature of the rat. In separate experi-ments, renin release by isolated afferent arterioles in vitro was 9 ng/hr/mgunder control conditions. Ang 11(0.1 jiM) inhibited renin secretion by20%, whereas the adenylyl cyclase activator forskolin (10 jiM) stimulatedrenin secretion by 50%. In arterioles isolated from rats chronically treatedwith a converting enzyme inhibitor (perindoprilate) to reduce endogenousformation of Ang II, renin release increased 20-fold under controlconditions in vitro and was further stimulated by forskolin. These resultsdemonstrate that this preparation is a useful tool to study the functionalrole of Mg II and the control of renin release in the afferent arterioles.The renin-angiotensin system (RAS) plays a major role in therenal function. Although several investigations attempted tolocalize the exact sites of angiotensin II (Ang II) action on therenal vasculature, controversy still remains on whether Ang II canact on the preglomerular vessels [1]. Micropuncture and clearancetechniques and studies on microdissected vessels suggest that AngII constricts preferentially the efferent arteriole [2, 3] On theother hand, data obtained from in vitro visualization models(juxtaglomerular perfused nephron, hydronephrotic kidney) sup-port the view that Ang II cnn act on afferent arterioles [4, 5].Previous studies from our laboratories indirectly supported thelatter hypothesis. Ang II receptors have been identified onisolated glomeruli and cultured mesangial cells, a structure that isthought to share several common characteristics with preglomeru-lar vascular smooth muscle cells [6, 7]. Furthermore, injection ofAng II directly into the renal artery decreased renal blood flow by© 1994 by the International Society of Nephrology50 to 60% [8]. Renal vasoconstriction of this magnitude is difficultto be achieved without participation of preglomerular vessels.The purpose of the present study was to provide convincingevidence demonstrating the existence of functional receptor sitesof Ang II in the preglomerular vasculature. For this reason wedeveloped a technique to isolate in vitro pure and viable prepa-rations of preglomerular vessels from the rat kidney, and wesubsequently studied the regulation and expression of the renin-angiotensin system in these vessels.MethodsIsolation of afferent arteriolesThe technique to isolate afferent arterioles from the rat kidneywas the same as previously described [9]. Briefly, a magnetizediron oxide suspension (1% Fe3O4 in isotonic saline) was perfusedinto the renal arteries of seven-week-old Wistar-Kyoto (receptorbinding studies) or Sprague Dawley (renin release studies) rats (6rats per experiment) (Fig. 1A). Thereafter the kidneys wereremoved, decapsulated, and the cortex was dissected from themedulla. Cortical tissue was homogenized with a Polytron homog-enizer, and the iron oxide-loaded tissues (renal vessels andglomeruli) were removed from the crude homogenate with the aidof a magnet (Fig. 1B). Afferent arterioles were separated fromlarger vessels and glomeruli using repetitive passages throughneedles and sieves of decreasing sizes (20 to 23 gauge and 150 to90 jxm, respectively). The microvessels were recovered from thetop of the 125 jim sieve and harvested with collagenase (55 U/mI)for 30 minutes at 37°C to remove the surrounding connectivetissue. Only vascular preparations containing >90% of afferentarterioles were retained for subsequent experiments.Receptor binding studiesAliquots of microvessels (40 j.tg of protein) were incubated withincreasing concentrations of 3H-Ang II as previously described[9]. Nonspecific binding was determined in the presence of anexcess of unlabeled Ang 11(10 jim). In competitive inhibitionstudies, the AT1 receptor antagonists Dup-753 and Dup-532(DuPont) and the AT2 receptor antagonists PD-123319 (Parke-Davis) and CGP-42112 (Ciba-Geigy) were used to displace 3H-Mg II. Analyses of the data using the LIGAND program gaveestimates of the maximum specific binding (Bmj and dissocationconstant (Kd).15704/t*j --4Longitudinal section of a kidney after'perfusion. B. Representative examplerent arteriole attached to aus. Note that iron oxide particles doletely fill glomerular capillaries.Renin release evaluationAliquots of microvessels (150 to 200 tg of protein) wereincubated at 37°C in a final volume of 1 ml DMEM/F12 (1:1)medium containing 1 mcvi Ca2. Incubations lasted one and threehours in the absence or presence of 10 .tM of forskolin or 0.1 iMof Ang II. Renin release was measured by radioimmunoassay asthe amount of Ang I produced by incubation of supernatants withan excess of rat renin substrate (plasma of binephrectomized rats)Chatziantoniou et al: RAS in afferent arterioles 15711572 Chatziantoniou et al: RAS in afferent arteriolesRenin release was expressed in nanograms of Ang I gener-ated per hour of incubation per milligram of protein of afferentarterioles.Angiotensin converting enzyme (ACE) inhibitionIn separate experiments, rats were treated with a converting-enzyme inhibitor (perindoprilate, 40 mg/kg/day) for five days toeliminate endogenous Ang II. Isolation of afferent arterioles andrenin release assay were performed as described above.ResultsReceptor binding studiesThe properties of Ang II receptor sites were characterized inafferent arterioles from seven-week-old rats using equilibriumradiolabeled techniques. Scatchard analysis revealed one class ofhigh affinity receptors (Kd = 1 flM) with a density of 200 fmollmgof protein.To identif' the Ang II subtype(s) expressed in these renalvessels, the AT1 antagonists Dup-753 and Dup-532, and the AT2antagonists PD-123319 and CGP-42112 were used to displaceunlabeled Ang II. The AT1 antagonists displaced almost com-pletely the Ang II binding. Analysis of the displacement curve bythe LIGAND program indicated that a two-site model produceda significantly better fit than the one-site model (P < 0.00 1). In thetwo-site model, Dup-753 binds to one site with high affinity (3 nM),whereas it displays 500-fold lower affinity for the other site (1.5tkM; (Fig. 2A). The high-affinity site is the most abundant andoccupies 80% of the total Ang II receptor sites. The low-affinityAng II site occupies the remaining 20% of the total receptor sites.The putative AT2 antagonist PD-123319 displaced up to 25% ofthe Ang II binding, with a rather medium affinity (100 nM; Fig.2B). The other AT2 antagonist, CGP-42112, showed very lowaffinity to the Ang II receptor sites (> 10 tM).To determine whether or not the high affinity site that Dup-753bound to is distinct from the medium affinity site recognized byPD-123319, competitive inhibition curves were performed byadding a standard concentration of PD-123319 (1 jiM) to increas-ing concentrations of Dup-753, and alternatively, by adding astandard concentration of Dup-753 (10 nM) to increasing concen-trations of PD-123319. As shown in Figure 2A, PD-123319provided an additive displacement (15 to 20%) that was uniformin all tested concentrations of Dup-753. Similarly, Dup-753 dis-placed an additional 20 to 25% of the sites in all PD-123319 testedconcentrations (Fig. 2B).Renin release studiesThe viability of the afferent arterioles was checked by measure-ments of creatine kinase activity (CKA) in the medium. Nochanges in the release of CKA were observed between 0 and 6hours after isolation of the vessels. On the contrary, CKA wasincreased eightfold at 24 hours compared to 0 hours. For thisreason, renin release measurements were performed at 1 and 3hours after isolation of afferent arterioles. During this period, therate of secretion of renin was increased significantly with time.After 1 hour renin release in supernatants was 10 2 ng/hr/mg,and after 3 hours it was 22 2 ng/hr/mg (Table 1).To investigate whether renin release can be regulated in ourvascular preparation in vitro, we used forskolin and Ang II, twoagents that are known to stimulate and inhibit renin release,Normal Perindoprilatelhr 3hr lhr 3hrControl 10 2 22 2 153 16 242 19Forskolin 15 2 34 4 217 16 394 42Ang II 8 1 15 2 125 10 160 14Values are expressed in nglhr/mg and are means SE of 4 independentexperiments.P < 0.05 vs. controlrespectively. Addition of forskoljn (10 jiM) increased renin releaseby 40% after 1 hour and 50% after 3 hours compared to controlvalues. In contrast, when exogenous Ang 11(0.1 jiM)was added,renin release by afferent arterioles was decreased by 35% at 3hours (Table 1).To examine if in vivo regulation of the expression of theA12010080604020010_il lO_lO 10-s 10-8 10 10-6 10 10DUP 753, MB11010090807060VC0.00)CIC0.00)CIFig. 2. Displacement curves of 3H-Ang II binding to preglomerular vesselsby increasing concentrations of the AT2 antagonist Dup 753 in absence andpresence of the AT2 antagonist PD 123319 (1 jiM) (A) and inversely, byincreasing concentrations of PD 123319 in absence and presence of Dup 753(10 nM) (B). Values are means SE for 4 experiments of each condition.Table 1. Renin release from afferent arterioles freshly isolated fromnormal and perindoprilate-treated rats5010_il 10-s 10-8 10-i 10-8 10-s 10-iPD 123319,Chatziantoniou et al: RAS in afferent arterioles 1573renin-angiotensin system can induce changes in renin release thatcan be detected in isolated afferent arterioles, a group of rats wastreated chronically with a converting enzyme inhibitor (perindo-prilate) to reduce production of endogenous Ang IL Afferentarterioles from rats treated with perindoprilate were found torelease renin at a rate 15-fold higher than control. Addition offorskolin further increased renin release (50 and 70% abovecontrol at 1 and 3 hr, respectively), whereas addition of exogenousAng II inhibited renin release (30% below control at 3 hr).DiscussionIn the present studies, we isolated afferent arterioles of highpurity, and in sufficient quantity to perform several biochemicalstudies such as receptor characterization or responses to a varietyof stimuli. This vascular preparation is viable in vitro, and it wasused to study Ang II receptor sites in biochemical (bindingcharacteristics) and functional (renin release regulation) terms.The use of nonpeptide antagonists of Ang II indicated theexistence of two subtypes of Ang II receptors in the afferentarterioles of rats. One site appears to be a typical vascular AT1subtype since it displays high affinity to the AT1 antagonistsDup-753 and Dup-532 and very low affinity to the AT2 antagonistsPD-123319 and CGP-42112. This subtype is predominant andoccupies 80% of the total number of Ang II receptor sites. Theother receptor subtype displays intermediate affinity to the AT2antagonist PD-123319 and low affinity to AT1 antagonists. How-ever, it does not appear to be the AT2 subtype found in theadrenal gland, because it shows low affinity to CGP-421 12. Severalrecent studies using molecular biology techniques indicated theexpression of two AT1 subtypes (designated as ATia and AT1b) inthe rat renal kidney [11, 121. Of particular interest, use ofquantitative PCR suggested that the relative ratio of the AT1,,versus ATIb subtype in the renal cortex was 4:1 [12].The characterization of Ang II receptor sites in freshly isolatedafferent arterioles does not necessarily mean that these sites arefunctional. To verify the adequacy of this vascular preparation forfunctional studies, we investigated the responsiveness of theafferent arterioles to agents that control the expression of therenin-angiotensin system, such as Ang II and forskolin. In controlrats, renin release was increased with time, it was inhibited byexogenous Ang II, and it was stimulated by forskolin. It would beinteresting to investigate whether the two Ang II receptor sub-types found in our preparation influence renin release differently.In rats chronically treated with an ACE inhibitor, baseline reninrelease was increased 15-fold. Such a large increase in reninrelease is in agreement with studies indicating that in response toACE inhibition vascular smooth muscle cells of the renal vascu-lature from afferent arteriole up to interlobar and arcuate arteriesexpress and release renin to a rate several times higher than thenormal [131. Interestingly, in the perindoprilate-treated rats,isolated afferent arterioles continued to be responsive to exoge-nous stimuli since renin release was increased or decreasedfollowing exposure to forskolin or Ang II, respectively (Table 1).In conclusion, we have described a reproducible technique toisolate from the rat kidney afferent arterioles of high purity and insufficient quantities to perform biochemical studies such as Ang IIreceptor characterization and regulation. The metabolic respon-siveness of our vascular preparation to mediators of Ang IIreceptor expression and renin release suggests that this model is auseful tool to study the autocrine and paracrine regulation of therenin-angiotensin system at the cellular level.AcknowledgmentsThis work was financially supported by the "Institut National de Ia Santeet de Ia Recherche Medicale" and the "Faculté de Médecine St Antoine",and by a grant from the National Heart, Lung, and Blood Institute(HL-02334). Dr. C. Chatziantoniou is recipient of a European Commu-nities fellowship (Human Capital and Mobility).Reprint requests to Pr. Raymond Ardaillou, INSERM U.64, Tenon Hos-pital, 4 rue de Ia Chine, 75020 Paris, France.References1. NAVAR LG: Physiological role of the intrarenal renin-angiotensinsystem. Fed Proc 45:1411—1413, 19862. EDWARDS RM: Segmental effects of norepinephrine and angiotensinII on isolated renal microvessels. Am J Physiol 244 (Renal FluidElectrol Physiol 13):F526—F534, 19833. I-L&u. JE: Regulation of glomerular filtration rate and sodium excre-tion by angiotensin II. Fed Proc 45:1431—1437, 19864. CARMINES PK, MORRISON TK, NAVAR LG: Angiotensin II effects onmicrovascular diameters of in vitro blood-perfused jwctaniedullarynephrons. Am J Physiol 251 (Renal Fluid Electrol Physiol 20):F610—F618, 19865. LOUTZENHISER R, EPSiruN M, HAYASHI K, TAKENAKA T, FOR5TER H:Characterization of the renal microvascular effects of angiotensin IIantagonist Dup 753: Studies in isolated perfused hydronephrotickidneys. Am J Hypertens 4:309S—314S, 19916. SRAER J, SRAER JD, ARDAILLOU R, MIMOUNE 0: Evidence for renalglomerular receptors for angiotensin II. Kidney mt 6:241—246, 19747. FOIDART J, SRAER J, DELARUE F, MAHIEU P, ARDAILLOU R: Evidencefor mesangial glomerular receptors for angiotensin II linked tomesangial cell contractility. FEBS Lett 121:333—339, 19808. Cn.TzIAroNIou C, DANIELS FH, ARENDSHORST WI: Exaggeratedrenal vascular reactivity to angiotensin and thromboxane in younggenetically hypertensive rats. Am J Phyciol 259 (Renal Fluid ElectrolPhysiol 28):F372.—F382, 19909. CHATZIANToNIOU C, ARENDSHORST WJ: Angiotensin receptor sites inrenal vasculature of rats developing genetic hypertension. Am JPhysiol 265 (Renal Fluid Electrolye Physiol 34):F853—F862, 199310. MENARD J, C&n KJ: Measurement of renin activity concentrationand substrate in rat plasma by radioimmunoassay of angiotensin I.Endocrinology 90:422—430, 1972ii. YE QM, HEALY DP: Characterization of an angiotensin type-ireceptor partial eDNA from rat kidney: Evidence for a novel ATibreceptor subtype. Biochem Biophys Res Commun 185:204—210, 199212. BOUBY N, CoRvoL P, BANIuR L, LLORENS-CORTES C: Localizationand quantification of angiotensin II receptor subtypes in the kidney ofSprague Dawley and Sabra rats. (abstract) JAm Soc Nephrol 4:482,199313. CASELLAS D, DUPONT M, KASKEL FJ, INAGAMI T, MOORE LC: Directvisualization of renin-cell distribution in preglomerular vascular treesdissected from the rat kidney. Am J Physiol 265 (Renal Fluid ElectrolPhysiol 34):F151—F156, 1993

Angiotensin II receptors and renin release in rat glomerular afferent arterioles

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Angiotensin II receptors and renin release in rat glomerular afferent arterioles

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