(Hypertension. 2001;37:110.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Differential Distribution of Bradykinin B2 Receptors in the Rat and Human Cardiovascular System
From the Instituto de Histologia y Patologia (C.D.F., A.M., U.N.), Universidad Austral de Chile, Valdivia, Chile; Department of Pharmacology (U.F.), Johannes Gutenberg University, Mainz, Germany; Iconix Pharmaceuticals (K.J.), Mountain View, Calif; Aventis (B.S.), Frankfurt, Germany; and Institute for Biochemistry II (W.M.-E.), Johann Wolfgang Goethe University, Frankfurt, Germany.
Correspondence to Werner Müller-Esterl, PhD, Institute for Biochemistry II, Building 75, University Hospital, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. E-mail wme{at}biochem2
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Abstract—Bradykinin, a major vasodilator peptide, plays an important role in the local regulation of blood pressure, blood flow, and vascular permeability; however, the cellular distribution of the major bradykinin B2 receptor in the cardiovascular system is not precisely known. Immunoblot analysis with an anti-peptide antibody to the bradykinin B2 receptor or chemical cross-linkage with [125I]Tyr0-bradykinin revealed a band of 69±3 kDa at varying intensity in the homogenates of the endothelium and tunica media of the rat aorta and endocardium. Immunostaining showed that the B2 receptor is abundant in the endothelial linings of the aorta, other elastic arteries, muscular arteries, capillaries, venules, and large veins, where it localizes preferentially to the luminal face of the endothelial cells. In marked contrast, small arterioles (ie, the principal blood–pressure regulating vessels) of the mesenterium, heart, urinary bladder, brain, salivary gland, and kidney had a different staining pattern in which B2 receptor was prominent in the perivascular smooth muscle cells of the tunica media. A similar distribution pattern was found in mouse as well as in human tissues, indicating that the particular distribution pattern of the B2 receptor in arterioles is not a species-specific phenomenon. During development, the distribution of B2 receptor in the heart changes; for example, in the heart of newborn rats, the B2 receptor was abundant in the myocardium, whereas in the adult heart, the receptor was present in the endocardium of atria, atrioventricular valves, and ventricles but not in the myocardium. Thus, B2 receptors are localized differentially in different parts of the cardiovascular system: the arterioles have smooth muscle–localized B2 receptors, and large elastic vessels have endothelium-localized receptors.
Key Words: receptors, bradykinin • endothelium • endocardium • nitric oxide synthase • histochemistry • antibodies
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Introduction |
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The variety of biological effects produced by kinins in mammals are mediated by two types of receptors: B1 and B2.1 Both kinin receptors belong to the superfamily of G protein–coupled receptors characterized by 7-transmembrane spanning helices, although they differ in their primary structures, regulation of expression, tissue distributions, and ligand profiles.2 3 The B1 receptor is stimulated by des-Arg9-bradykinin and blocked by des-Arg9-Leu8-bradykinin, whereas the B2 receptor is stimulated by bradykinin and inhibited by HOE140.4 Under physiological conditions, most biological effects produced by kinins are mediated through the B2 receptor, which appears to be constitutively expressed by many cell types.5
The known cardiovascular effects of kinins include the induction of hypotension, regulation of local blood flow, and increased vascular permeability.5 The hypotensive effects of kinins are mediated at least in part by the release of NO from endothelial cells, because endothelial denudation of large arteries abrogates their vasodilator response to kinins.6 In addition, autacoids, such as prostaglandin I2,7 are synthesized and released due to bradykinin stimulation. In other situations, the endothelium-derived hyperpolarizing factor may mediate the vasodilator effects of kinins.8 Recent experimental evidence indicates that kinins may also have cardioprotective effects during ischemia, although this issue has been disputed.9 10 11
Several components of the kinin-generating system have been localized in the cells of the cardiovascular system. For example, Nolly et al12 found a tissue kallikrein-like kininogenase in rat blood vessels. Kallikrein activity and kallikrein gene expression were found in the rat and human heart.13 14 15 Cultured rat vascular smooth muscle cells have been shown to contain tissue kallikrein, kininogen, and kininase activity.16 Moreover, kininogen immunoreactivity was found on human endothelial cells17 and neutrophils.18
Many previous studies have provided evidence for bradykinin receptors in the cardiovascular system, such as, in endothelial cells of the aorta,19 20 21 of the pulmonary artery,22 of postcapillary venular vessels,23 of coronary arteries,24 and of cerebral microvessels.25 Kinin receptors were also found in aortic smooth muscle cells,26 in heart valves,27 and in the myocardium.28 Most of these studies demonstrated the presence of B2 receptors by functional assays after second messenger release,6 by autoradiographic techniques,28 by Northern blot analyses,2 or by RT-PCR.29 However owing to the limited resolution inherent in these methods, the precise cellular distribution of the kinin receptors in the cardiovascular system has remained unknown. For example, the mRNA for the B2 receptor was found in the heart,2 whereas radioreceptor assays revealed the absence30 or presence28 31 of bradykinin-binding sites in the adult myocardium and neonatal cardiomyocytes. Autoradiographic studies demonstrated bradykinin-binding sites in rat heart valves but not in other portions of the heart.27 The reasons for these discrepancies are unknown.
We sought to study the distribution of the B2 receptor in the cardiovascular system of the rat. By applying complementary techniques with anti-receptor antibodies, radioligands, anti-ligand antibodies, and binding site–directed immunoglobulins, we identified and mapped the B2 receptor in the various portions of the vascular bed in vitro and in vivo. Our findings indicate that the principal bradykinin receptor is differentially distributed with respect to developmental stage (neonatal versus adult), vessel caliber (arterial versus arteriolar), and cellular face (luminal versus basolateral), suggesting that the distinct distribution of B2 receptors may contribute to the differential effects of kinins in the various parts of the cardiovascular system.
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Methods |
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Collection of Tissues and Crude Membrane Preparation
Adult Holtzman (Sprague-Dawley) rats (350 to 400 g; Charles River Laboratories) used throughout this study were killed while under ether anesthesia. The thoracic and abdominal portions of the aorta were removed and immediately washed in freshly prepared Hanks’ balanced salt solution (HBSS). The vessels were placed in cold HBSS, stripped of fat and surrounding connective tissues, and opened with scissors, and the endothelium was removed using a Teflon policeman. Endothelial crude membranes were prepared as detailed later. The tunica media was mechanically separated from the adventitia under a stereoscopic microscope and monitored with hematoxylin-eosin staining of corresponding tissue sections. Hearts were removed from the killed rats, washed in freshly prepared HBSS at room temperature, and placed in an ice-cold Petri dish. Heart compartments were opened, and heart valves were removed. The endocardium was scraped off, and the myocardium was prepared from the left ventricle wall by removing the inner and the outer layers. The tissues were homogenized in 1 mmol/L NaHCO3 containing 0.1 mmol/L PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, and 0.1 µg/mL antipain (Sigma) and centrifuged at 13 000g for 45 minutes at 4°C. The pellets were resuspended in 50 mmol/L Tris-HCl, pH 7.6, containing 0.25% sucrose (immunoblotting) or in 20 mmol/L HEPES, pH 7.0 (cross-linking); we refer to them as "crude membrane" preparations.
Western Blotting and Chemical Cross-Linking
Crude membranes (80 µg protein/lane) were dissolved in sample
buffer,32 run on 12.5% SDS-PAGE in the presence of 2.5%
mercaptoethanol, and electrotransferred to nitrocellulose. Blots were
incubated overnight with antiserum AS351 to the synthetic peptide MLN33
of the extracellular domain-1 (ED1) of rat B2
receptor (Table). Bound antibody was visualized with an
125I-labeled secondary antibody or with the
peroxidase/antiperoxidase method.33 For chemical
cross-linking, 80 µg membrane protein in 20 mmol/L HEPES, pH
7.0, was incubated for 1 hour at 4°C with 10 nmol/L
[125I]Tyr0-bradykinin
(see later) in the same buffer containing 5 mmol/L EDTA, 20
µmol/L captopril (Sigma), and 0.5 mmol/L PMSF. The
homobifunctional cross-linker ethylene glycol
bis(succinimidylsuccinate) was used at a final concentration of
0.25 mmol/L. Nonspecific binding was determined in the presence of
10 to 20 µmol/L HOE140 (Icatibant).
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Autoradiography With
[125I]Tyr0-Bradykinin
The bradykinin analog Tyr0-bradykinin
(Sigma) was radioactively labeled with
[125I]NaI (specific activity 17 Ci/mg; Comision
de Energia Nuclear).34 Freshly removed hearts and vessels
were rapidly frozen in liquid nitrogen, and sections of 15 to 20
µm were prepared in a cryostat at -30°C, mounted on slides
precoated with poly(lysine) (Sigma), and stored at -70°C. Before
use, the slides were warmed to room temperature and washed with cold
50 mmol/L Tris-HCl, pH 7.4, containing 0.1% bovine serum
albumin (Sigma), 1 mmol/L EDTA, 1 mmol/L EGTA, and
20 µmol/L captopril ("washing buffer"). Incubation was
performed at 4°C overnight with 5 nmol/L
[125I]Tyr0-bradykinin
dissolved in washing buffer containing 140 µg/mL bacitracin (Sigma)
and 1 mmol/L dithiothreitol.35 The sections were
rinsed with cold washing buffer, rapidly cool-dried, and exposed to a
Biomax film (Kodak) for 72 hours at -20°C. Binding specificity was
probed in the presence of 5 µmol/L unlabeled
Tyr0-bradykinin or HOE140.
Light Microscopy Immunohistochemistry
Rat blood vessels and hearts were frozen in liquid nitrogen and
maintained at -70°C or fixed in
periodate-lysine-paraformaldehyde35 and
embedded in Histosec (Merck, Germany). For a comparison of the
distribution of B2 receptors among different
species, various tissues from mouse (SVE 129/J strain; Taconic) and
human origins were processed as described. The human tissues
corresponded to histologically normal segments obtained
from organs surgically removed due to inflammation or cancer and were
kindly provided by the Department of Pathology at the Regional Hospital
(Valdivia, Chile). The fixative for NO synthase (NOS)-III staining was
1% (wt/vol) paraformaldehyde in 0.1 mol/L borate
buffer, pH 9.5.36 Tissue sections prepared from frozen
materials and used for B2 receptor localization
were fixed with cold acetone for 20 minutes. For immunohistochemistry,
the sections from frozen or embedded tissues were washed 3 times with
50 mmol/L Tris-HCl, pH 7.8, or with PBS for 5 minutes each and
incubated overnight with the relevant antibody in the same buffer
including 1% immunoglobulin-free bovine serum albumin (Sigma).
Rabbit antibodies to peptides derived from the rat
B2 receptor sequence were used as a mixture
diluted 1:500 to 1:1000 or applied individually at 1:100
(Table). After an overnight incubation with the primary
antibody, the sections were sequentially incubated with swine
anti-rabbit immunoglobulin (1:80; DAKO) and the preformed
peroxidase/antiperoxidase complex (1:100; DAKO) for 30 minutes
each.35 In the case of murine antibodies, the sections
were incubated with rabbit anti-mouse immunoglobulin (1:500; DAKO)
before incubation with anti-rabbit immunoglobulin. Each incubation was
followed by the washing step. Bound peroxidase complex was visualized
with 0.1% 3,3'-diaminobenzidine-HCl (Sigma), 0.03% (v/v)
H2O2 in 50 mmol/L
Tris-HCl, pH 7.8. The sections were counterstained with Harris’
hematoxylin for 5 seconds, dehydrated, and mounted with balsam.
Alternative FITC-labeled swine anti-rabbit immunoglobulin (DAKO) was
applied at 1:30, and sections were mounted with Mowiol (Polysciences
Inc) containing 0.83%
p-phenylenediamine.
Ligand Probing of the B2 Receptor
To probe for ligand binding in situ, we used affinity-purified
anti-peptide antibodies (anti-ED3N) to the
amino-terminal portion of ED3 of the B2
receptor.37 Thoracic aortas were prepared as described
and, cut into rings 
10 mm long, and the sections were incubated
with 5 nmol/L
[125I]Tyr0-bradykinin in
HBSS for 2 hours at 4°C in the absence or presence of bradykinin,
HOE140, des [Arg9]brady-kinin (100
µmol/L each), or anti-ED3N or anti-ID4 (300
nmol/L each; Table). After incubation for 15 minutes at 4°C,
the radioactivity present in each ring was measured in a
-counter (Packard). For autoradiography, the treated
aortic rings were frozen in liquid N2, and
15-µm sections were prepared, dried, and exposed to a Biomax film
(Kodak). For immunostaining, unfixed frozen sections
were incubated overnight at 4°C with 300 nmol/L
anti-ED3N in the presence or the absence of
100 µmol/L bradykinin, HOE140, or
des[Arg9]bradykinin in HBSS containing 20
µmol/L captopril, followed by an FITC-labeled secondary antibody as
described. For in vivo labeling of the receptors, the high-affinity
antagonist HOE140 was applied in a single dose of 500
µg/kg body wt in 0.9% NaCl via injection into the tail vein of rats.
For control, the vehicle alone was used. At 5 to 10 minutes after the
injection, the rats were killed under ether anesthesia, and
the hearts were fixed through immersion in
periodate-lysine-paraformaldehyde as described. A
portion of each sample was embedded in Histosec, and the remainder was
rapidly frozen and stored in liquid nitrogen. Tissue sections were
incubated with antibodies to HOE140 (Table) and further
processed for immunohistochemistry as described.
Immunogold Electron Microscopy
Aortic rings and atria fixed with
periodate-lysine-paraformaldehyde for 1
hour35 were incubated with a mixture of anti-peptide
antibodies (1:200) directed to ED1-4 of the B2
receptor (Table) in PBS, pH 7.4, supplemented with 1%
immunoglobulin-free bovine serum albumin. Controls were
performed with preimmune rabbit serum at 1:200. The tissue sections
were washed and incubated with anti-rabbit immunoglobulin coupled to
gold particles of 10 nm (DAKO) or 30 nm (Amersham International). The
washed sections were postfixed with 3% glutaraldehyde
followed by 1% osmium tetroxide and embedded.18
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Results |
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Biochemical Characterization of the Vascular B2 Receptor
Crude membrane preparations from the endothelium and tunica media of the adult rat aorta were subjected to SDS-PAGE and Western blotting with an anti-peptide antiserum to the rat B2 receptor (Figure 1). A major band of 69±3 kDa was observed for both the endothelium (Figure 1, lane 1) and media (lane 3), although the intensities differed between the 2 sites. Preimmune serum applied at the same dilution failed to produce significant staining (lanes 2 and 4), thus demonstrating the specificity of the antibody. To monitor the completeness of the separation of endothelium and media, we applied an antibody to von Willebrand factor and found that only the endothelial preparation was positive for this marker protein (lanes 13 and 14). In a parallel experiment, B2 receptors from the same membrane preparations were chemically cross-linked to [125I]Tyr0-bradykinin by ethylene glycol bis(succinimidylsuccinate) and subjected to SDS-PAGE. The corresponding autoradiography revealed a major band of 69 kDa each for endothelium and media (lanes 5 and 7). A 1000-fold molar excess of the unlabeled B2 antagonist, HOE140, completely displaced the radioligand (lanes 6 and 8), thus proving the specificity of the ligand-receptor interaction.
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Distribution of B2 Receptors in the Rat Aorta
Identification of B2 receptors at the
cellular level was made with 2 independent techniques.
Immunohistochemistry with antibodies to the various intracellular and
extracellular domains of the B2 receptor revealed
a strong immunoreactivity of the aortic
endothelium (Figure 2a).
Less intense staining was seen for the smooth muscle cells interspersed
in the tunica media (Figure 2a), whereas the tunica externa was
free of staining except for vasa vasorum, which showed a strong
labeling (not shown). Application of the corresponding preimmune serum
or preabsorption of the antibodies with their corresponding antigens
failed to produce a significant immunostaining (Figures 2j and 2k). Autoradiography with
[125I]Tyr0-bradykinin
revealed a similar distribution pattern for bradykinin-binding sites
localized in the endothelium and in the media layer
(Figure 3a). Specific labeling was
abrogated by a 1000-fold molar excess of unlabeled bradykinin (Figure 3b) but not of the B1 receptor agonist
des[Arg9]- bradykinin (not shown).
Densitometric analyses revealed that bradykinin-binding sites
were more dense in the endothelial layer than in the
tunica media (Figure 3, insets). Hence, the aortic
endothelium is rich in B2
receptors, whereas the smooth muscle cells interspersed in the tunica
media of the aortic wall have a lower density of
B2 receptors.
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Immunoprobing for Kinin-Binding Sites
Autoradiographic studies with radiolabeled bradykinin
are complicated by the fact that other high-affinity binding sites for
bradykinin, such as ACE or endopeptidase EP24.15,
present in the same tissues may produce false-positive staining. To
exclude this possibility, we combined the autoradiographic
and immunoanalytic approaches and applied an affinity-purified antibody
to extracellular domain ED3 of the B2 receptor
(anti-ED3N). This antibody docks to a ligand
binding of the receptor, thereby preventing bradykinin attachment to
its cognate receptor.37 Rings of thoracic aorta were
incubated with 5 nmol/L
[125I]Tyr0-bradykinin in
the presence or absence of anti-ED3N. At a 300
nmol/L concentration of the antibody (60-fold molar excess over
radioligand), anti-ED3N effectively
displaced
[125I]Tyr0-bradykinin
from the aortic binding sites (Figure 4, bottom and middle). Similarly, 100 µmol/L bradykinin or HOE140
displaced the radioligand, whereas control antibody to an
intracellular domain ID4 of B2 receptor or
des[Arg9]bradykinin had no effect (Figure 4, bottom).
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This finding prompted the question of whether anti-ED3N could reversibly bind to B2 receptors in situ and thereby provide a unique specificity control that selectively probes for intact ("functional") kinin-binding sites. To test this possibility, we used frozen sections prepared from nonfixed aorta and incubated them with 300 nmol/L anti-ED3N in the presence or the absence of 100 µmol/L bradykinin, HOE140, or des[Arg9]bradykinin overnight at 4°C (Figure 4, top). A strong immunostaining of the endothelial layer was seen with anti-ED3N in the absence of competing ligands (Figure 4a), whereas coincubation with 100 µmol/L HOE140 (Figure 4b) or bradykinin (Figure 4c), but not with des[Arg9]bradykinin (Figure 4d), markedly reduced the specific staining. The failure of anti-ED3N to stain smooth muscle cells in the media likely reflects the lower affinity of the monospecific antibody to a single sequence segment as opposed to a polyvalent antiserum used for other immunolocalizations in this study. Collectively, these data stress the capacity of our anti-peptide antibodies to specifically detect the B2 receptor in the various layers of the rat aorta in situ.
Differential Distribution of B2 Receptors in Blood
Vessels
Immunovisualization of B2 receptors in blood
vessels other than aorta revealed that large muscular arteries such as
the carotid, mesenteric artery (Figure 2c), iliac and femoral
arteries, vena cava (Figure 2h), small capillaries (Figure 2d), and venules (Figure 2g) from various organs show a
rather uniform pattern, that is, prominent staining of the
endothelial cell layer and little, if any, staining of
the media and externa. Unexpectedly, an "inverse"
immunostaining pattern was seen for small arterioles:
immunoreactivity was most prominent in the muscularis surrounding the
arterioles, and only a thin rim of immunostaining was
visible at the arteriolar endothelium (Figures 2e and 2i). The replacement of the B2
receptor antiserum by nonimmune serum and the use of an excess of the
same peptides used for immunization, during the incubation of the
antiserum, demonstrated the specificity of the
immunostaining pattern observed in arterioles (Figures 2j and 2k). To confirm this strikingly differential staining
pattern, we followed the distribution pattern of NOS-III (ie, the major
effector enzyme of the B2 receptor in the
cardiovascular system). NOS-III was invariably
present in the endothelial layer of the aorta and
of small arterioles, whereas the media of vessels of varying caliber
was free of immunoreactivity (Figures 2b and 2f), thus
demonstrating the selectivity of our antibody probes. We made
qualitatively similar observations through
autoradiography using
[125I]Tyr0-bradykinin
(not shown), although the low-resolution power of this method did not
allow an unequivocal identification of the labeled cells. Hence,
B2 receptor appeared to be prominent in the
endothelium of the entire vascular system of Holtzman
rats, with the notable exception of resistance vessels, where
B2 receptors were abundant in the
muscularity.
Distribution Pattern of B2 Receptor in Rat, Mouse, and
Human Arterioles
Given this striking pattern of B2 receptor
distribution in rat mesenteric arterioles, we asked whether our finding
reflects a more generalized phenomenon. Indeed, rat urinary bladder
arterioles produced a similar staining pattern as mesenteric arterioles
(Figure 5a). In addition,
immunostaining of human breast (Figure 5c),
myometrium (Figure 5d), and skin (Figure 5g), as well as
mouse salivary gland (Figure 5f), showed essentially the same
arteriolar distribution for the B2 receptor:
intense staining of the tunica media and minor staining of the
endothelium. Controls with preimmune serum (Figure 5e) were negative. Hence, the characteristic distribution
pattern of the B2 receptor in rat arterioles is
also found in other mammalian arterioles, although the intensity of
staining may vary. In some cases, smooth muscle cells of the arteriolar
wall that express immunoreactive B2 receptors
were intermingled with cells devoid of any specific staining (Figure 5c). By contrast, large distributing arteries of human thyroid
exhibited the typical distribution pattern of B2
receptors in rat aorta: prevalent staining of the
endothelium with minor staining of the smooth muscle
cells interspersed in the vessel wall (Figure 5b). Thus, the
"inverse" distribution pattern of B2
receptors in arterioles is a general phenomenon across organs and
species.
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Localization of B2 Receptors in the Heart
Previous studies have produced conflicting results regarding the
presence of B2 receptors in the rat
heart.28 31 38 Our initial Western blotting experiments
revealed a weak band of 69 kDa indicative of the
B2 receptor in ventricular and
valvular endocardium, whereas the myocardium of the
adult heart was virtually free of immunoreactivity, as were controls
with preimmune sera (data not shown).
Immunofluorescence staining demonstrated
B2 receptors in endocardium of ventricles and
atria (Figures 6a and 6b), in the
endothelium of coronary arteries and veins (not
shown), and, less prominently, in heart capillaries (Figure 6b).
B2 receptors were also present in the
endocardium of tricuspid and mitral valves (Figure 6c).
Autoradiography with
[125I]Tyr0-bradykinin
confirmed these findings (Figure 6d). Hence, the adult rat heart
shows a distinct localization of B2 receptors
prominent in the endocardium and scarce or even absent in the
myocardium. Because recent work had demonstrated
bradykinin-binding sites of cultured neonatal myocytes,28
we wondered whether the newborn heart would differ in its expression
pattern of B2 receptors.
Immunostaining clearly revealed positive
immunoreactivity for both the endothelial cells of the
endocardium and the cardiomyoctes of the myocardium of the
newborn heart (Figure 7a; preimmune serum
control, Figure 7b), indicating that the expression of the
B2 receptor may vary during development and
growth of the rat heart.
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In Vivo Labeling of B2 Receptors
B2 receptors are subject to rapid
sequestration, swift desensitization, and possibly extensive
downregulation.39 40 Therefore, we asked whether the
relative localizations of the receptor demonstrated in vitro are
congruent with the patterns observed in vivo. We took advantage of the
facts that (1) the principal B2
antagonist HOE140 binds very tightly to the
B2 receptor,4 (2) unbound HOE140 is
rapidly cleared from the plasma via glomerular
filtration,41 and (3) antibodies to HOE140, a kinin-like
peptide, do not cross-react with the endogenous ligand
bradykinin.38 Crude membrane preparations from the
endothelium and tunica media of the rat aorta were
chemically cross-linked with HOE140, and the resultant products
were analyzed by SDS-PAGE and Western blotting. A major band of
69 kDa was observed for both endothelium (Figure 1, lane 9) and media (lane 11), whereas an isotype-matched
control antibody failed to produce significant staining (lanes 10 and
12), demonstrating the specificity of the detection method. HOE140 was
administered at 500 µg/kg of rat for 10 minutes via the tail vein;
thereafter, the animals were killed. Immunohistochemistry of the heart
atrium (Figure 7c) and ventricles (Figure 7e) with
anti-HOE140 revealed patterns of immunostaining that
were almost indistinguishable from those obtained with anti-receptor
antibodies. No specific immunostaining was observed
with isotype-matched nonimmune mouse immunoglobulin (Figure 7d).
Likewise, when rats were injected with vehicle alone, we did not
observe a specific tissue staining (not shown). Collectively, these
findings indicate that the cellular distribution of the
B2 receptors in vitro adequately reflects the
situation in vivo.
Subcellular Distribution of B2 Receptors
Autoradiography and immunocytochemistry at the
light microscopy level do not reveal the details of a differential
receptor distribution at the cellular level. Indeed, previous studies
of the B2 receptor in the rat distal nephron had
demonstrated that this receptor is abundant on both the basolateral
face and the luminal side of tubular cells,35 and studies
with recombinant kinin receptors revealed that the bulk of the receptor
protein resides in a perinuclear compartment that overlaps the
endoplasmic reticulum of overexpressing cells.42
Therefore, we used immunoelectron microscopy with anti-peptide
antibodies to the extracellular domains of the B2
receptor and gold-labeled secondary antibodies to probe for the
receptor on the various faces of endothelial cells. The
vast majority of B2 receptors were associated
with the luminal face of the plasma membrane of
nonpermeabilized aortic endothelial
cells (Figure 8), whereas the abluminal
side exposed the receptor at much lower frequency. We have made
similar observations for endothelial cells of the
endocardium (data not shown).
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Discussion |
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Cardiovascular functions are regulated by numerous factors, such as the sympathetic nervous system, endocrine hormone systems, and paracrine autacoid systems. In recent years, the role of local mediators, in particular those released by endothelial cells, has attracted significant attention. Due to its strategic anatomic position, the endothelium can respond to mechanical forces, such as shear stress,43 and to mediators from other cells, such as leukocytes, by releasing contracting and/or relaxing factors, which in turn modulate the activity of the vascular smooth muscle cells.44 45 Accumulating evidence suggests that many of the endothelium-mediated effects are strictly regulated with respect to time and space; that is, the signals are transient and local rather than sustained and systemic.44 One prototypic system that intimately interacts with the endothelium is the kallikrein-kinin system. Endothelial cells expose specific docking sites that serve to assemble the constituents of the kinin-generating pathway on the endothelium, thereby allowing the circumscribed release of short-lived kinins.46 47 The findings indicate that the structural characterization and spatial mapping of the terminal effectors of the system, kinin receptors, are critical for a deeper understanding of the mechanisms that limit the cardiovascular effects of bradykinin with respect to time and space.48
Using site-directed antibodies, we demonstrate that the bradykinin B2 receptor is differentially distributed in the rat cardiovascular system. The most unexpected finding is the variation in B2 receptor distribution in the muscularis of arterial vessels with caliber: elastic, and muscular arteries abundantly express B2 receptors in their endothelial lining and have much less or even no receptor in the smooth muscle cells of their walls. In marked contrast, small arterioles express large numbers of B2 receptors in the smooth muscle cells of their tunica media and only a few receptors in their endothelial layer. Many studies have demonstrated the presence of bradykinin B2 receptors in cerebral, renal, and coronary arteries and in muscular arterioles49 50 51 52 53 of vertebrate species; however, owing to the limitations inherent to receptor autoradiography, intracellular signaling, and/or ligand binding studies, none of them have revealed the precise in situ distribution of kinin receptors.
The presence of B2 receptors in the smooth muscle cells of the arteriolar wall may well explain the endothelium-independent relaxation observed for omental and coronary arterioles.54 55 In accord with these findings, pharmacological studies have demonstrated that vasodilator effects of kinins in large-bore vessels are primarily mediated by endothelial NO,45 56 whereas the inhibition of NO formation by NG-methyl-L-arginine has only a minor effect on bradykinin action in arterioles.57 58 Because smooth muscle cells of the arteriolar vessel wall do not express NOS-III (endothelial NOS), it is tempting to speculate that bradykinin triggers a NO-independent signaling cascade or cascades in these perivascular smooth muscle cells.59 60 This notion is in line with previous findings that the effects of bradykinin on small arterioles are at least in part mediated by signaling pathways that bypass endothelial cells.61 The differential distribution of B2 receptors in the resistance vessels may also help explain the biphasic response of renal afferent arterioles to increasing concentrations of bradykinin.50
An issue not solved in the present study is the considerable variation in B2 receptor expression levels during cardiac growth and development,28 30 31 which may have important (patho)physiological consequences. Functional studies have demonstrated the presence of bradykinin B2 receptors in cultured neonatal cardiomyocytes28 and the adult heart of the rat,31 whereas others failed to detect kinin receptors in adult cardiomyocytes.30 Our results clearly show the presence of B2 receptors in the heart of newborn rats; thus, the finding of kinin receptor expression in cultured neonatal cardiomyocytes is not an in vitro artifact.28 31 Because functional studies revealed only small copy numbers of B2 receptors in the adult heart,31 it is possible that the density of kinin receptors is too low to be detected through our immunological approach. Our findings seem to indicate that the myocardial B2 receptor is downregulated during growth and development of the rat heart. This observation correlates with the frequently seen "loss" of B2 receptors in the first few passages of human umbilical vein endothelial cell culture (W. Müller-Esterl, unpublished results). The B2 receptor gene could be the target of an extensive transcriptional regulation,52 and differential modulation of mRNA and/or protein stability may further contribute to the observed phenomena.
Differential B2 receptor expression may have important implications for the application of B2 receptor agonists in brain tumor therapy.62 Owing to the large number and/or efficient coupling of B2 receptors present in the endothelium lining of newly formed vessels, novel kinin receptor agonists such as RMP-7 have been developed63 that transiently and selectively open the blood-brain barrier, thereby promoting the extravasation of coadministered anticancer drugs en route to tumor tissues.64 It is tempting to speculate that the overexpression of B2 receptors in newly formed tumor vessels may translate into differential susceptibility to kinin agonists and therefore enhanced vulnerability to coadministered cytostatic drugs.65
In summary, we have shown that the distribution of the bradykinin B2 receptor is affected by 3 factors: age (newborn versus adult heart), vessel type (arteries versus arterioles), and cell polarity (luminal versus basolateral). Our results may help to underpin future studies that address the effects of kinins on growth, development, and protection of the heart; the beneficial effects of ACE inhibitors in hypertension and cardiac ischemia; and the role of kinins in blood-brain barrier opening and their applications to tumor therapy.
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Acknowledgments |
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This work was supported by grants from the FONDECYT (1940890 and 8980002), Direccion de Investigacion Universidad Austral, Volks-wagen Foundation (I-69637), Deutsche Forschungsgemeinschaft (Mu 598/4-2), and Fonds der Chemischen Industrie (163323). The authors are indebted to Dr C. Gonzalez (Valdivia) for providing [125I]Tyr0-bradykinin and to the members of the Mainz laboratory for help with the preparation and characterization of anti-peptide antibodies.
Received June 19, 2000; first decision July 5, 2000; accepted July 17, 2000.
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