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Vol. 280, Issue 2, 1109-1116, 1997
Hoechst-Marion-Roussel, Disease Group Cardiovascular, Frankfurt, Germany (P.W., K.W., B.A.S., G.W.), and University of Mainz, Institute for Physiological Chemistry and Pathobiochemistry, Mainz, Germany (J.D.)
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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In bovine aortic endothelial cells (BAECs), we previously demonstrated
B1 and B2 kinin receptor-mediated increases in
intracellular guanosine-3
,5-cyclic monophosphate (cGMP). In this
study, the B2 kinin receptor agonist bradykinin increased
cGMP in rat microvascular coronary endothelial cells (RMCECs) and human
umbilical vein endothelial cells (HUVECs), which could be prevented
with the specific B2 kinin receptor antagonist icatibant
but not with the B1 kinin receptor antagonist
des-Arg9-[Leu8]bradykinin or with the
nonpeptide kinin receptor antagonist WIN 64338. B2 kinin
receptor mRNA could be detected in all three cell types using reverse
transcription-polymerase chain reaction and subsequent Southern
blotting. The B1 kinin receptor agonist
des-Arg9-bradykinin increased cGMP in RMCECs but not in
HUVECs. The response in RMCECs could be prevented by
des-Arg9-[Leu8]bradykinin as well as by WIN
64338 but not by icatibant. In BAECs, the B1 kinin
receptor-mediated cGMP synthesis could be prevented by icatibant and
desensitized by preincubation with des-Arg9-bradykinin as
well as bradykinin. We detected B1 kinin receptor mRNA in
RMCECs and HUVECs but not in BAECs. In HUVECs, the detection of
B1 kinin receptor mRNA is in contradiction to the cGMP
measurements. In BAECs, the atypical B1 kinin receptor
pharmacology, the heterologous desensitization of the receptor and the
failure to detect B1 kinin receptor mRNA cannot be
explained by a typical B1 kinin receptor subtype. Thus,
B2 kinin receptors with similar pharmacology are constitutively expressed in each of the three endothelial cell types.
However, the endothelial cell types are heterogeneous in the expression
of typical B1 kinin receptors and the pharmacology of the
B1 kinin receptor-mediated responses.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Kinins are a group of
structurally similar peptides cleaved from large precursors, the
kininogens, by limited proteolysis by tissue and plasma kallikreins.
Kinins act in paracrine and autocrine manners by activating cell
surface receptors mediating a number of biological processes, including
regulation of vascular tone, moderation of pain and neurotransmission
and cell proliferation (for review, see Bhoola et al.,
1992
).
Receptors for kinins are classified into two subtypes, B1
and B2, according to the relative potency of kinin agonists
(Regoli and Barabé, 1980; Burch and Kyle, 1992; Hall, 1992). The
B1 kinin receptor is stimulated by des-Arg9-BK
and des-Arg10-kallidin, whereas bradykinin and kallidin are
more potent agonists for the B2 kinin receptor.
The B2 kinin receptor is constitutively expressed in
different cell types and tissues, and most of the actions of kinins are mediated by this receptor. The B1 kinin receptor, on the
other hand, is thought to be induced under certain pathophysiological conditions such as tissue injury and inflammation (Marceau, 1995). However, constitutive expression of the B1 kinin receptor
has been demonstrated in vivo in the dog coronary system
(Nakhostine et al., 1993) and in the cat pulmonary vascular
bed (DeWitt et al., 1994).
In every endothelial cell type examined thus far, a B2
kinin receptor has been demonstrated, the activation of which leads to
an increase in intracellular calcium and the production of nitric oxide
and prostacyclin (Schini et al., 1990; Boulanger et
al., 1990; Wiemer et al., 1991). In a CPAE cell line,
both the B1 and B2 kinin receptors could be
demonstrated using binding experiments and measurement of intracellular
calcium levels and nitric oxide release (Sung et al., 1988;
Smith et al., 1995). We previously found some indications of
both kinin receptor subtypes in primary cultured BAECs, by measuring
agonist-induced increases in intracellular cGMP (Wiemer and Wirth,
1992; Wirth et al., 1994). In the latter studies, however,
the des-Arg9-BK-induced increase in cGMP could be prevented
by pretreatment with icatibant, a kinin receptor antagonist known to be
selective for the B2 kinin receptor subtype (Rhaleb
et al., 1992), or WIN 64338, a nonpeptide kinin receptor
antagonist initially reported to be specific for the B2
kinin receptor (Marceau et al., 1994; Sawutz et
al., 1994).
The intention of this study was to characterize the kinin receptors in two additional endothelial cell types, HUVECs and RMCECs. A complete pharmacological classification in these endothelial cells, possibly expressing a mixture of kinin receptor subtypes, proved to be impracticable on the basis of intracellular cGMP measurements. Therefore, we included detection of kinin receptor subtype-specific mRNA by RT-PCR coupled with Southern blotting.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cell cultures.
HUVECs were isolated by modification of the
method of Jaffe et al. (1973). Freshly obtained umbilical
cords were stored (until use) at 4°C in sterile DPBS containing
glutathione and L-(+)-ascorbic acid (each 5 µg/ml,
Biotect protection medium; Biochrom, Berlin, Germany). Segments (10-25
cm long) were perfused with cold DPBS to wash out blood cells. The vein
was filled with Dispase II (2.4 U/ml in DPBS; Boehringer, Mannheim,
Germany) and incubated at 37°C for 60 min. Detached cells were washed
out and collected by centrifugation at 50 × g for 10 min. The cell pellet was resuspended in Iscove's minimal essential
medium containing glutamine (2 mM), penicillin (100 IU/ml),
streptomycin (100 µg/ml) and Biotect protection medium. Cells
isolated from one vein were plated at a density of 105
cells/well on six to eight 12-well plates (Falcon, Heidelberg, Germany)
coated with collagen I (from rat tail; Becton Dickinson, Heidelberg,
Germany). The purity of the endothelial cell cultures was checked by
uptake of fluorescently labeled, acetylated, low-density lipoprotein
(Voyta et al., 1984) or by immunostaining against van
Willebrand factor (Boehringer) or endothelial constitutive nitric oxide
synthase (Affiniti, Nottingham, UK).
(10 U/ml; Genzyme, Cambridge, MA)
for 1 to 24 hr or recombinant human tumor necrosis factor-
(10-100
ng/ml; Genzyme) for 1 to 24 hr.
RMCECs were isolated from rat hearts by modification of the methods of
Piper et al. (1990) and Stoll et al. (1995). The
hearts of male Wistar rats (120-150 g b.wt.) were removed and perfused in a noncirculating Langendorff system, at 80 mm Hg, with Krebs Henseleit buffer equilibrated at 37°C with carbogen. After 5 min the
system was switched to recirculating perfusion for 20 min with Krebs
Henseleit buffer containing bovine serum albumin (0.27 mg/ml), calcium
chloride (2.5 × 10
5 M), dispase II (0.06 IU/ml),
trypsin (120 µg/ml) and collagenase D (30 IU/ml; all from
Boehringer). The ventricles were trimmed, cut into small pieces and
further digested with the same protease solution for an additional 15 min. The resulting suspension was filtered through nylon mesh (200-µm
pore size) and adjusted with sterile Percoll solution (Pharmacia,
Freiburg, Germany) to a final density of 1.05 g/ml. Endothelial cells
were purified by two subsequent density gradient centrifugations
(0.45-0.876 and 0.28-0.62 g/ml) at 840 × g. The
cells were washed with Dulbecco's modified Eagle medium/Ham's F-12
medium (1:1) supplemented with glutamine (1 mM), penicillin (50 IU/ml), streptomycin (50 µg/ml), endothelial cell growth supplement
(50 µg/ml; Sigma), heparin (8.8 IU/ml; Boehringer), Biotect
protection medium and 20% heat-inactivated fetal calf serum. Cells
pooled from three hearts were plated at a density of 3 × 105 cells/well in four to six six-well plates (NUNC
Intermed, Wiesbaden, Germany) precoated with collagen A (Boehringer
Ingelheim Bioproducts, Heidelberg, Germany). The purity of the
endothelial cell cultures was confirmed by positive immunostaining for
the constitutive endothelial nitric oxide synthase (Affiniti,
Nottingham, UK) and lack of immunostaining for smooth muscle actin
(Sigma). BAECs were isolated and cultured as previously described
(Wiemer and Wirth, 1992).
cGMP measurements.
Measurements were made as described on
primary confluent cells, 6 to 8 days after seeding (Wiemer and Wirth,
1992). Briefly, after being washed twice with warm (37°C) HEPES
Tyrode's solution, the endothelial cells were preincubated for 15 min
with IBMX (0.1 mM) and SOD (20 U/ml) and stimulated with
bradykinin or des-Arg9-BK (Sigma) at the concentrations and
for the times indicated in "Results." In some experiments the
bradykinin and des-Arg9-BK stimulations were preceded by
preincubation for 5 min with single concentrations of icatibant
(HOE-140, Pharma synthesis; Hoechst AG), WIN 64338 (kindly provided by
Dr. G. Sawutz, Sterling Winthrop) or
des-Arg9-[Leu8]BK (Sigma). At the
concentrations chosen for icatibant and
des-Arg9-[Leu8]BK, these antagonists exhibit
kinin receptor subtype specificity in cellular models (Hock et
al., 1991; Hall, 1992; Menke et al., 1994). Each
agonist or antagonist used was added from freshly prepared 100×
concentrated stock solutions. The reactions were stopped by rapidly
removing the incubation medium and extracting the cells with an
ice-cold mixture of 1 N formic acid/acetone (15:85, v/v). After the
solvent mixture was removed, cGMP was determined using a specific
radioimmunoassay (NEN DuPont, Bad Homburg, Germany).
7 M, for 10 min) or des-Arg9-BK (106 M, for 10 min) in
HEPES/Tyrode's solution without IBMX/SOD. After this pretreatment the
kinin-containing supernatants were replaced by fresh buffer containing
IBMX/SOD, and the stimulations were carried out as described above.
RNA isolation and cDNA synthesis.
HUVECs, BAECs or RMCECs
(1-2 × 106 cells) were washed in ice-cold DPBS and
lysed in 4 M guanidinium isothiocyanate, 0.5% (w/v) sarcosyl, 2.5 mM
sodium citrate, 0.1 M 2-mercaptoethanol. mRNA was extracted with
phenol/chloroform as described (Chomczynski and Sacchi, 1987).
Contamination of total RNA preparations by traces of chromosomal DNA
was removed using RNase-free DNase I (Boehringer). cDNA synthesis was
performed in a 20 µl total volume containing 1 µg of total cellular
RNA, 200 U of Moloney mouse leukemia virus reverse transcriptase (New
England Biolabs, Schwalbach, Germany), 1 mM deoxynucleotide
triphosphates, 10 U of RNAsin (Boehringer), manufacturer's reverse
transcriptase buffer and 100 ng of oligo-dT16 (Roth,
Karlsruhe, Germany). RT was performed at 37°C for 2 hr. The reaction
volume was scaled up to 1 ml with 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA.
Oligonucleotide synthesis and PCR.
Oligonucleotides were synthesized either by Roth
(Karlsruhe, Germany) or by Hoechst AG (Frankfurt, Germany). The
following primers were used for PCR: B2Ru+
(5-GTCCATGGGCCGGATGCGCGG-3), derived from intracellular domain 2;
B2Ru
(5- CGATGCAGCGTATCCAGGAAGGTGC-3), from extracellular domain 4 of the human B2 kinin receptor (both oligonucleotide
sequences are highly conserved among different species,
i.e., human, rat, mouse and rabbit); B1Ra+
(5-CAGAAATCTACCTGGCCAACCTG-3), derived from transmembrane domain 2;
B1Ra (5-CTCCGCAGGGAGGCCAGGATGTGG-3), from intracellular domain 3;
B1Rb+ (5-GGTGGTGGCCATCAGCCAGGACC-3), from transmembrane domain 3;
B1Rb (5-GGTAAGGGGCCCAGCAGACCAGG-3), from transmembrane domain 6;
B1Rc+ (5-CTGCCACATCCTGGCCTCCCTGC-3), from intracellular domain 3;
B1Rc (5-CTTGGTCCTGAAGAGCCGGCCCAC-3), from intracellular domain 4 of
the human B1 kinin receptor (these sequences are highly
conserved among human and rabbit); B1Ru+ (5-GGCAGAAATCTACCTGGCCAACC-3), derived from transmembrane domain 2;
B1Ru (5-GCCAGTGGTAGGAGGAAACCCAG-3), from transmembrane domain 5 of
the human B1 kinin receptor (in other seven-transmembrane receptors, both sequence regions are highly conserved among different species).
and 3 primers, 250 µM deoxynucleotide triphosphates, 10 mM
Tris-HCl, pH 8.3, 50 mM KCl and 1.5 mM MgCl2. Samples were
overlaid with 70 µl of mineral oil and amplified using the following
protocol: 45 sec at 94°C, 45 sec at 48°C and 1 min at 72°C for 40 cycles on a thermal cycler.
Southern blotting.
Ten microliters of the PCR samples were
run in 1% agarose gels and transferred to Hybond N nylon filters
(Amersham-Buchler, Braunschweig, Germany) by standard techniques
(Sambrook et al., 1989). Hybridizations were performed
overnight under medium stringency conditions (60°C in 3× saline
sodium citrate) using either radiolabeled human B1 kinin
receptor cDNA (generous gift from F. Hess, Merck Research Laboratories,
Rahway, NJ) or human B2 kinin receptor cDNA. Filters were
washed twice in 2× saline sodium citrate/0.1% sodium dodecyl sulfate
at 60°C for 20 min. Probes for hybridizations were labeled with
[-32P]dCTP (ICN, Meckenheim, Germany) using the random
priming method developed by Feinberg and Vogelstein (1983).
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effects of bradykinin and des-Arg9-BK on
cGMP synthesis in RMCECs, HUVECs and BAECs.
The exposure of RMCECs
to bradykinin and des-Arg9-BK led to a concentration-
and time-dependent increase in intracellular cGMP synthesis. After 1 min of incubation, maximal increases were obtained with 1 × 108 to 3 × 108 M bradykinin, with a
threshold concentration of about 1 × 109 M (fig.
1A). In contrast, an approximately 30-fold higher
concentration (3 × 107 M) was needed for maximum
stimulation by des-Arg9-BK. The increases in cGMP induced
by both agonists were transient, with a maximum being reached between 1 and 3 min (fig. 1B). Preincubation with the B2 kinin
receptor antagonist icatibant (107 M) abolished the cGMP
increase induced by bradykinin (108 M) but not that
induced by des-Arg9-BK (107 M) (fig.
2). The B1 kinin receptor antagonist
des-Arg9-[Leu8]BK (3 × 106 M) prevented the cGMP increase elicited by
des-Arg9-BK but not that elicited by bradykinin. WIN 64338 (107 M), initially reported to be a nonpeptide
B2 kinin receptor antagonist, inhibited the
des-Arg9-BK-induced but not the bradykinin-induced cGMP
synthesis. Even at higher concentrations (up to 104 M),
WIN 64338 could not antagonize the bradykinin-induced cGMP synthesis in
RMCECs (data not shown).
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7 M bradykinin, with the
threshold concentration being 3 × 109 M (fig.
3A). The bradykinin response was transient and peaked at
3 min after application (fig. 3B). At no time was any effect of
des-Arg9-BK observed. The increase in intracellular cGMP
stimulated by a 3-min incubation with bradykinin (108 M)
could be prevented with icatibant (107 M) but not
des-Arg9-[Leu8]BK (3 × 106 M) or WIN 64338 (107 M) (fig.
4). As in RMCECs, higher concentrations of WIN 64338 (up
to 104 M) were ineffective to suppress the
bradykinin-induced cGMP synthesis (data not shown).
|
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6 M) in BAECs could be suppressed by
des-Arg9-[Leu8]BK (3 × 106 M), WIN 64338 (107 M) and icatibant
(107 M) (fig. 6). The icatibant-mediated
suppression of the des-Arg9-BK response could be abolished
by higher concentrations of des-Arg9-BK (105
M) (data not shown).
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7 M)
reduced the bradykinin (108 M)-dependent cGMP synthesis
by 65%. The same pretreatment, however, also led to suppression of the
des-Arg9-BK (107 M)-dependent cGMP formation
by >55%. After pretreatment with des-Arg9-BK
(106 M), the ability of the cells to respond to a
subsequent challenge with bradykinin (108 M) or
des-Arg9-BK (107 M) was reduced by 36 and
73%, respectively. Lower concentrations of agonists during the
pretreatment or shorter pretreatment times failed to reduce the
subsequent bradykinin and des-Arg9-BK responses (data not
shown).
|
Kinin receptor subtype-specific mRNA in RMCECs, HUVECs and
BAECs.
To substantiate the pharmacological results, we analyzed
the endothelial cells for specific mRNA for the B1 and
B2 kinin receptors. Isolation and RT of total RNA, as well
as amplification of cDNA with specific primers, was followed by
Southern blotting against radiolabeled human B1 and
B2 kinin receptor cDNA (fig. 8). Using this
method, B2 kinin receptor-specific PCR fragments with the expected molecular size of 339 base pairs could be amplified from HUVECs, RMCECs and BAECs cDNA (fig. 8A). Despite the DNase digestion, some background signals resulting from genomic DNA could be observed in
total RNA preparations from RMCECs and BAECs (fig. 8A, lanes 5 and 7).
|
) derived from a sequence region of the human
B1 kinin receptor where various other seven-transmembrane,
G protein-coupled receptors show high homology among different species.
With this primer pair, B1 kinin receptor-specific PCR
fragments of the expected size (437 base pairs) could be amplified from
HUVEC and RMCEC cDNA (fig. 8B, lanes 2 and 6) but not from BAEC cDNA
(fig. 8B, lane 4).
Because of our inability to detect B1 kinin receptor mRNA
in BAECs, we extended the RT-PCR using different sets of primers derived from sequence comparison of the human and rabbit B1
kinin receptors (table 1). With every pair of primers,
PCR fragments of the expected sizes could be detected in HUVECs. Some
of these primers could be used to amplify B1 kinin
receptor-specific PCR fragments from RMCEC cDNA, but none of them
resulted in amplification of B1 kinin receptor-specific
fragments from BAEC cDNA, thus confirming the result obtained with the
primer pair B1Ru+/B1Ru.
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In the present study, we have pharmacologically characterized in
three endothelial cell types the kinin receptor-mediated increases in
the intracellular cGMP concentration, which has been proven to be an
useful index of nitric oxide synthesis (Wiemer et al.,
1996). Furthermore, we included detection of kinin receptor subtype-specific mRNA by RT-PCR and Southern blotting. We found, independently of the endothelial cell type investigated, hints for the
expression of B2 kinin receptors, which exhibited similar pharmacological properties. However, differences between the
endothelial cell types could be observed with respect to the expression
of B1 kinin receptor mRNA and the pharmacological response
to B1 kinin receptor agonists and antagonists (table
2).
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In primary RMCECs, HUVECs and BAECs, bradykinin induced a transient,
concentration-dependent increase in intracellular cGMP, which could be
prevented by a short preincubation with icatibant (HOE-140), a
selective B2 kinin receptor antagonist. An antagonist specific for the B1 kinin receptor subtype,
des-Arg9-[Leu8]BK, was without effect.
Consistent with these pharmacological results, we could demonstrate
mRNA for the B2 kinin receptor in all three endothelial
cell types by RT-PCR coupled with Southern blotting using a
radiolabeled human B2 kinin receptor cDNA probe. Species-independent pairs of PCR primers were successfully selected based on the known sequences of the human, rat, mouse and rabbit B2 kinin receptors (McEachern et al., 1991; Hess
et al., 1992, 1994; Bachvarov et al., 1995).
Thus, in accordance with the results of other investigators, this
receptor subtype seems to be identically expressed and
pharmacologically coupled in all of the endothelial cell types
examined.
With respect to the B1 kinin receptor, the situation is
more complex. B1 kinin receptors are normally thought not
to be expressed by endothelial cells in intact blood vessels and to be
up-regulated only after vascular trauma (Pruneau et al.,
1994), in vitro incubation (Regoli et al., 1981;
Pruneau and Belichard, 1993) or treatment with lipopolysaccharide
(Marceau et al., 1980; Regoli et al., 1981;
Bouthillier et al., 1987; deBlois et al., 1989).
Furthermore, in some vascular preparations cytokines such as tumor
necrosis factor- or interleukin-1 were potent inducers of
B1 kinin receptor-mediated effects (deBlois et
al., 1991). However, in two in vivo studies, in dogs
and cats, constitutive expression of the B1 kinin receptor has been demonstrated (DeWitt et al., 1994; Belichard
et al., 1996). Furthermore, in a CPAE cell line, the
constitutive expression of the B1 and B2 kinin
receptors could be demonstrated, with both subtypes being coupled to
calcium signaling pathways (Smith et al., 1995).
Under our experimental conditions, we found that des-Arg9-BK induced cGMP synthesis in primary RMCECs and BAECs but not in HUVECs. In primary RMCECs, the B1 response could be prevented by preincubation with des-Arg9-[Leu8]BK but not with icatibant. In addition, B1 kinin receptor mRNA could be detected by RT-PCR using pairs of primers based on the human and rabbit sequences. Therefore, in addition to the B2 kinin receptor, these cardiac microvascular endothelial cells seem to express a typical B1 kinin receptor subtype.
In primary HUVECs, no des-Arg9-BK-induced cGMP increase
could be detected. A similar lack of effect was observed using
des-Arg10-kallidin (data not shown), a B1 kinin
agonist reported to be more potent in binding to the cloned
B1 kinin receptor (Menke et al., 1994) and in
relaxing porcine coronary arteries (Pruneau et al., 1996).
However, in an apparent contradiction to the pharmacological data, we
could clearly demonstrate B1 kinin receptor mRNA in this human endothelial cell type. A simple explanation could be that HUVECs
synthesize mRNA for this receptor but are unable to translate it. An
alternative explanation could be that the B1 kinin receptor molecules expressed are contained within intracellular compartments or
pools and are therefore unavailable to extracellularly applied agonists
(Pruneau et al., 1996). Such an intracellular localization of G protein-coupled receptors has been demonstrated for the endothelin ETA receptor subtype in a stably transfected cell line
(Chun et al., 1994) and for the AMPA-receptor type in rat
hippocampus (Henley, 1995). Only after "up-regulation" due to
unknown mechanisms might these receptor molecules be transferred to the
plasma membrane to form pharmacologically active receptors.
Efforts to up-regulate a des-Arg9-BK-mediated increase in
cGMP in HUVECs by pretreatment with different cytokines and modulators proved unsuccessful. Pretreatment of HUVECs with lipopolysaccharide or
tumor necrosis factor-, both applied for up to 24 hr, was ineffective, as was pretreatment with interleukin-1 for up to 48 hr
(data not shown). An up-regulation of des-Arg10-kallidin
binding sites has been described in interleukin-1-treated rabbit
mesenteric artery smooth muscle cells (Galizzi et al., 1994). We cannot exclude the possibility that human endothelial cells
express pharmacologically active B1 kinin receptors only under the influence of a cocktail of different cytokines and/or other
modulators.
In contrast to results with the human endothelial cells but in
agreement with data obtained using our rat endothelial cells, we
reported that in cultures of primary BAECs des-Arg9-BK
induced a transient cGMP increase. This response could be somewhat
unexpectedly attenuated by preincubation with icatibant (Wiemer and
Wirth, 1992). A similar icatibant-mediated B1 kinin antagonism has been observed in porcine coronary arteries (Pruneau et al., 1996) but not in bovine and human coronary arteries
(Drummond and Cocks, 1995, 1996). In CPAE cells, a pulmonary
endothelial cell line from the same species as BAECs, icatibant is only
a very weak B1 kinin receptor antagonist (Smith et
al., 1995). Thus, it remains unclear whether this B1
kinin receptor antagonism by icatibant is determined by the species or
tissue of origin or by some other, as yet unidentified, factors.
In an attempt to further characterize the B1 kinin receptor in BAECs, we performed analysis for B1 kinin receptor mRNA. However, using the same pairs of primers for the RT-PCR that could successfully amplify B1 kinin receptor mRNA in HUVECs and RMCECs, we were unable to detect any signal in BAECs. Furthermore, we have demonstrated in the present study that the bradykinin and des-Arg9-BK responses in BAECs can be heterologously desensitized. To our knowledge, this is the first time that such a heterologous bradykinin receptor desensitization could be observed in endothelial cells.
Although it is not possible to state an unifying concept capable of
accounting for heterologous desensitization, icatibant-mediated suppression of the des-Arg9-BK response and the lack of a
RT-PCR signal for a typical B1 kinin receptor, two
different explanations can be proposed. First, bradykinin and
des-Arg9-BK may act at separate receptors in BAECs, which
exhibit the characteristics of a B2 kinin receptor and an
atypical B1 kinin receptor. In that case, however, to
explain the heterologous desensitization it must be assumed that
receptor cross-talk occurs. Second, both agonists (bradykinin and
des-Arg9-BK) may bind to and activate a single receptor
subtype, which mainly exhibits the characteristics of a classical
B2 kinin receptor but differs in its increased affinity for
B1 kinin receptor agonists and antagonists. Such an
hypothesis could easily explain the heterologous desensitization
observed. However, only a single finding of a kinin receptor subtype
with such an atypical agonist-sensitivity profile has been described to
date; it was demonstrated that the cloned murine B2 kinin
receptor exhibits a mixed B1 and B2 kinin pharmacology after expression in Xenopus laevis oocytes or
COS-7 cells (McIntyre et al., 1993; Brown et al.,
1995). Only cloning of all kinin receptor subtypes expressed in BAECs
will help to elucidate this matter.
The cellular basis for the differing B1 kinin responses of
HUVECs, BAECs and RMCEC is not not known, but it seems not to be attributable to a difference between microvascular and macrovascular endothelial cells; both HUVECs and BAECs are macrovascular models. Other differences between HUVECs and BAECs, e.g., in the
dependency of flow- or bradykinin-induced nitric oxide synthesis on the
transmembrane potential, have been described (Gooch and Frangos, 1996).
Finally, the subtype specificity of the nonpeptide kinin receptor
antagonist WIN 64338 in vascular cell types and tissues is still
controversial. In both rabbit jugular and human umbilical veins, WIN
64338 exhibits competitive antagonism of bradykinin-induced contractions, with pA2 values of 6.14 and 5.99, respectively (Marceau et al., 1994). In addition, WIN 64338 is inactive in a rabbit aorta bradykinin B1 kinin receptor
assay (Sawutz et al., 1994). In IMR-90 cells, a human
fibroblast cell line, competitive inhibition of bradykinin-stimulated
45Ca2+ efflux could be observed, with a
pA2 of 7.1 (Sawutz et al., 1994). Taken together, these data suggest that WIN 64338 is a B2
and not a B1 kinin receptor antagonist. In contrast, in our
primary endothelial cell models, WIN 64338 appeared to act
species-independently as a B1 and not a B2
kinin receptor antagonist, because it prevented the
des-Arg9-BK-induced cGMP increase in BAECs (Wirth et
al., 1994) and RMCECs. Additionally, even at the highest
concentrations (104 M), the bradykinin-induced cGMP
synthesis could not be antagonized by WIN 64338 in all three
endothelial cell types (data not shown). The reasons for this
discrepancy in the subtype selectivity of WIN 64338 in our models,
compared with that described in the literature are unclear.
In the present study, we have demonstrated expression of pharmacologically similar, active, B2 kinin receptors in three endothelial cell models. In contrast to the B2 kinin receptor, the expression and pharmacology of the B1 kinin receptor-dependent response is dependent on the endothelial cell type. In RMCECs, a classical B1 kinin receptor appears to be constitutively expressed, whereas no classical B1 kinin receptor could be identified in BAECs. The receptor underlying the latter constitutive B1 kinin response in BAECs remains to be identified and may represent a new, atypical, B1 kinin receptor subtype. However, our observations may also be accounted for by a modified classical B2 kinin receptor that has a higher affinity for B1 kinin receptor agonists and antagonists. In HUVECs, mRNA for the classical B1 kinin receptor was found but no pharmacological activity could be detected. It remains to be elucidated whether these observations indicate the differential expression of partially unidentified B1 kinin receptors in endothelial cells.
| |
Acknowledgments |
|---|
We thank Anke Hullmann, Manuela Schatter, Elke Keler, Susanne
Romwalter and Marion Jorge for excellent technical assistance. We also
thank Prof. Werner Müller-Esterl (University of Mainz, Mainz,
Germany) for many helpful discussions and Dr. Ingrid Fleming (University of Frankfurt, Frankfurt, Germany) for invaluable help in
critical reading of this manuscript.
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Footnotes |
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Accepted for publication October 18, 1996.
Received for publication December 27, 1995.
Send reprint requests to: Dr. Paulus Wohlfart, Hoechst-Marion-Roussel, Disease Group Cardiovascular, Building H 825, 65926 Frankfurt, Germany.
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Abbreviations |
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BAEC, bovine aortic endothelial cell;
CPAE, calf pulmonary artery endothelial;
cGMP, guanosine-3,5-cyclic
monophosphate;
des-Arg9-BK, des-Arg9-bradykinin;
DPBS, Dulbecco's phosphate-buffered
saline;
HUVEC, human umbilical vein endothelial cell;
IBMX, 3-isobutyl-1-methylxanthine;
PCR, polymerase chain reaction;
RMCEC, rat
microvascular coronary endothelial cell;
RT, reverse transcription;
SOD, superoxide dismutase;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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in isolated smooth muscle cells: Correlation with B1 agonist-induced PGI2 production.
Br. J. Pharmacol.
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) and
des-Arg9-BK (
) on the intracellular accumulation of cGMP
in RMCECs after 1-min incubation, as a function of concentration. B,
time course of effects of bradykinin (10
), compared with
control values (
