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Centre Hospitalier Universitaire de Québec, Centre de Recherche du Pavillon l’Hôtel-Dieu de Québec, Quebec, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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75%) and selectively inhibited the
spontaneous sensitization to des-Arg9-BK over 6 h. SB
203580 also significantly reduced the development of the response to
des-Arg9-BK as stimulated by IL-1 or EGF. Both spontaneous
and IL-1ß-stimulated up-regulation of responsiveness to
des-Arg9-BK were significantly inhibited by the MAP kinase
extracellular signal-regulated kinase kinase 1 inhibitor PD 98059
(40%). The protein kinase inhibitors failed to inhibit protein
synthesis and to acutely inhibit the contractile effect of
des-Arg9-BK, suggesting that they do not influence
B1 receptor transduction mechanisms. In cultured aortic
smooth muscle cells stimulated with EGF, MAP kinase activation preceded
B1R mRNA induction. Protein kinase inhibitors reveal the
role of cell injury-controlled MAP kinase pathways, and singularly of
the p38 pathway, in the induction of B1R. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B1 type receptors are selectively sensitive to
fragments of kinins without the C-terminal arginine (e.g.,
des-Arg9-BK and Lys-des-Arg9-BK, also called
des-Arg10-kallidin); the prototype selective antagonist is
[Leu8]des-Arg9-BK (3, 4). B1
receptors (B1Rs) have a special interest due to their
strong up-regulation following some types of tissue injury. A large
body of evidence indicates that the B1Rs are generally
absent from normal tissues and animals, but are rapidly induced
following some types of injuries (4). A time- and protein
synthesis-dependent up-regulation of B1R-mediated
mechanical responses from a null initial level has been described in
the rabbit aorta and several other smooth muscle preparations (4),
including some of human origin (5, 6), following isolation and in vitro
incubation in simple physiologic solutions. The postulated
up-regulation of B1Rs by tissue injury may explain several
observations of in vivo enhanced functional responses to the
corresponding agonists in systems pertaining to hemodynamics, smooth
muscle contractility, pain perception, and leukocyte recruitment (4, 7, 8, 9). For instance, bacterial products sensitize the whole
cardiovascular system of rabbits, rats, or pigs to the B1R
agonist, des-Arg9-BK (10, 11, 12). Accordingly, the maximal
binding capacity (Bmax) of the B1R population
is increased in rabbit vascular smooth muscle cells by treatment with
LPS (13), and transcription of the B1R gene has been shown
in hearts from rabbits pretreated with endotoxin, but not in organs
from control animals (14). Thus, unlike the constitutively expressed BK
B2 receptor, the B1R appears to be dynamically
regulated. The sequence analysis of the 5'-flanking region of the human
B1R gene revealed the presence of a consensus TATA box and
numerous candidate transcription factor binding sequences, including
some associated with cytokine-induced gene expression, such as AP-1 and
nuclear factor-
B (15), which is consistent with a highly
regulated gene.
While the B1R promoter is currently being dissected to explain its inducibility, a parallel immunologic and pharmacologic approach was followed to study the mechanism of B1R induction. The role of inflammatory cytokines, particularly IL-1, in the vascular up-regulation of B1 receptors, has been suspected for some time (4). Recombinant IL-1 can reproduce the B1R up-regulation in vivo in the cardiovascular system and in a hyperalgesia model (7, 16) and accelerate the in vitro sensitization of the rabbit aorta to B1R agonists (17) or increase the B1R Bmax in binding assays in cultured cells (1, 13, 18). However, several other cytokines and growth factors are active in this respect in one or several of these experimental systems: epidermal growth factor (EGF), oncostatin M, nerve growth factor, IL-2, and IL-8 (7, 10, 17, 19, 20, 21). While some of these factors may recruit autocrine or paracrine IL-1 (7) and others may exert postreceptor potentiating effects in functional assays (20, 22), the redundancy and multiple signaling pathways recruited by these factors prompted us to probe the role of downstream intracellular biochemical pathways that can be common to several stress situations. We investigated the roles of different protein kinase pathways, namely tyrosine kinases and the multiple mitogen activated-protein (MAP) kinases ERK, SAPK/JNK, and p38 MAP kinase, in the regulation of expression of B1Rs. Direct kinase assays on aortic tissue extracts and selective inhibitory compounds (genistein, a tyrosine kinase inhibitor; SB 203580, an inhibitor of p38 MAP kinase activity; and PD 98059, a MEK1 inhibitor) in the contractility assay were used for this purpose. Rabbit aortic tissue was used under an experimental condition of B1R up-regulation that has been pharmacologically well characterized and serves as a model of tissue injury: the spontaneous appearance of a contractile responsiveness to B1R agonists from a null level in aortic rings following tissue isolation, as amplified or not by exogenous cytokines (10, 20). In addition, the correlation between MAP kinase activation by the cytokines and the transcriptional activity of B1R has been verified in cultured aortic smooth muscle cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The thoracic aorta was isolated from New Zealand White rabbits
of either sex (20) (1.5–2 kg). The vessels were cut into rings (4
mm in diameter, 3–4 mm in length) and were suspended between a metal
hook and a thread loop under a tension of 2 g in 5-ml tissue
baths containing oxygenated (95% O2/5% CO2)
Krebs solution. The composition of Krebs was 117.5 mM NaCl, 4.7 mM KCl,
1.2 mM KH2PO4, 1.18 mM MgSO4, 2.5
mM CaCl2, 25.0 mM NaHCO3, and 5.5 mM
D-glucose (20). Isometric changes in vascular tone were
measured by force transducers (model 52–9545, Harvard Apparatus, South
Natick, MA) coupled to LKB chart recorders (model 2210 or REC 102, LKB,
Rockville, MD).
Contractility studies were always based on the construction of five
full cumulative concentration-effect curves. The ones for the
B1 receptor agonist, des-Arg9-BK, were
constructed after 1, 3, and 6 h of incubation. The repeated
stimulations with des-Arg9-BK were performed to monitor the
progressive increase in the responsiveness of aortic strips to the
kinin metabolite, thus revealing the up-regulation of receptors by
monitoring their effects. The concentration-effect curves for the
-adrenoceptor agonist phenylephrine (Pe) were established at 1.5 and
7.5 h as more stable contractile responses (20); in addition, the
late response to Pe was depressed in tissues exposed to IL-1 (IL-1),
and there is ample functional evidence that nitric oxide mediates this
nonspecific decrease in contractility in this system (23, 24), thus
presumably representing the cytokine up-regulation of inducible nitric
oxide synthase (i-NOS). Tissues were amply washed with fresh Krebs
buffer between stimulations.
Contractility studies pursued several objectives. Firstly, the selected inhibitory drugs for protein kinase pathways, postulated to exert no direct myotropic effect and no overt toxicity on the contractile mechanisms, were introduced in the bathing fluid of some tissues (continuous application) to analyze the mechanism of the spontaneous (isolation-induced) sensitization to the B1 receptor agonist, des-Arg9-BK. As some exogenous cytokines and growth factors, such as IL-1 and EGF, respectively, can up-regulate B1 receptors in functional (10, 17) and binding assays based on rabbit arterial smooth muscle (13, 18, 19), other tissues were exposed to human rIL-1ß (5 ng/ml) or human rEGF (100 ng/ml) for the first 3 h of incubation as described previously (17, 20). These treatments do not interfere with the construction of the concentration-effect curves to des-Arg9-BK or Pe, which were performed as described above. The inhibitory drugs were combined with the cytokine treatment in some tissues to analyze the pathways used by these substances to potentiate B1 receptor regulation. The late response to Pe in these tissues may also provide an insight into another independent functional response induced by IL-1 in blood vessels, i.e., the induction of i-NOS (revealed as a nonspecific depression of the contractility), and inhibitory drugs may also reveal the mechanism of this gene activation. Vascular rings from each animal were assigned randomly to each of the treatments and to the control group, so that the statistical weight of each animal is equal in each group.
Contractions are expressed as the percentage of the maximum Pe-induced contraction recorded at 1.5 h, an internal standard for each tissue. This contractility value is equivalent to 4.47 ± 0.17 g of weight in the control groups (n = 30) and did not vary as a function of pharmacologic treatments (not shown). Sigmoidal concentration-effect curves are characterized by the half-maximal effective concentration (EC50) and the maximal absolute contraction amplitude (Emax; percentage of internal standard). Statistical analysis was performed by Kruskal-Wallis test followed by Mann-Whitney test or Student’s t test, using the InStat 2.0 computer program (GraphPad Software, San Diego, CA).
MAP kinase assays in aortic tissue
Aortic rings were prepared from normal rabbits as described above (ring weight, 40–50 mg). The rings were incubated for 15 min in 5% CO2 at 37°C in equilibrated Krebs buffer containing, or not, MAP kinase inhibitory compounds in a 12-well plate. The cut tissues were then stimulated with a cytokine (5 ng/ml IL-1ß or 100 ng/ml EGF) or the saline vehicle for 15 min or 3 h. The rings were quickly wiped on gauze and frozen in liquid N2. Later, tissues were pulverized in a mortar containing liquid N2, and the powder was resuspended in 250 µl of a lysis buffer (20 mM (3-[N-morpholino]propanesulfonic acid) (MOPS) myelin basic protein, pH 7.0, containing 10% glycerol, 80 mM ß-glycerophosphate, 5 mM EGTA, 0.5 mM EDTA, 1 mM Na3VO4, 5 mM Na4P2O7, 50 mM NaF, 1% Triton X-100, 1 mM benzamidine, 1 mM DTT, and 1 mM PMSF). The homogenates were vortexed and centrifuged at 17,000 x g for 12 min at 4°C. The supernatants were either immediately used for immunoprecipitation or stored at -80°C.
The immunoprecipitations and kinase assays were performed as previously described (25). From here on, all steps were performed at 4°C. The supernatants from the aortic rings were diluted four times in buffer I (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1 mM EGTA, 1 mM MgCl2, 1 mM Na3VO4, 1% Triton X-100, and 1 mM PMSF); undiluted anti-p38, anti-ERK2, or anti-MAPKAP kinase 2/3 were added in limiting concentrations; and the mixtures were incubated for 1 h. Ten or fifteen microliters of protein A-Sepharose (50%, v/v; Pharmacia, Piscataway, NJ) diluted in buffer I was added, and the mixtures were incubated for 30 min, centrifuged for 15 s, and washed three times with 300 µl of buffer I. These immmunoprecipitates were used directly for kinase assays.
The ERK2 and MAPKAP kinase 2/3 assays were performed in 25 µl of
kinase buffer K (100 µM ATP, 1–3 µCi of [
-32P]ATP
(3000 Ci/mmol), 40 mM p-nitrophenyl phosphate, 20 mM MOPS
(pH 7.0), 10% glycerol, 10 mM (15 mM for MAPKAP) MgCl2,
0.05% Triton X-100, 1 mM DTT, 1 µM leupeptin, 0.1 mM PMSF, and 0.3
µg protein kinase A inhibitor). Immunoprecipitated ERK2, p38, or
MAPKAP kinase 2/3 activities were determined using myelin basic
protein, ATF-2/GST, or recombinant heat shock protein-27, respectively,
as substrates (26, 27). The p38 assay buffer contained 100 µM ATP, 15
mM MgCl2, 50 mM HEPES (pH 7.4), 50 mM ß-glycerophosphate,
50 mM MgCl2, 0.2 mM Na3VO4,
ATF-2/GST, and 1 to 3 µCi [-32P]ATP. ERK2, p38, and
MAPKAP kinase 2/3 activities were assayed for 30 min at 30°C and
stopped by the addition of 10 µl of SDS-PAGE loading buffer. For the
SAPK/JNK assay, the supernatants were adsorbed on GST/c-Jun beads, and
the kinase was tested using the same beads as substrate (28). Briefly,
the GST/c-Jun fusion protein bound to glutathione/Sepharose beads was
incubated for 30 min at 4°C with the extracts in buffer I. The beads
were then pelleted, washed with buffer I, and incubated for 30 min at
30°C with 3 µCi [-32P]ATP in kinase buffer K. The
phosphorylated GST/c-Jun was boiled in SDS sample buffer to stop the
reaction. The activity of the various kinases was quantified by
measuring the incorporation of radioactivity onto the specific
substrate after SDS-PAGE.
[3H]leucine incorporation by rabbit aortic tissue
As continuous exposure to protein synthesis inhibitors prevents the sensitization to kinins in the rabbit aortic preparation (10, 16), we further validated the specificity of other drugs that inhibit this phenomenon using a [3H]leucine incorporation assay to exclude a global depression of protein synthesis. Rings of rabbit aorta (average wet weight, 20 mg) were prepared as described above and further divided into four or five pieces with fine scissors. The effect of each drug on the incorporation of [3H]leucine was tested at the same concentration as that used in organ baths, and cycloheximide (71 µM) was used as a positive control. The tissue fragments from each ring were incubated together for 6 h at 37°C (95% air/5% CO2) in 3 ml of Krebs buffer containing 0.5 µCi/ml of [3H]leucine (New England Nuclear Corp., Boston MA; sp. act., 144 Ci/mmol). Incubated fragments were rinsed three times in PBS, pH 7.4. Tissue fragments exposed to [3H]leucine were homogenized in 2 N NaOH, and the associated radioactivity was determined.
Assays based on cultured smooth muscle cells
Rabbit aortic smooth muscle cells were isolated, cultured and
characterized as described previously (29), and used at passage 5.
Groups of five 75-cm2 flasks (5 x
106 cells) were used for Northern blot analysis of
B1 receptor expression as affected by EGF (100 ng/ml).
Seventy-five to eighty percent confluent cells, grown in medium 199
supplemented with 10% FBS, fresh L-glutamine, and
antibiotics, were fed a reduced serum medium (0.4%) 24 h before a
3-h cytokine stimulation. Total RNA was extracted from the tissues
according to the method of Chomczynski and Sacchi (30). The samples
were denatured and electrophoresed in a 1.2% agarose gel containing
2% formaldehyde. The gel was transferred to a nylon membrane, and the
membrane was hybridized at 65°C with a 0.8-kb 32P-labeled
probe corresponding to a PstI fragment of the coding
sequence of the rabbit B1 receptor (31) (a gift from Dr.
Fred Hess). The final wash was with 0.1 x SSC/0.1% SDS at
65°C. The membrane was then subjected to autoradiography. The filter
was further washed with boiling 0.1% SDS and rehybridized with a probe
corresponding to GAPDH, a housekeeping gene. Results are reported as
the ratio of B1R signal to the GAPDH one. Individual
75-cm2 flasks of rabbit aortic smooth muscle cells
maintained in reduced serum (0.4%) medium for 24 h were also used
for extraction and quantification of basal and EGF-stimulated MAP
kinase activities (15-min stimulation; assayed as described above) to
monitor whether increased activities of these enzymes precede the
receptor up-regulation.
Materials
SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole) and its less active structural analogue, SKF 106978, were gifts from Dr. John C. Lee, SmithKline Beecham Pharmaceuticals (King of Prussia, PA). SB 203580 is a specific inhibitor of the p38 MAP kinase activity (32). PD 98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) was purchased from Research Biochemicals International (Natick, MA). This compound is a specific inhibitor of the MAP kinase kinase (MEK1) (33). Genistein, a tyrosine kinase inhibitor (34), was obtained from Biomol (Plymouth Meeting, PA), and des-Arg9-BK was obtained from Bachem (Torrance, CA). Recombinant IL-1ß and EGF (both of human sequences) were donated by Dr. D. E. Tracey (Upjohn Co., Kalamazoo, MI) and purchased from Calbiochem (San Diego, CA), respectively. Cycloheximide was purchased from Sigma Chemical Co. (St. Louis, MO), and Pe was obtained from Winthrop (Aurora, Canada). Anti-ERK2 is a rabbit polyclonal Ab raised against a synthetic peptide corresponding to the 14 carboxyl-terminal amino acids of rat ERK2 (35). Anti-p38 is a rabbit polyclonal Ab raised against the C-terminal sequence PPLQEEMES of murine p38 (25). Anti-GST-MAPKAP kinase 2/3 is a rabbit polyclonal Ab raised in rabbits after injecting a GST fusion protein containing the 223 C-terminal amino acids of Chinese hamster MAPKAP kinase 2 (35). This Ab immunoprecipitates both p45 and p54 isoforms of MAPKAP kinase 2, one of which probably corresponds to MAPKAP kinase 3 (25, 35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aortic rings isolated from normal rabbits were incubated in Krebs
buffer in the presence of cytokines (5 ng/ml IL-1ß or 100 ng/ml EGF)
or saline vehicle. The kinase assays showed a high level of basal
activity of all the MAP kinases tested (Fig. 1
, insets), most notably of p38 MAP
kinase (Fig. 1A), whose activity in control rings was
about 10- to 20-fold higher than that in untreated cultured HUVECs
(25), IMR-90, or CCL 39 cells (data not shown). Treatment of the rings
with either cytokine did not significantly augment the observed
activity of p38 (Fig. 1A) or that of the downstream
MAPKAP kinase 2/3 in separate tests (Table I). Activation of the later kinase did
not decline significantly after the 3-h incubation (Table I). The
SAPK/JNK assay also revealed that the basal activity of this kinase was
elevated and was further stimulated by the addition of cytokines to the
medium (Fig. 1B, statistical analysis in Table I). Also,
even though the ERK2 basal activity was high in the control rings, it
was inducible in presence of IL-1ß (1.5-fold increase) and EGF
(2.5-fold increase; Fig. 1C and Table I). SAPK/JNK and
ERK2 activities generally declined as a function of incubation time, as
assessed at 3 h (Table I). The decrease in ERK2 activity was less
pronounced in the EGF-treated tissues. These findings suggest that upon
tissue isolation or animal death, the p38 MAP kinases are persistently
activated to a high level, which is probably maximal, since the
addition of cytokines did not affect it. The other MAP kinases are also
activated, but more transiently and to a lesser extent, as indicated by
the induction of their activity by IL-1ß or EGF.
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Control aortic rings isolated from normal rabbits exhibited the
well-characterized passage from a null response to a time- and protein
synthesis-dependent increase in the maximal response to the
B1 receptor agonist des-Arg9-BK (Figs. 2 and 3). In these graphic
representations of the concentration-effect curves, C and
D represent the responses recorded after 3 and 6 h of
incubation, respectively. The maximal level of response to the kinin
recorded at 1 h was always close to zero (see Table II, which also contains statistical
analyses). The inhibitor of the p38 MAP kinase, SB 203580 (10 µM),
massively (75%) inhibited the spontaneous, isolation-induced
sensitization to des-Arg9-BK over 6 h (Figs. 2 and 3, C and D).
Yet, the compound exerted no overt toxicity on the contractile
mechanisms in the tissues, as the early (1.5 h) or late (7.5 h)
responses to Pe were not affected by the drug (Figs. 2 and 3,
A and B). In addition, SB 203580 failed to
inhibit des-Arg9-BK-induced contraction if it was applied
only 30 min before constructing the concentration-effect curve (Table III), suggesting that the drug did not
act as a kinin receptor antagonist or did not interfere with the
B1R transduction systems leading to muscle contraction.
Temporary exposure to exogenous IL-1ß or EGF considerably increased
the process of sensitization to the kinins, affecting both the maximal
effect and the sensitivity (EC50 values), as reported
previously (Figs. 2 and 3, respectively; Table II). SB 203580 also
significantly reduced by 50% (at 6 h) the development of the
response to des-Arg9-BK when stimulated with IL-1 or EGF.
In addition, IL-1, reduced the late (7.5 h) effect of Pe, as assessed
by both the maximal effect and the EC50 values; EGF was
much less active in this respect, increasing the EC50 by
only a small, but significant, margin (Fig. 2 and Table II). The
depressing effect of IL-1 on the apparent potency of Pe was partially
and significantly reversed by SB 203580. A structural analogue of SB
203580, much less active as a p38 kinase inhibitor, SKF 106978 (25),
did not alter the most representative contractility parameters of the
kinin recorded at 6 h, as assessed with or without cytokine
stimulation (Table II); some partial inhibition was recorded at earlier
times of observation.
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). The activity
of MAPKAP kinase 2/3, a kinase specifically activated by p38, was used
to monitor SB 203580-induced inhibition of p38 activity as described
previously (25). The drug reduced the activity of the downstream kinase
MAPKAP kinase 2/3 under all conditions, significantly for the control
or IL-1ß-treated tissues. SB 203580 exerted no overt effect on other
MAP kinase activities, except for a marginal increase in SAPK/JNK in
control tissues.
Both the spontaneous and the IL-1ß-stimulated up-regulation of
responsiveness to des-Arg9-BK were significantly inhibited
(by about 40%) by PD 98059 (25 µM) at 6 h (Table II); however,
the drug did not affect des-Arg9-BK-induced responses in
tissues treated with EGF or the late depression of the response to Pe
in tissues treated with IL-1ß. An acute exposure to PD 98059 30 min
before the 6-h recording of the concentration-effect curve of
des-Arg9-BK failed to influence the responses (Table III).
PD 98059 is an inhibitor of MEK1 MAP kinase (the upstream activator of
ERK). Accordingly, the drug selectively and significantly inhibited
ERK2 activity in cytokine-treated aortic rings, but the inhibition was
not as profound as that obtained by SB 203580 against the p38 pathway
(Table I).
Continuous exposure to the tyrosine kinase inhibitor, genistein (40
µM), markedly and selectively inhibited the response to the kinin
des-Arg9-BK in a manner similar to that of SB 203580,
except for the cytokine-induced depression of the 7.5 h response
to Pe, which was not prevented by genistein. The acute exposure to
genistein failed to significantly depress the 6 h response to
des-Arg9-BK (Table III), as reported previously
(22).
Inhibition of protein synthesis by drugs
The drugs SB 203580, PD 98059, and genistein failed to inhibit
[3H]leucine incorporation into fragments of rabbit
aortic tissues incubated for 6 h in Krebs solution (Fig. 4). Cycloheximide was effective in this
respect.
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Under culture conditions, rabbit aortic smooth muscle cells expressed a basal population of B1Rs that seems to be up-regulated more efficiently by exogenous EGF than by IL-1, as assessed by binding assays (18, 19). Northern blot analysis, performed on total RNA extracted 3 h after cytokine stimulation, showed that EGF treatment (100 ng/ml) determined a 1.7-fold increase in the concentration of B1R mRNA in cells, relative to the control value. The effect of EGF on receptor mRNA was preceded by an activation of all three tested MAP kinase pathways, as assessed at 15 min (2.1-fold for MAPKAP kinase 2/3, 3.5-fold for ERK2, and 1.4-fold for SAPK/JNK).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rabbit isolated aorta contractile response to kinins, mediated only by the B1R, was instrumental in defining its pharmacologic profile (40). The contraction is independent of prostanoids, although they are produced and released under the effect of des-Arg9-BK (20). The evidence for the de novo synthesis of B1R in the rabbit aorta is based on the selective abolition of the up-regulation of responses to des-Arg9-BK by protein synthesis inhibitors in isolated tissues (10, 16) and on the induction of both a pharmacologic responsiveness to des-Arg9-BK (41) and of the B1R mRNA (data not shown) by LPS pretreatment applied in vivo before sacrifice. The Northern blot approach notably shows that the basal level of B1R mRNA is very low or null in the normal rabbit aorta (not shown), as recently shown in the normal rabbit heart (14). Aortic rings that have been isolated and incubated in vitro for some time are not a source of mRNA of sufficiently high quality for Northern blot analysis.
The present data suggest that specific protein kinases are recruited
following tissue isolation to trigger the de novo synthesis of
B1Rs. In particular, p38 MAP kinase activity is very high
in control aortic rings (10- to 20-fold higher level of stimulation
in the immunoprecipitated fractions) relative to that in various types
of untreated cultured cells. The spontaneous up-regulation of
B1Rs is preceded by an activation of p38 catalytic
activity, as also reflected by the high and persistent activation of
its downstream substrate, MAPKAP kinase 2/3, in extracts of aortic
rings (Fig. 1 and Table I). At a concentration that effectively
inhibits MAPKAP kinase 2/3 activation, SB 203580 massively (75%)
inhibited the spontaneous up-regulation of responsiveness to the kinin,
indicating that p38 MAP kinase is an important determinant of
B1R expression. The p38 MAP kinase pathway is one of three
known homologous pathways that transduce the messages generated by
stressing agents or cytokines and is notably activated by oxidants,
LPS, IL-1, heat shock, and hyperosmolarity in various cell types (25, 26). The p38 inhibitor, SB 203580, inhibits the production of
inflammatory cytokines in response to LPS (42). The p38 kinase and the
downstream MAPKAP kinase 2/3 activities were only moderately increased
by cytokines (Fig. 1A and Table I), but SB 203580 could
still effectively block the sensitization of cytokine-stimulated rings
to des-Arg9-BK (Figs. 2 and 3, and Table II); this further
supports an important role for the p38 pathway in the expression of
B1R. Several transcription factors are known to be
activated more or less selectively by this pathway (growth arrest and
DNA damage inducible 153, cAMP response element binding protein, ATF-1,
and ATF-2) (43), and this may represent a link between tissue injury
and B1R induction. On the other hand, the p38 pathway may
not be recruited by B1R agonists to contract the tissue
when the receptors are formed, as SB 203580 failed to acutely influence
the response to des-Arg9-BK. SB 203580 exerts profound
anti-inflammatory effects in several animal models of
immunopathology (44), and repression of the expression of
B1R may represent one of the modes of action of this drug.
Accordingly, a peptide B1R antagonist clearly has an
analgesic and anti-inflammatory potential in animal models (8, 45).
Other cytokine-regulated genes may contribute to the therapeutic effect
of SB 203580, as suggested by the partial prevention of IL-1ß-induced
loss of Pe contractile efficacy at the late time of 7.5 h (Table II). From our previous work, we interpret this late effect of IL-1 on
Pe-induced contractility as the induction of a NOS isoform (i-NOS) (23, 24). The nonselective, agonist- and endothelium-independent depressant
effect of NO on contractility in IL-1-treated tissues was indirectly
identified by its complete reversal by nitro-L-arginine, a
guanylyl cyclase inhibitor, or hemoglobin. The effect of IL-1 was
dependent on protein synthesis and was suppressed by dexamethasone.
Thus, the p38 pathway may be recruited by IL-1 to induce i-NOS in the
rabbit aorta.
PD 98059 is a specific inhibitor of both tyrosyl and threonine protein
phosphorylation directed by MEK1, thus stopping the downstream
activation of the ERK MAP kinase pathway (33). The partial inhibition
of B1R induction of the contractile effect in rabbit aortic
rings by this drug suggests a modulatory function of this pathway on
receptor expression, possibly of a less critical nature than the role
played by the p38 pathway. An incidental finding in our study is that
ERK2 is strongly activated by EGF in fresh aortic tissue (Fig. 1C and Table I). EGF up-regulates the number of
B1R on rabbit vascular smooth muscle (19), but also acutely
potentiates the effect of B1R agonists (22), suggesting
interactions at multiple levels between B1 and EGF
receptors.
A practical limitation of the use of the inhibitory drug is the partial
nature of the inhibition obtained, as assessed by the MAP kinase
assays, especially for PD 98059 (Table I). For instance, contractility
tests showed that addition of exogenous EGF abrogated the inhibitory
effect of PD 98059 (Table II), but ERK2 assays in aortic extracts
suggests that the drug inhibition was partially surmountable by the
growth factor action. Other cases of partial surmountability of drug
inhibition by cytokines were observed (Table I) and may explain why SB
203580 and PD 98059 were proportionally less effective in
cytokine-stimulated aortic rings to prevent the development of
contractile responses to des-Arg9-BK (Figs. 2 and 3, and
Table II). In the fresh, compactly organized aortic tissue, the access
of drugs to their molecular targets might have been less optimal than
in cultured cells, where the same concentrations were proportionally
more effective (25).
The drugs used in the present study do not cover specifically the c-Jun
NH2-terminal kinase pathway, which is, however, activated
by the isolation procedure and IL-1 (Fig. 1B and Table I).
We cannot exclude that this alternate MAP kinase pathway may also
influence B1R expression. Activation of the three families
of serine and threonine kinases depends on both tyrosyl and threonyl
phosphorylation of the MAP kinase proteins. Genistein, a tyrosine
kinase inhibitor, was the least specific of the three inhibitors used
in the present study. Its potent and selective inhibitory effect on the
development of tissue response to des-Arg9-BK is consistent
with the role of MAP kinase pathways in the regulation of
B1R.
Cultured rabbit aortic smooth muscle cells express a relatively high basal population of B1Rs relative to the inducible levels following cytokine treatments. The regulation of B1Rs in these cells is conceivably influenced by the proliferative phenotype, the MAP kinase response to serum, and the alien environment (plastic vessels, culture medium, endotoxin traces, etc.). A 6-fold stimulation of Bmax by EGF was reported under experimental conditions that differed from ours (19). The B1R mRNA level is null or very low in the aorta immediately isolated from normal rabbits (data not shown), but is measurable in cultured aortic smooth muscle cells and is increased 1.7-fold 3 h after EGF stimulation. This effect was preceded by a stimulation of all measured MAP kinase in these cells, consistent with the role of MAP kinase pathways in the regulation of B1Rs.
Protein kinase inhibitors reveal the role of cell injury-controlled MAP kinase pathways, singularly of the p38 pathway, in the induction of B1R by tissue injury in rabbit aortic tissue. It is expected that specific inhibitory drugs will be useful in dissecting the promoter/enhancer function of the B1R gene.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. F. Marceau, Centre Hospitalier Universitaire de Québec, Centre de Recherche, Pavillon HDQ, 11 Côte-du-Palais, Quebec, Quebec, Canada G1R 2J6.
3 Abbreviations used in this paper: BK, bradykinin; B1R, B1 receptor; Bmax, maximal binding capacity; EGF, epidermal growth factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK/SAPK, c-Jun-N-terminal kinase/stress-activated protein kinase; Pe, phenylephrine; i-NOS, inducible nitric oxide synthase; EC50, half-maximal effective concentration; Emax, maximal effect amplitude; MAPKAP kinase 2/3, mitogen-activated protein kinase-activated protein kinase 2/3 ATF-2, activating transcription factor; GST, glutathione-S-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase MEK1, MAP kinase extracellular signal-regulated kinase kinase 1.
Received for publication May 5, 1997. Accepted for publication October 20, 1997.
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-thrombin in rabbit aortic rings. Br. J. Pharmacol. 105:959.[Medline]
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