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(Circulation Research. 2004;94:1375.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Bradykinin B₁ Receptor Expression Induced by Tissue Damage in the Rat Portal Vein

A Critical Role for Mitogen-Activated Protein Kinase and Nuclear Factor-B Signaling Pathways

Rodrigo Medeiros, Daniela A. Cabrini, Juliano Ferreira, Elizabeth S. Fernandes, Marcelo A.S. Mori, João B. Pesquero, Michael Bader, Maria C.W. Avellar, Maria M. Campos, João B. Calixto

From the Department of Pharmacology (R.M., D.A.C., J.F., E.S.F, M.M.C., J.B.C), Universidade Federal de Santa Catarina, Brazil; Department of Pharmacology (M.C.W.A) and Department of Biophysics (M.A.S.M., J.B.P.), Universidade Federal de São Paulo, Brazil; Max Delbrück Centre for Molecular Medicine (D.A.C., M.B.), Berlin-Buch, Germany.

Correspondence to João B. Calixto, Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, Rua Ferreira Lima, 82, 88015-420, Florianópolis, SC, Brazil. E-mail calixto{at}farmaco.ufsc.br or calixto3{at}terra.com.br

	Abstract

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The bradykinin B₁ receptor (B₁R) is normally absent under physiological conditions, but is highly inducible during inflammatory conditions or following tissue damage. The present study attempted to determine some of the mechanisms underlying B₁R upregulation following tissue injury in rat portal vein. Damage induced by tissue isolation and in vitro incubation caused a significant and time-dependent increase in des-Arg⁹–bradykinin (des-Arg⁹–BK) responsiveness that paralleled the B₁R mRNA expression, as confirmed by real-time quantitative PCR. In vitro incubation of rat portal vein also induced the activation of some members of the mitogen activated protein kinase (MAPK) family, namely, extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 MAPK, an effect accompanied by degradation of the inhibitory protein IB and translocation of nuclear transcription factor-B (NF-B) to the nucleus. The blockade of p38 MAPK, JNK or NF-B, but not ERK pathways with selective inhibitors, resulted in a significant reduction of the upregulated contractile response caused by the selective B₁R agonist des-Arg⁹–BK, and largely prevented the induction of B₁R mRNA expression in the rat portal vein. Together, these results demonstrate that in vitro tissue damage induces activation of several intracellular signaling pathways that have a key role in the control of B₁R expression. B₁R could exert a pivotal role in the development of the cardiovascular response associated with vascular damage.

Key Words: receptor, bradykinin B₁tissue damage • mitogen-activated protein kinases • portal vein • rats

	Introduction

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It is now well recognized that kinins are involved in a series of physiological and pathological cardiovascular responses.^1–3 Most of their actions are mediated by the activation of 2 G protein-coupled receptors, named B₁ and B₂.⁴ The activation of kinin receptors may play a key role in the modulation of atherosclerotic risk through the promotion of microangiogenesis,⁵ inhibition of vascular smooth muscle cell growth,⁶ coronary vasodilatation,⁷ increased local nitric oxide synthesis,⁸ or by exerting antithrombotic actions.⁹ The bradykinin B₂ receptor (B₂R) is normally constitutively expressed, mediating most of the physiological actions evoked by kinins and exhibiting high affinity for BK and kallidin. By contrast, the bradykinin B₁ receptor (B₁R) shows higher affinity for the kinin metabolites des-Arg⁹–BK and des-Arg¹⁰–kallidin, and is normally absent in nontraumatized tissues, but may be strongly upregulated during inflammatory processes or following tissue trauma.¹⁰

B₁R expression has also been associated with some pathologies such as atheromatous disease,¹¹ myocardial ischemia or infarction,^12,13 and septic shock.¹⁴ The induction or overexpression of B₁R, observed after tissue injury or following inflammatory and infectious stimuli in vascular tissues, has been described by several reports.^15–19 However, the mechanisms involved in the expression of B₁R in cardiovascular diseases are not completely known. It has been reported that the upregulation of B₁R is associated with tissue injury, thus indicating that B₁R could exert a critical role in the cellular response in certain cardiovascular diseases.

It has been suggested that B₁R expression may involve activation of a number of signaling pathways including the transcriptional factor NF-B^20–26 and some MAPKs.^21,25,27 In the present study, we examined the requirement of NF-B and MAPKs for the upregulation of the B₁R in the rat portal vein following tissue damage. We also evaluated the temporal profile for the activation of these molecules before B₁R upregulation.

Data obtained indicate that prolonged in vitro incubation of rat portal vein activates MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 MAPK, as well as the NF-B. Moreover, the activation of these pathways appears to be linked to the functional and molecular upregulation of B₁R in the rat portal vein. Collectively, the present results provide new molecular and functional insights into the role played by B₁R in the cardiovascular system.

	Methods

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Our investigation was in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Pharmacological Studies
The rat portal vein was isolated and set up in organ chambers as previously described.¹⁶ Contractile responses are expressed as a percentage of KCl-induced contraction (80 mmol/L). Following a 60-minute period of stabilization, preparations were exposed to a single concentration of KCl (80 mmol/L) as the standard stimulus. The B₁R agonist, des-Arg⁹–BK (1 µmol/L) was added to the preparations at 2, 4, and 6 hours following the set-up of the tissues. Repeated stimulation with des-Arg⁹–BK was performed to evaluate the time-dependent upregulation of the contractile response of the rat portal vein.

To analyze the role of MAPK or NF-B pathways in the time-dependent increase in responsiveness to des-Arg⁹–BK, preparations were continuously exposed to one of the following inhibitors: MG132 (1 µmol/L); BAY117082 (5 µmol/L) or PDTC (10 µmol/L) (NF-B inhibitors) or SB203580 (30 µmol/L); and PD98059 (30 µmol/L) or SP600125 (10 µmol/L) (p38, MAPK kinase 1/2 and JNK inhibitors, respectively). In another set of experiments, the receptor was induced and the contractile response to des-Arg⁹–BK was confirmed at 5.5 hours after establishment of the preparations. Then, preparations were incubated with 1 of the inhibitors (for 30 minutes, acute incubation) and again exposed to the B₁R agonist des-Arg⁹–BK.

To determine any possible nonspecific effect of inhibitors in the contractile response to des-Arg⁹–BK, a contractile response to noradrenaline (1 µmol/L) was obtained in the absence or in the presence of inhibitors 7 hours after in vitro incubation of the rat portal vein.

Preparation of Tissues for Molecular Studies
In these experiments, tissues were collected as previously described.²⁶ Rat portal vein was incubated for different periods of time in 24-well plates filled with serum-free Dulbecco’s modified Eagle culture medium in the presence or absence of MAPK or NF-B inhibitors. After the incubation period, tissues were frozen in liquid N₂. Some tissues were frozen immediately after isolation (basal group).

Western Blot Analysis
Cytoplasm protein extraction and Western blot analysis were performed as previously described.^28,29 Membranes were incubated with antibodies for total and phosphorylated forms of p38 MAPK, ERK, and JNK, or with anti-IB (Santa Cruz Biotechnology). The proteins were visualized by the ECL detection system (Amersham Biosciences).

Electrophoretic Mobility Shift Assay
Nuclear protein extraction and electrophoretic mobility shift assay (EMSA) were performed as described before.²⁸ NF-B consensus oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3) was end-labeled with [-³²P]ATP (Amersham Biosciences).

Real-Time Quantitative RT-PCR
Total RNA was extracted from rat portal vein using the TRIzol reagent (Invitrogen) according to the manufacturer’s recommendation. Two micrograms of RNA were reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen). First-strand cDNA synthesized from total RNA with random hexamerprimers was used as the template for each reaction. The iCycle iQ Real Time PCR Detection System (Bio-Rad) was used for the signal detection, and the PCR was performed using QuantiTec SYBR Green PCR (QIAGEN GmbH, Germany) using 300 nmol/L of each primer. For standardization and quantification, rat ß-actin was amplified simultaneously. The following primer sequences were employed: 5'-CCAAGACAGCAGTCACCATCAA-3' (forward) and 5'-CAGCAGGTCCCAGTCTTCTAG-3' (reverse) for the amplification of rat B₁ receptor, 5'-CCTCTGAACCCTAAGGCCAA-3' (forward) and 5'-AGCCTGGATGGCTACGTACA-3' (reverse) for the amplification of rat ß-actin. PCR conditions were 95°C for 10 minutes, followed by 45 cycles at 95°C for 15 seconds, 62°C or 60°C for 20 seconds, and 72°C for 20 seconds. Fluorescence was detected at the end of every extension phase (72°C). Data generated from SYBR Green were analyzed according to Winer et al³⁰ and Livak and Schmittgen.³¹ Calculation of the fold change in B₁R was relative to the ß-actin endogenous control using 2 ^–Ct.

Statistical Analysis
Results are expressed as mean values±SEM. Statistical significance was analyzed by means of paired or unpaired Student t test, or ANOVA and Dunnett test. Differences were considered significant at P<0.05.

	Results

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B₁R Functional and Molecular Upregulation
The induction of B₁R in the rat portal vein was assessed by analysis of time-related des-Arg⁹–BK-induced contraction and the mRNA expression using quantitative real time PCR.

In vitro incubation of rat portal vein increased the sensitivity to des-Arg⁹–BK (1 µmol/L) in a time-dependent manner (Figure 1A and 1B). In this tissue, no contractile response was observed after 1 hour of incubation. However, a significant increase in des-Arg⁹–BK-induced contraction was observed as early as 3 hours, reaching the maximal response at 6 hours and persisting until at least 12 hours later. No time-dependent change was observed for noradrenaline-induced contraction when tissues were incubated up to 12 hours, indicating the specificity of increased responsiveness to des-Arg⁹–BK.

View larger version (20K): [in this window] [in a new window]   Figure 1. Time-course of B1R receptor upregulation. A, Typical record showing the time-dependent increase in the contractile response to des-Arg9–BK (top) and sustained contractile response to noradrenaline (bottom) when rat portal vein was continuously incubated for 12 hours. B, Graph illustrating the temporal profile of the contractile response induced by des-Arg9–BK and noradrenaline in rat portal vein (data are the means±SEM of 5 preparations). C, Time-dependent expression of B1R mRNA in rat portal vein (data are the means±SEM of 3 preparations). *P<0.05 and **P<0.01, as compared with basal values.

As shown in Figure 1C, the portal vein obtained from the basal group expressed no detectable levels of mRNA for B₁R. Time-course analysis revealed that B₁R mRNA expression was minimal at 0.5 hour, but increased in a time-dependent manner, peaking at 6 hours (about 150-fold increase over basal), and being reduced 12 hours later.

Effect of MAPKs and Nuclear Factor-B Inhibitors on Des-Arg⁹–BK-Mediated Functional Responses in Rat Portal Vein
Continuous exposure of tissue preparations (for 6 hours) to the inhibitors of p38 MAPK (SB203580, 30 µmol/L) or JNK (SP600125, 10 µmol/L) largely prevented the functional B₁R upregulation in the rat portal vein, as indicated by a significant decrease in the des-Arg⁹–BK-induced contractile response (64 and 52% inhibition, respectively). By contrast, the incubation of preparations with PD98059 (30 µmol/L), an inhibitor of an upstream activator of ERK (MAPK kinase 1/2), had no significant effect on the des-Arg⁹–BK-induced time-dependent contractile response in rat portal vein (Figure 2A).

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of MAPK inhibitors on B1R upregulation following rat portal vein damage. Tissues were continuously incubated with (A) PD98059, (B) SB203580, (C) SP600125 or vehicle (control) for 7 hours, while des-Arg9–bradykinin (1 µmol/L) was applied at 2, 4, and 6 hours, and noradrenaline (NA, 1 µmol/L) at 7 hours. Data are the means±SEM of 4 or 5 preparations. *P<0.05 and **P<0.01 indicate a significant difference when comparing inhibitors and vehicle-treated preparations.

The possible involvement of NF-B in the time-dependent contraction caused by des-Arg⁹–BK was evaluated by the use of 3 different inhibitors. As shown in Figure 3, the continuous incubation with PDTC (10 µmol/L), MG132 (1 µmol/L), or BAY117082 (5 µmol/L) resulted in a significant inhibition of this response (29%, 75%, and 50% inhibition, respectively).

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of nuclear factor-B inhibition on the B1R upregulation response following rat portal vein damage. Tissues were continuously incubated with (A) PDTC, (B) MG132, (C) BAY117082 or vehicle (control) for 7 hours, while des-Arg9–bradykinin (1 µmol/L) was applied at 2, 4 and, 6 hours, and noradrenaline (NA, 1 µmol/L) at 7 hours. Data are the means±SEM of 4 or 5 preparations. *P<0.05 and **P<0.01 indicate a significant difference when comparing inhibitors and vehicle-treated preparations.

No alteration of noradrenaline-induced contraction was observed following the continuous exposure to the MAPK or NF-B blockers incubated under the same conditions described before (Figures 2 and 3 ). Furthermore, the same inhibitors failed to reduce des-Arg⁹–BK-induced contraction of the rat portal vein, when applied for 30 minutes, 5.5 hours after the beginning of tissue incubation (results not shown).

Effect of MAPK and NF-B inhibitors on B₁R mRNA Expression
To extend the functional evidence indicating the relevance of MAPK and NF-B pathways for the B₁R upregulation following tissue damage, we analyzed the effect of selective inhibitors of these pathways on B₁R mRNA expression in the rat portal vein. The incubation of either SB203580 or SP600125 markedly reduced the increase in B₁R mRNA expression after 6 hours of incubation (85% and 98% inhibition, respectively). However, treatment with PD98059 caused no significant change in B₁R mRNA levels (Figure 4A). The effect of NF-B inhibitors on B₁R mRNA expression was also examined. Continuous incubation of the rat portal vein with PDTC (30 µmol/L), MG132 (1 µmol/L) or BAY117082 (5 µmol/L), promoted a significant inhibition of B₁R mRNA expression (38%, 92%, and 68%, respectively) (Figure 4B).

View larger version (20K): [in this window] [in a new window]   Figure 4. Effect of protein kinase and nuclear factor-B inhibitors on the bradykinin B1 receptor mRNA expression. Total RNA was extracted from portal vein preparations submitted or not (basal) to incubation for 6 hours in DMEM in the absence (induced control) or presence of (A) protein kinases or (B) nuclear factor-B inhibitors. Specific B1 receptor mRNA was determined by SYBR Green real-time RT-PCR assay and normalized to ß-actin mRNA concentrations. Results are expressed as means±SEM (n=3). **P<0.01, as compared with the induced control.

Activation of MAPKs by Damage to Rat Portal Vein
Western blot analysis was used to evaluate the activation of ERK, JNK, and p38 MAPK pathways after rat portal vein incubation. Data in Figure 5 indicate that no detectable activation of p38 MAPK and ERK, and only very low levels of JNK activation, was observed under basal conditions. The isolation of the rat portal vein resulted in a marked and time-dependent activation of ERK (1.7-fold), JNK (2.2-fold), and p38 (1.6-fold). The maximal increase in the phosphorylation of p38 was reached at 5 minutes, and at 15 and 30 minutes for ERK and JNK, respectively. The activation of all MAPKs was found to be diminished after 60 minutes.

View larger version (51K): [in this window] [in a new window]   Figure 5. Time course of MAPKs activation after rat portal vein trauma. Rat portal vein preparations were incubated for the indicated times and cytosolic extracts were then prepared. Cytosolic levels of (A) phosphorylated p38 MAPK (top) and total p38 MAPK (bottom), (B) phosphorylated ERK (top) and total ERK (bottom), or (C) phosphorylated JNK (top) and total JNK (bottom) were determined using specific antibodies. D, Results were normalized by arbitrarily setting the densitometry of the basal group (Bs) and are expressed as mean±SEM (n=4). Data presented are representative of at least 3 similar experiments. *P<0.05 and **P<0.01, as compared with basal values.

Damage to Portal Vein Induces IB Degradation and NF-B Activation
To assess the effect of rat portal vein isolation on NF-B signaling activation, we evaluated the IB expression and NF-B/DNA binding activity by Western blot analysis and EMSA, respectively. The in vitro incubation of the portal vein resulted in a rapid onset and time-dependent IB degradation, which was maximal at 15 minutes, returning to basal values after 120 minutes (Figure 6A).

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of rat portal vein damage on IB degradation and NF-B–specific DNA-protein complex formation. Rat portal veins were incubated for the indicated times, then cytosolic and nuclear extracts were prepared. A, Cytosolic levels of IB were determined using a specific antibody. B, Specific NF-B/DNA binding activity in nuclear extracts was determined by electrophoretic mobility shift assay (EMSA). Results were normalized by arbitrarily setting the densitometry of the basal group, and are expressed as mean±SEM (n=3). *P<0.05 and **P<0.01, as compared with basal values.

As depicted in Figure 6B, NF-B activity was undetectable in nuclear proteins obtained from basal portal vein preparations. DNA binding activity was found to be significantly increased in nuclear extracts following 1 to 3 hours of in vitro incubation. After 6 hours, levels of nuclear NF-B returned toward baseline.

	Discussion

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Several reports have demonstrated the functional upregulation of B₁R-mediated responses by means of both in vitro and in vivo approaches. B₁R is usually absent from normal tissues, but its synthesis can be rapidly induced following some types of stimuli.¹⁰ As demonstrated previously,¹⁶ des-Arg⁹–BK-induced contraction of the rat portal vein increases progressively from initial low levels as a function of in vitro incubation time. This phenomenon was shown to be sensitive to the protein synthesis inhibitor cycloheximide, indicating its dependence on de novo protein synthesis. The same profile of B₁R induction has been described in other vascular smooth muscle preparations including the rabbit aorta,³² the human umbilical vein,³³ and the rabbit mesenteric artery,³⁴ among others.

Damage-induced B₁R upregulation can be mimicked by the in vitro incubation of infectious agents or by some inflammatory mediators (eg, endotoxins and IL-1ß).^10,35 IL-1ß has been shown to increase B₁R mRNA levels in rat aorta smooth muscle cells,²⁰ and to induce the enhancement of B₁R binding sites in human lung fibroblasts.^25,36 In addition, IL-1ß and TNF- were found to increase B₁R-mediated responses in human umbilical vein^18,37 and in rabbit aorta.³² Evidence suggests that B₁R induction by tissue damage could be related to the production of inflammatory cytokines. In fact, it has been demonstrated that upregulation of B₁R induced by tissue isolation in the rabbit aorta could be prevented by an IL-1 receptor antagonist.³⁸ Similarly, it was previously shown that the incubation of proinflammatory cytokines (eg, IL-1ß, TNF, or IL-6) induces a leftward shift of the des-Arg⁹–BK-induced concentration-dependent contractile response, whereas anti-inflammatory cytokines (such as IL-4) have an opposite effect in the human umbilical vein preparation.^18,37,39 The upregulation of B₁R following tissue damage and/or mechanical stress could also be associated with the production of other inflammatory mediators, with the stimulation of inflammatory cells, and, finally, with the activation of several intracellular signaling pathways, although the exact mechanisms involved in this process still remain unclear. For this reason, the present study was designed to investigate the possible mechanisms involved in the upregulation of B₁R induced by damage to the rat portal vein.

Extending previous data,¹⁶ our results demonstrated a marked and time-related increase of functional responsiveness involving B₁R activation, which was found to be sustained, remaining elevated at least until 12 hours following incubation of the rat portal vein. Our data also indicate that functional B₁R induction correlates with the increase of B₁R mRNA expression in this preparation. To our knowledge, this is the first evidence indicating that tissue trauma may lead to the increased expression of B₁R mRNA in a venous preparation using real-time quantitative RT-PCR.

Transcriptional factors of the NF-B family are regulated through interactions with inhibitory proteins called IBs. In most cell types, NF-B dimers are found in the cytoplasm associated with IBs, which prevents their nuclear localization.^40,41 This pathway is activated on appropriate cell stimulation by, for example, proinflammatory cytokines, viral infection, oxidants, phorbol esters, and ultraviolet irradiation. Cell stimulation leads to the rapid phosphorylation and polyubiquitination of the IBs, targeting these proteins for degradation by the 26S proteasome.^40–42 Degradation of IB allows the NF-B to translocate to the nucleus, where it regulates the transcription of several genes,^40,41 including the B₁R gene.^10,35 In fact, it was shown that in cultured human lung fibroblasts, IL-1ß-induced B₁R upregulation is controlled at the transcriptional level through NF-B activation.²² In addition, the postulated NF-B binding site on the B₁R promoter seems to be mainly responsible for its inducibility in response to IL-1ß, TNF-, and lipopolysaccharide.²⁰ In the present study, we have demonstrated that tissue damage produces an increase in NF-B DNA-binding activity, through a mechanism involving degradation of IB and subsequent translocation of NF-B into the nucleus. Furthermore, we have shown that treatment of rat portal vein with the IB kinase inhibitor BAY117082⁴³ or with the proteasome activity inhibitor MG132,^44,45 consistently blocked the upregulation of the B₁R-mediated contractile response as well as the increase in B₁R mRNA expression. These results confirm and extend previous studies showing that NF-B activation constitutes an important step in the regulation of B₁R gene expression.

The present study also provides evidence for the involvement of MAPK in the induction of B₁R in the rat portal vein after tissue damage. It is well recognized that MAPKs constitute one of the most important mammalian signaling pathways. In particular, 3 subfamilies of MAPKs (ERK, JNK, and p38 MAPK) have been extensively characterized. The stimulation of MAPKs cascades occurs in response to several stimuli,⁴⁶ such as mechanical stress, inflammatory cytokines (IL-1ß and TNF-), heat and chemical shock, or bacterial products, all of them also capable of stimulating B₁R induction.^10,35 Recent reports indicated that p38 MAPK and ERK pathways may be involved in the B₁R upregulation both in vitro and in vivo.^21,25,27 In the present study, we clearly confirmed these findings by demonstrating for the first time that tissue damage is able to induce the time-dependent activation of all three MAPKs (ERK, JNK, and p38 MAPK) in the rat portal vein. However, p38 MAPK and JNK, but not ERK inhibitors, were effective in suppressing the increase in both the des-Arg⁹–BK-contractile response and the expression of B₁R mRNA in the rat portal vein. In this tissue, the activation of ERK may exert other cellular effects unrelated to the increase in B₁R expression.

Several studies have indicated the participation of p38 MAPK and JNK in the regulation of inflammatory gene expression, acting both at transcriptional and post-transcriptional levels.^47–53 Likewise, the activation of these MAPKs has been implicated in the regulation of the half-lives of many inflammatory mediator RNAs, including cyclooxygenase 2 (COX-2), TNF, and IL-1ß.^51,54–56 Potential mRNA destabilizing AU-rich elements, often consisting of 1 to several copies of the sequence AUUUA, have been reported to exist in the 3'-untranslated regions (3'-UTR) of genes.⁵⁷ Moreover, other regions of the mRNA, such as the coding region, may also be involved in the regulation of mRNA stability.⁵⁸ Recently, the presence of instability regions was demonstrated in 3'-UTR B₁R mRNA.⁵⁹ In addition, 2 AUUUA motifs are found in the coding region of the B₁R, but their relevance to B₁R mRNA stability remains to be determined.⁵⁹ In this context, MAPKs activation could contribute to B₁R induction by increasing the stability of some proinflammatory proteins mRNAs (eg, IL-1ß and TNF), or even by enhancing the stability of B₁R mRNA. However, additional studies are necessary to confirm this hypothesis.

The activation of p38 MAPK and JNK has been linked to the increased activation of the transcription factors AP-1 and NF-B,^{47,49,60–62} which are known to be important regulators of B₁R gene expression.^20,22,63,64 The possible mechanisms involved in the crosstalk between MAPKs and NF-B are not yet completely understood. A recent study⁶² revealed that the p38 MAPK pathway does not affect NF-B binding and transcriptional activity directly, but rather it acts by enhancing NF-B recruitment into a subset of genes. Conversely, Tsai et al⁶⁵ reported that p38 MAPK signaling could promote the IB phosphorylation and degradation, directly activating the NF-B pathway. On the other hand, the mechanisms by which p38 MAPK and JNK modulate the AP-1 transcriptional activity are better understood and have been shown to be relevant for multiple cell responses.⁴⁷ Additional studies are necessary to clarify whether an interaction between MAPKs and NF-B could mediate trauma-induced B₁R upregulation. However, one could suggest that these pathways would work in a synergistic manner, resulting in the increased expression of B₁R in this preparation.

The current results provide clear functional and molecular evidence indicating that tissue injury of rat portal vein induces the activation of p38 MAPK and JNK as well as the transcriptional factor NF-B. Furthermore, our data also clearly show that activation of these pathways has a critical role in modulating functional and molecular upregulation of B₁R in rat portal vein, a process that could be of great relevance to the pathophysiology of the cardiovascular system.

	Acknowledgments

 
This study was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos, and by Programa de Apoio aos Núcleos de Excelência, Brazil. R.M. is a MSc student and holds a grant from CNPq. E.S.F., J.F., and M.A.S.M. are PhD students receiving grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CNPq, and Fundação de Amparo à Pesquisa do Estado de São Paulo, respectively. D.A.C. and M.M.C. are holders of postdoctoral fellowships from CNPq and CAPES, respectively.

	Footnotes

 
Original received February 16, 2004; revision received April 1, 2004; accepted April 7, 2004.

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