Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Vascular bradykinin (BK) B₁ receptors were first described in isolated rabbit anterior mesenteric vein by Regoli et al. (1978) after a long in vitro incubation. These authors postulated the de novo formation of BK B₁ receptors to account for this phenomenon. Thereafter, induction of BK B₁ responses was documented in different isolated tissue preparations (Marceau et al., 1998). BK B₁ receptors are not normally present in nontraumatized tissues, but their synthesis can be induced under certain physiopathological conditions such as tissue injury or inflammation, or during trauma tissue isolation and incubation (Marceau et al., 1998).

In isolated human umbilical vein (HUV), the BK B₁ receptor-mediated contractile response develops from an initial null level and increases in magnitude as a function of the in vitro incubation time (Sardi et al., 1997). This up-regulation process is dependent on the de novo synthesis of receptors since it is abolished by translation, transcription, and protein trafficking or glycosylation inhibitors (Sardi et al., 2000a). On the other hand, in isolated HUV, BK B₂ receptors are constitutively expressed and do not undergo additional induction (Sardi et al., 1997, 1998).

In vitro and in vivo studies have demonstrated a close link between inflammatory mediators and the expression of BK B₁ receptors (Campos et al., 1998; Marceau et al., 1998). In HUV, it has been reported that interleukin-1 (IL-1) or tumor necrosis factor- (TNF-) treatment potentiates BK B₁ receptor-mediated responses (Sardi et al., 1998, 1999). These cytokines have been linked to nuclear factor-B (NF-B) pathway activation (Baldwin, 1996). Furthermore, in this human tissue, the development of BK B₁ receptor-sensitized responses has been inhibited by anti-inflammatory agents that could be linked to NF-B pathway inactivation, such as dexamethasone, pyrrolidine-dithiocarbamate (PDTC), all-trans-retinoic acid or 9-cis-retinoic acid (Sardi et al., 1998, 1999, 2000b).

NF-B is a ubiquitously expressed transcription factor that consists of homodimers or heterodimers of a family of structurally related proteins (Baldwin, 1996; Ghosh et al., 1998). In most cell types, it is present as a heterodimer comprising p65 and p50 subunits, which is held in an inactive form in the cytosol by interaction with a member of the I-B family of inhibitory proteins (Verma and Stevenson, 1997; Karin, 1998). NF-B is activated by the phosphorylation and subsequent degradation of I-B in the proteasome, which results in translocation of the liberated NF-B to the nucleus where it induces transcription of target genes that mediate an acute inflammatory response (Baldwin, 1996; Baeuerle and Baichwal, 1997). A wide variety of noxious stimuli, such as viral and bacterial infection, UV light, ionizing radiation, and free radicals, as well as a variety of lymphokines and cytokines, activate the NF-B pathway.

The aim of this study was to obtain further pharmacological evidence of NF-B pathway involvement on the BK B₁ receptor sensitization process in HUV and to evaluate the effects of some pro- and anti-inflammatory cytokines in this human model. Therefore, we evaluated the effects of an I-B kinase inhibitor, Bay 11-7082 (3-(4-methylphenylsulfonyl)-2-propenenitrile), a proteasome activity inhibitor, MG-132 (Z-Leu-Leu-Leu-CHO), and cytokines known to affect the NF-B pathway, such as interleukin-6 (IL-6), interleukin-4 (IL-4) and tumor growth factor-1 (TGF-1) on concentration-response curves to the selective BK B₁ receptor agonist des-Arg⁹-BK in this tissue.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Preparation of Tissues for Tension Measurements. Human umbilical cords excised midway between the placenta and infant were obtained from normal full-term deliveries. Immediately, cords were placed in modified Krebs' solution at 4°C (of the following composition: 119 mM NaCl, 4.7 mM KCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.0 mM MgSO₄, 0.004 mM EDTA, 11 mM D-glucose). Written informed consent was obtained from each parturient woman. The time from delivery until the tissue was set up in the organ bath was approximately 3 h. The cords were placed onto dissecting dishes containing Krebs' solution. Veins were carefully dissected free from Warthon's jelly using micro-dissecting instruments and cut into rings of approximately 3 mm width. The equilibration period started when the tissues were suspended in 10-ml organ baths. The veins were stretched with an initial tension of 3 to 5 g as described previously (Errasti et al., 1999). Changes in tension were measured with Grass isometric transducers (FT-03C; Grass Instruments, Quincy, MA) and displayed on a Grass polygraph (model 7D). During the incubation period, Krebs' solution was maintained at 37°C and at pH 7.4 by constant bubbling with 95% O₂/5% CO₂. Bath solution was replaced every 15 min, and test agents were added after every wash. After 70 min of equilibration, each preparation was contracted with KCl (40 mM) to test its functional state. Optimal passive tension was adjusted throughout the equilibration period.

Cumulative Concentration-Response Curves. After a 5-h equilibration period, cumulative concentration-response curves were obtained for des-Arg⁹-BK, BK B₁ receptor-selective agonist, or serotonin (5-HT; 5-hydroxytryptamine), a BK unrelated agonist. Only one agonist concentration-response curve was performed on a single ring. Tissues were incubated with captopril (1 µM) 30 min before the BK receptor stimulation to avoid peptide degradation by kininase II (angiotensin-converting enzyme).

Chemicals and Solutions. BK and captopril were purchased from Sigma-Aldrich (St. Louis, MO). Bay 11-7082 and MG-132 were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA), and des-Arg⁹-BK was obtained from Bachem California (Torrance, CA). Human recombinant IL-6, IL-4, and TGF-1 were a generous gift from Dr. Miguel Mamone, Productos Roche (Buenos Aires, Argentina).

Expression of Results and Statistical Analysis. All data are presented as mean ± S.E.M. Responses are expressed as grams of developed contraction. The pEC₅₀ values, negative logarithm of the agonist concentration that produces 50% of the maximum, were determined using ALLFIT (National Institutes of Health, Bethesda, MD), a nonlinear curve-fitting computer program (De Lean et al., 1978). The pEC₅₀ values between control and treated tissues were compared only when their maximal responses were not significantly different. Statistical analysis was performed by means of paired Student's t test, and p < 0.05 were taken to indicate significant differences between means.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of Bay 11-7082 on BK B₁ Receptor-Sensitized Responses in HUV. Some HUV rings were exposed to Bay 11-7082 (3, 10, and 30 µM) during 5 h to examine the possible effect of this inhibitor of I-B kinase on the BK B₁ receptor sensitization process. Bay 11-7082 demonstrated a dose-dependent depressor effect (Table 1). Only Bay 11-7082 (10 µM) produced a rightward shift and a significant reduction of the maximal response of the concentration-response curve to des-Arg⁹-BK (Fig. 1A; Table 1), without affecting the maximal response to BK (0.1 µM) at the end of each experiment (control: 17.9 ± 1.4 g, treated: 16.6 ± 1.7 g, n = 6). On the other hand, when tissues were exposed to Bay 11-7082 (30 µM), the maximal response to des-Arg⁹-BK was significantly depressed (Table 1), as well as the tissue maximal response (BK, 0.1 µM) at the end of each experiment (control, 11.9 ± 2.6 g; treated, 1.0 ± 0.5 g; p < 0.01, n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Concentration-response curves for des-Arg9-BK (A and B) or 5-HT (C) on control HUV rings () and on tissues exposed to the I-B kinase inhibitor, Bay 11-7082 (10 µM, ). Tissues were incubated with Bay 11-7082 for 5 h (A, n = 6; C, n = 7) or for the last 30 min (B, n = 6). The points represent the mean of n determinations made after a 5-h equilibration period, and the vertical lines show S.E.M. #, significant differences (p < 0.05) between maximal responses.

Effect of MG-132 on BK B₁ Receptor-Sensitized Responses in HUV. To evaluate the possible involvement of the proteasome on the BK B₁ receptor sensitization phenomenon in isolated HUV, rings were exposed to MG-132 (0.3, 1, and 10 µM) during 5 h. The proteasome inhibitor demonstrated a dose-dependent depressor effect on this process (Table 1). Only MG-132 (1 µM) produced a rightward shift and a significant reduction of the maximal response of the concentration-response curve to des-Arg⁹-BK (Fig. 2A; Table 1) without affecting the maximal response to BK (0.1 µM) at the end of each experiment (control, 17.2 ± 1.5 g; treated; 14.8 ± 1.7 g; n = 7). When tissues were exposed to a higher concentration of this proteasome inhibitor (10 µM), the maximal response to the BK B₁ receptor agonist was significantly diminished (Table 1), as was the tissue maximal response (BK, 0.1 µM) at the end of each experiment (control, 22.2 ± 2.3 g; treated, 16.3 ± 2.1 g; p < 0.01, n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for des-Arg9-BK (A and B) or 5-HT (C) on control HUV rings () and on tissues exposed to the proteasome inhibitor, MG-132 (1 µM, ). Tissues were incubated with MG-132 for 5 h (A, n = 7; C, n = 6) or for the last 30 min (B, n = 7). The points represent the mean of n determinations made after a 5-h equilibration period, and the vertical lines show S.E.M. * and #, significant differences (p < 0.05) between pEC50 and maximal responses, respectively.

Effect of Recombinant Human Interleukin-6 on BK B₁ Receptor-Sensitized Responses in HUV. The possible potentiating effect of this pro-inflammatory cytokine on the BK B₁ receptor sensitization process at 5 h was evaluated by incubating HUV rings with recombinant human IL-6 (10 ng/ml) for the initial 3 h. The cytokine treatment produced a significant leftward shift of the concentration-response curve to des-Arg⁹-BK at 5 h (Fig. 3; Table 1). However, maximal response to the BK B₁ receptor-selective agonist was unaffected by IL-6 treatment (Fig. 3; Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves for des-Arg9-BK on control HUV rings () and on tissues exposed to interleukin-6 for the initial 3 h (10 ng/ml, , n = 6). The points represent the mean of n determinations made after a 5-h equilibration period, and the vertical lines show S.E.M. *, significant differences (p < 0.05) between pEC50 values.

Effect of Recombinant Human Interleukin-4 on BK B₁ Receptor-Sensitized Responses in HUV. Rings were exposed to the anti-inflamatory cytokine IL-4 (20 ng/ml) for 5 h to evaluate its possible depressor effect on the BK B₁ receptor-sensitized responses. This cytokine produced a significant rightward shift of the concentration-response curve for des-Arg⁹-BK without reduction of the maximal response (Fig. 4A; Table 1). On the other hand, when HUV rings were exposed to IL-4 (20 ng/ml) for the last 30 min, the curve for the BK B₁ receptor agonist was not modified, thus discarding any acute effect of this treatment (Fig. 4B; Table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves for des-Arg9-BK on control HUV rings () and on tissues exposed to interleukin-4 (20 ng/ml, ). Tissues were incubated with interleukin-4 for 5 h (A, n = 5) or for the last 30 min (B, n = 4). The points represent the mean of n determinations made after a 5-h equilibration period, and the vertical lines show S.E.M. *, significant differences (p < 0.05) between pEC50 values.

Effect of Recombinant Human Tumor Growth Factor-1 on BK B₁ Receptor-Sensitized Responses in HUV. The possible depressor effect of this anti-inflammatory cytokine on the BK B₁ receptor-sensitized responses was evaluated by incubating HUV rings with TGF-1 (3 ng/ml) for 5 h. This treatment produced a significant rightward shift of the concentration-response curve for BK B₁ receptor agonist, without affecting the maximal response (Fig. 5A; Table 1). On the other hand, the curve for des-Arg⁹-BK was not modified when HUV rings were exposed to TGF-l (3 ng/ml) for the last 30 min (Fig. 4B; Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Concentration-reponse curves for des-Arg9-BK on control HUV rings () and on tissues exposed to tumor growth factor-1 (3 ng/ml, ). Tissues were incubated with tumor growth factor-1 for 5 h (A, n = 6) or for the last 30 min (B, n = 5). The points represent the mean of n determinations made after a 5-h equilibration period, and the vertical lines show S.E.M. *, significant differences (p < 0.05) between pEC50 values.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Since the cloning of the BK B₁ receptor in 1994 by Menke et al., several groups have begun to determine the transduction mechanisms involved in its induction (Bachvarov et al., 1996; Yang and Polgar, 1996; Ni et al., 1998; Schanstra et al., 1998; Phagoo et al., 2001). The 5'-flanking region of the human BK B₁ receptor gene bears putative NF-B as well as activator protein-1 binding motifs, a promoter organization consistent with an activation by cytokines, such as IL-1 or TNF- (Bachvarov et al., 1996). Using transfected cultured cells, Ni et al. (1998) have demonstrated that NF-B is involved in the inducible expression of the human BK B₁ receptor gene during inflammatory processes. In resting cells, NF-B is held inactive in the cytosol by association with inhibitory proteins of the I-B family (Baldwin, 1996). When the NF-B pathway is activated by agents such as lipopolysaccharide, IL-1, and TNF-, a phosphorylation-dependent proteolytic degradation of I-B is initiated, allowing NF-B to translocate into the nucleus (Baldwin, 1996). In HUV, it has been proposed that NF-B is involved in the expression of BK B₁ receptor-mediated contractions after a prolonged in vitro incubation (Sardi et al., 1999, 2000b), since these responses are inhibited by continuous exposure to agents with NF-B pathway inhibitory activity such as PDTC, dexamethasone, or retinoids (Traenckner et al., 1994; Gille et al., 1997; Ni et al., 1998). Pierce et al. (1997) have reported that Bay 11-7082 prevents I-B phosphorylation as well as its subsequent degradation, thus inhibiting NF-B activation. Moreover, MG-132 has also been described to inhibit NF-B translocation into the nucleus by interfering with the proteasome I-B degradation (Palombella et al., 1994; Grisham et al., 1999). In HUV, contractile responses to des-Arg⁹-BK were inhibited in a dose-dependent manner by continuous incubation with either Bay 11-7082 or MG-132 (Table 1). When both agents were used in effective depressor doses (MG-132, 1 µM; Bay 11-7082, 10 µM) for the last 30 min of incubation, the BK B1-sensitized response was not modified, thus discarding any toxic effects. Furthermore, these actions are apparently selective, because the maximal responses obtained at 5 h to BK (a BK B₂ receptor agonist) or to 5-HT (a BK receptor unrelated agonist) were not modified. Taken together, the present inhibitory effects of Bay 11-7082 and MG-132, and the previously published effects of PDTC, dexamethasone, and retinoids, on the BK B₁ receptor responses support the notion that NF-B pathway activation may play a role in the development of this sensitization process in HUV.

Studies in different tissues provide evidence that several pro-inflammatory cytokines, such as IL-1 and TNF-, are involved in BK B₁ receptor induction (Marceau et al., 1998). IL-1 and TNF- have been shown to increase BK B₁ receptor mRNA levels in rat aorta smooth muscle cells through NF-B activation (Ni et al., 1998). IL-1 induces the expression of BK B₁ binding sites in human lung fibroblasts (Zhou et al., 1998). IL-1 or TNF- produces potentiation on the BK B₁ receptor sensitization process in HUV (Sardi et al., 1998, 1999). IL-6 belongs to a group of structurally related cytokines involved in inflammatory responses, and its signaling pathway is still unclear. However, it has been shown that this pro-inflammatory cytokine can activate the NF-B pathway (Middleton et al., 2000). We therefore consider it interesting to examine the possible effects of this pro-inflammatory cytokine on the BK B₁ receptor sensitization process. In HUV rings, IL-6 treatment potentiated the contractile responses induced by the BK B₁ receptor-selective agonist, des-Arg⁹-BK. To our knowledge, this is the first report showing that IL-6 induces potentiation on the BK B₁ receptor sensitization phenomenon, in accord with previous results using other pro-inflammatory cytokines, as indicated above.

The anti-inflammatory activity of IL-4 has been shown to target NF-B-dependent gene expression (Donnelly et al., 1993; Takeshita et al., 1996; Bennett et al., 1997). In several cases this has been reported to involve either the inhibition of NF-B-specific DNA binding activity or competition between signal transducer and activator of transcription-6 (STAT6) and NF-B for a shared or overlapping binding site. Recently, Ohmori and Hamilton (2000) have proposed an alternative mechanism for the antagonistic effect of IL-4 on NF-B-dependent transcription. These authors postulated that IL-4-activated STAT6 and NF-B might compete for a limited supply of transcriptional coactivator cyclic AMP response element-binding protein. In our preparations of HUV rings, contractile-sensitized responses to the selective BK B₁ receptor agonist were inhibited by continuous incubation with this anti-inflammatory cytokine (Fig. 4A).

In human salivary gland cells and in murine B-lymphocytes, TGF-1 has been shown to inhibit NF-B activity through induction of I-B expression (Arsura et al., 1996; Azuma et al., 1999). In HUV, the concentration-response curves to des-Arg⁹-BK at 5 h were antagonized by continuous exposure to TGF-1. Moreover, when tissues were incubated with the anti-inflammatory cytokines, IL-4 or TGF-1, for the last 30 min, the curves for des-Arg⁹-BK were not different from that of controls, thus showing the lack of toxic effect of these cytokines (Figs. 4B and 5B).

In summary, the depressor effects observed with the cytokines IL-4 and TGF-1 on BK B₁ receptor sensitization process are in accord with its anti-inflammatory properties. Moreover, to our knowledge, this is the first report showing these effects.

BK B₁ receptor sensitization in the HUV is a well characterized model of the up-regulation process in a human tissue (for review see Sardi et al., 2000). The present data obtained in this tissue with the inhibitors of the NF-B pathway, Bay 11-7082 and MG-132, provide additional pharmacological evidence that this pathway may play a role in BK B₁ receptor-sensitized responses, and the observations with IL-6, IL-4, and TGF-1 support the role of inflammatory mediators in modulating BK B₁ sensitization. On the other hand, the results obtained with these cytokines, promoting a leftward or a rightward shift of the concentration-response curve to des-Arg⁹-BK without affecting the maximal response, support the presence of BK B₁ spare receptors in the HUV under control conditions after a 5-h incubation period, as previously suggested (Sardi et al., 1998, 1999). The assessment of NF-B activity/level modulation should be performed to further support the hypothesis of the involvement of the NF-B pathway on the BK B₁ receptor up-regulation process in HUV.