Elsevier

Regulatory Peptides

Volume 97, Issues 2–3, 2 March 2001, Pages 121-130
Regulatory Peptides

The ‘in vivo’ and ‘ex vivo’ roles of cylcooxygenase-2, nuclear factor-κB and protein kinases pathways in the up-regulation of B1 receptor-mediated contraction of the rabbit aorta

https://doi.org/10.1016/S0167-0115(00)00186-5Get rights and content

Abstract

This study investigates some of the mechanisms involved in the up-regulation of the B1 receptor in the rabbit aorta. Pre-treatment of rabbit aorta with cyclooxygenase (COX) inhibitors 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsuphonyl) phenyl-2 (5H)-furanone (DFU), N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide (NS-398) or with indomethacin, but not with piroxicam, for 6 h, resulted in a significant inhibition of time-dependent contraction to the B1 selective agonist des-Arg9-Bradykinin (des-Arg9-BK), without affecting noradrenaline (NA) response. The kinase inhibitors bisindoylmaleimidine IX (RO 318220), staurosporine, genistein or tyrphostin B42 and the nuclear factor-κB (NF-κB) inhibitors pyrrolidinedithiocarbamate (PDTC), Nα-p-tosyl-l-lysine chloro-methyl ketone (TLCK) or sulfasalazine, incubated for 6 h each, resulted in similar inhibition of des-Arg9-BK-induced contraction. When these inhibitors were pre-incubated for only 30 min, 6 h after setting up the preparations, sulfasalazine was the only drug tested that inhibited des-Arg9-BK-induced contraction, an effect which was reverted after the washing-out of the preparations. In preparations obtained from animals treated with lipopolysaccharide i.v. (LPS) 12 h prior, the up-regulation of B1 receptor in the aorta was markedly increased. The treatment of rabbits with PDTC, dexamethasone (Dexa), genistein or an association of subliminal doses of Dexa or with PDTC 12 h prior, which alone had no effect, all caused significant inhibition of des-Arg9-BK-induced contraction in the rabbit aorta. These results indicate that the time-dependent up-regulation of des-Arg9-BK-mediated contraction in the rabbit aorta involves the activation of protein kinase C, tyrosine kinase, through participation of COX-2 and the NF-κB transcription factor pathways.

Introduction

Kinins are vasoactive peptides that are generated in the plasma and tissues from α2-globulins called kininogens, following tissue trauma or accompanying inflammation [1]. Once released, kinins produce many physiological and pathological actions, such as smooth muscle contraction, vasodilatation, increased vascular permeability and pain induction. All these actions are mediated by activation of their specific cell surface receptors [2]. Based on structure activity studies, it was first proposed by Regoli and Barabé [3] the existence of two kinin receptors, B1 and B2. The B2 receptors, widely distributed in the central and peripheral tissues, are known to be normally constitutive and are the predominant biological receptors, mediating most of the physiological activities commonly associated with kinin responses [4]. Differently, B1 receptors with rare exceptions are not normally present in non-traumatized tissues, but their synthesis can be induced over a period of time either in vitro or in vivo by a wide variety of noxious stimuli, such as pro-inflammatory cytokines, after long-term in vitro incubation of the tissue, or by in vivo treatments with E. coli lipopolysaccharide, Mycobacterium bovis bacillus Calmette-Guérion (BCG), chemical colitis or cystitis, immune complex arthritis, or following in vivo desensitization of B2 receptor [3], [5], [6], [7], [8]. It has been demonstrated that the up-regulation of B1 receptors can be inhibited either in vitro or in vivo by glucocorticoids, an effect that can be explained by the suppression of interleukin-1 (IL-1) synthesis and perhaps also by an additional action more proximal to the B1 receptor expression [9].

The B1 receptors exhibit higher affinity for des-Arg9-BK and for Lys-des-Arg9-BK (Lys-kallidin), the breakdown products of BK and Lys-BK, formed through the action of carboxypeptidase N, than for BK itself [3], [10], [11]. The genes encoding B1 receptors have already been cloned and sequenced from humans, rabbits, rats and mice, and its cDNA reveal that it is a member of the G-protein-coupled superfamily of receptors with seven transmembrane domains and that it is likely to be processed through the endoplasmic reticulum–Golgi pathway after its ribosomal synthesis [12], [13], [14], [15], [16], [17]. In all species studied, the B1 receptor showed a highly conserved homology, especially between rabbits and humans (68–78%) [18].

The isolated rabbit aorta is known to present only B1 receptors for kinins, and for this reason this selected bioassay is normally employed to enable study the functional up-regulation of B1 receptors [7], [19], [20]. One particular characteristic of this tissue is the fact that it does not respond to the typical B1 agonist, des-Arg9-BK, in the first hour of incubation, but the later time-dependent contractile responses are prevented if the tissues are continuously treated with a protein synthesis inhibitor (cycloheximide) or with inhibitors of protein trafficking (brefeldin A), or even with glucocorticoids [7], [8], [21], [22]. Recently, Larrivée et al. [23] showed, using the rabbit isolated aorta as model of injury, that different kinase pathways, including tyrosine kinase, mitogen-activated protein kinase (MAPK) and p38 MAPK have a critical role in controlling the spontaneous or cytokine up-regulation of the B1 agonist des-Arg9-BK-mediated contraction in this preparation. Schanstra et al. [24] reported that the expression of B1 receptor in cultured human lung fibroblasts, in response to IL-1β, is modulated at the transcriptional level through the activation of NF-κB pathway. In addition, the presence of NF-κB-like sequence on B1 receptor promoter, which seems to be the responsible for the process of B1 receptor induction in response to IL-1β, TNFα or LPS treatment, has been also demonstrated in the vascular smooth muscle cells [25]. In spite of the recent progress in understanding some of the mechanisms underlying the expression of B1 receptors in most of the tissues, many aspects involved in the up-regulation of B1 receptors still remain a mater of debate.

Therefore, the purpose of the present study was to investigate further the in vitro and ex vivo the effect of cyclooxygenases (COX1 and COX2), some kinases and the transcriptional factor NF-κB, on the up-regulation of B1 receptor-mediated contraction in the rabbit isolated aorta.

Section snippets

Tissue preparation

The thoracic aorta was isolated from New Zealand white rabbits of either sexes (2–3 kg). Animals were sacrificed with an overdose of pentobarbital (60 mg kg−1, i.v.; given through the ear vein) and exsanguinated from carotid arteries. The thoracic aorta was rapidly removed, carefully dissected from adhering tissues, and cut into rings (∼4 mm in diameter, 2–3 mm in length; 14–20 mg in weight). Preparations were suspended between a metal hook and thread loop under a basal tension of 2 g and were

In vitro experiments

As reported previously [7], a nearly maximal concentration of des-Arg9-BK (1 μM) exerted no contractile effect when applied 1 h after the beginning of the in vitro incubation of isolated rabbit aorta (results not shown). However, at the time points 2, 4 and 6 h a time-dependent increase in the contractile response was noted. When responses were calculated as percentage of KCl (40 mM)-mediated contraction, des-Arg9-BK contraction corresponds to approximate 8±2, 26±3 and 38±4% at 2, 4 and 6 h

Discussion

The data presented in the current study provides convincing evidence that the in vitro and ex vivo treatments with inhibitors of NF-kB and several kinase inhibitors, namely tyrosine kinase and protein kinase C, the COX-2 but not the COX-1 inhibitor, all significantly prevented the time-dependent up-regulation of the selective B1 agonist des-Arg9-BK-mediated contraction in the rabbit aorta in vitro. Such results further extend the molecular and available functional data concerning the mechanisms

Acknowledgements

The authors are grateful to Rosana Maria Ostroski, Eunice André and Ms. Maria Martha Campos for technical support. This work was supported by grants from CNPq and FINEP. We thank the pharmaceutical companies for the kind donation of some of the drugs used in this work. R. Medeiros is a pharmacist undergraduate student receiving a grant from CNPq (Brazil) and D.A. Cabrini is a PhD student in Pharmacology receiving a grant from CAPES (Brazil).

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