Characterization of dual agonists for kinin B1 and B2 receptors and their biased activation of B2 receptors

Kinin B1 and B2 receptors (kB1R and kB2R) play important roles in many physiological and pathological processes. In some cases, kB1R or kB2R activation can have overlapping or complementary beneficial effects, thus an activator of both receptors might be advantageous. We found that replacement of the C-terminal Arg in the natural kB2R activators bradykinin (BK) or kallidin (KD) with Lys (K(9)-BK or K(10)-KD) resulted in agonists that effectively stimulate the downstream signaling of both the kB1R and kB2R as measured by increased inositol turnover, intracellular calcium, ERK1/2 phosphorylation, arachidonic acid release and NO production. However, K(9)-BK and K(10)-KD displayed some characteristics of biased agonism for kB2Rs as indicated by the rapid kinetics of ERK1/2 phosphorylation induced by K(9)-BK or K(10)-KD compared with the prolonged response mediated by BK or KD. In contrast, kinetics of ERK phosphorylation stimulated by K(10)-KD activation of the kB1R was the same as that induced by known kB1R agonist des-Arg(10)-KD. Furthermore, the endocytosis of kB2Rs mediated by K(9)-BK and K(10)-KD was remarkably less than that induced by BK and KD respectively. K(10)-KD stimulated kB1R and kB2R-dependent calcium responses and ERK1/2 phosphorylation in bovine endothelial cells. In cytokine-treated human endothelial cells, K(10)-KD stimulated ERK1/2 phosphorylation and a transient peak of NO production that was primarily kB2R-dependent. K(10)-KD also stimulated prolonged NO production that was both kB1R and kB2R-dependent. These data provide the first examples of dual agonists of kB1R and kB2R, and a biased agonist of kB2R and may provide useful clues for developing dual modulators of kB1Rs and kB2Rs for potential therapeutic use.     

Differential role of kinin B1 and B2 receptors in ischemia-induced apoptosis and ventricular remodeling

We investigated the role of kinin receptors in cardiac remodeling after ischemia/reperfusion (I/R). Bradykinin injection improved cardiac contractility, diastolic function, reduced infarct size and prevented left ventricular thinning after I/R, whereas des-Arg(9)-BK injection had no protective effects. Bradykinin, but not des-Arg(9)-BK, reduced cardiomyocyte apoptosis and increased Akt and GSK-3beta phosphorylation. Furthermore, myocardial infarct size was similar between wild type and B2 knockout mice after I/R, but significantly reduced in kinin B1 receptor knockout mice. These results indicate that the kinin B2 receptor, but not the B1 receptor, protects against I/R-induced cardiac dysfunction by inhibiting apoptosis and limiting ventricular remodeling.     

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

We hypothesized that the inducible kinin B(1) receptor (B(1)R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B(1)Rs and related B(2) receptors (B(2)Rs) was investigated. Endocytosis of the B(1)R-yellow fluorescent protein (YFP) conjugate was more intense than that of B(2)R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B(1)R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B(2)R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B(1)R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B(2)R-GFP remained constant. Wild-type B(1)Rs were also cleared faster than B(2)Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B(1)Rs are more rapidly eliminated than B(2)Rs (decreased maximal effect of agonist over 2 h). The highly regulated B(1)R is rapidly degraded, relative to the constitutive B(2)R.     

Expression of tissue and plasma kallikreins and kinin B1 and B2 receptors in lung cancer

Abstract Tissue kallikrein (hK1) and plasma kallikrein (PK, hKB1) are serine proteases that produce biologically active kinin peptides from endogenous kininogen substrates. There is evidence linking the kallikreins and the mitogenic kinin peptides to carcinogenesis. The aim of this study was to investigate the expression of tissue prokallikrein (pro-hK1), plasma prekallikrein (PPK, pre-hKB1) and kinin B(1) and B(2) receptor proteins in different subtypes of lung cancer. Immunohistochemistry, using specific antibodies, was performed on archived normal lung sections and sections from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, small cell carcinomas and carcinoid tumours of the lung. Immunoperoxidase labelling was visualised by brightfield microscopy and immunofluorescence labelling by confocal microscopy. Extensive cytoplasmic expression of pro-hK1 and PPK was observed, which was similar in small cell and non-small cell tumours. However, nuclear labelling for the kallikreins was absent or limited. The kinin B(1) and B(2) receptors were highly expressed in the cytoplasm of all tumour types and in the nuclei of non-small cell tumours. Further studies are required to assess the functional significance of the expression of hK1, PK and kinin receptors in lung tumours, and whether any of these proteins may be potential biomarkers for specific subtypes of lung carcinoma.     

Carboxypeptidase M and kinin B1 receptors interact to facilitate efficient b1 signaling from B2 agonists

Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg(9)-bradykinin or des-Arg(10)-kallidin. Carboxypeptidase M (CPM) is a membrane protein potentially well suited for this function. Here we show that CPM expression is required to generate a B1R-dependent increase in [Ca(2+)](i) in cells stimulated with B2R agonists kallidin or bradykinin. CPM and the B1R interact on the cell membrane, as shown by co-immunoprecipitation, cross-linking, and fluorescence resonance energy transfer analysis. CPM and B1R are also co-localized in lipid raft/caveolin-enriched membrane fractions, as determined by gradient centrifugation. Treatment of cells co-expressing CPM and B1R with methyl-beta-cyclodextrin to disrupt lipid rafts reduced the B1R-dependent increase in [Ca(2+)](i) in response to B2R agonists, whereas cholesterol treatment enhanced the response. A monoclonal antibody to the C-terminal beta-sheet domain of CPM reduced the B1R response to B2R agonists without inhibiting CPM. Cells expressing a novel fusion protein containing CPM at the N terminus of the B1R also increased [Ca(2+)](i) when stimulated with B2R agonists, but the response was not reduced by methyl-beta-cyclodextrin or CPM antibody. A B1R- and CPM-dependent calcium signal in response to B2R agonist bradykinin was also found in endothelial cells that express both proteins. Thus, a close relationship of B1Rs and CPM on the membrane is required for efficiently generating B1R signals, which play important roles in inflammation.     

Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors

Bradykinin (BK) and des-Arg⁹-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B₂ (B₂R) and B₁ (B₁R) receptors, respectively. It was already shown that the deletion of kinin B₁R or of B₂R induces upregulation of the remaining receptor subtype [10], [12], [16], [28], [36]. However studies on overexpression of B₁R or B₂R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined [17], [19], [33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B₁R (TGR(Tie₂B₁)) exclusively in the endothelium. In another study, mice overexpressing B₁R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B₂R was investigated in the thoracic aorta isolated from TGR(Tie₂B₁) rats overexpressing the B₁R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B₂R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie₂B₁) rats could be due to the enhanced expression of B₂R associated with the overexpression of the B₁R.     

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.     

Plasma extravasation mediated by lipopolysaccharide-induction of kinin B1 receptors in rat tissues.

The present study was performed to: (a) evaluate the effects of kinin B1 (Sar[D-Phe8]-des-Arg9-BK; 10 nmol/kg) and B2 (bradykinin (BK); 10 nmol/kg) receptor agonists on plasma extravasation in selected rat tissues; (b) determine the contribution of a lipopolysaccharide (LPS) (100 microg/kg) to the effects triggered by B1 and B2 agonists; and (c) characterize the selectivity of B1 ([Leu8]desArg9-BK; 10 nmol/kg) and B2 (HOE 140; 10 nmol/kg) antagonists as inhibitors of this kinin-induced phenomenon. B1 and B2 agonists were shown to increase plasma extravasation in the duodenum, ileum and also in the urinary bladder of the rat. LPS pretreatment enhanced the plasma extravasation mediated only by the B1 agonist in the duodenum, ileum, trachea, main and segmentar bronchi. These effects were prevented by the B1. but not the B2 antagonist. In normal rats, the B2 antagonist inhibited the effect of B2 agonist in all the tissues analyzed. However, in LPS-treated rats, the B2 antagonist was ineffective in the urinary bladder. These results indicate that kinins induce plasma extravasation in selected rat tissues through activation of B1 and B2 receptors, and that LPS selectively enhances the kinin effect on the B1 receptor in the duodenum, ileum, trachea and main and segmentar bronchi, and may increase B1 receptor expression in these tissues.     

Mechanisms of angiotensin II-induced expression of B2 kinin receptors

Although the primary roles of the kallikreinkinin system and the renin-angiotensin system are quite divergent, they are often intertwined under pathophysiological conditions. We examined the effect of ANG II on regulation of B(2) kinin receptors (B2KR) in vascular cells. Vascular smooth muscle cells (VSMC) were treated with ANG II in a concentration (10(-9)-10(-6) M)- and time (0-24 h)-dependent manner, and B2KR protein and mRNA levels were measured by Western blots and PCR, respectively. A threefold increase in B2KR protein levels was observed as early as 6 h, with a peak response at 10(-7) M. ANG II (10(-7) M) also increased B2KR mRNA levels twofold 4 h after stimulation. Actinomycin D suppressed the increase in B2KR mRNA and protein levels induced by ANG II. To elucidate the receptor subtype involved in mediating this regulation, VSMC were pretreated with losartan (AT(1) receptor antagonist) and/or PD-123319 (AT(2) receptor antagonist) at 10 microM for 30 min, followed by ANG II (10(-7) M) stimulation. Losartan completely blocked the ANG II-induced B2KR increase, whereas PD-123319 had no effect. In addition, expression of B2KR mRNA levels was decreased in AT(1A) receptor knockout mice. Finally, to determine whether ANG II stimulates B2KR expression via activation of the MAPK pathway, VSMC were pretreated with an inhibitor of p42/p44(mapk) (PD-98059) and/or an inhibitor of p38(mapk) (SB-202190), followed by ANG II (10(-7) M) for 24 h. Selective inhibition of the p42/p44(mapk) pathway significantly blocked the ANG II-induced increase in B2KR expression. These findings demonstrate that ANG II regulates expression of B2KR in VSMC and provide a rationale for studying the interaction between ANG II and bradykinin in the pathogenesis of vascular dysfunction.     

Classification of kinin receptors

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1 receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK > or = LysBK > BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC50 8.4) > [Leu8]desArg9BK (pIC50 7.4) > HOE 140, a B2 receptor antagonist, pIC50<5.0), and the B2 receptor of the rabbit jugular vein (potency of agonists: LysBK = BK > LysdesArg9BK = desArg9BK and HOE 140 (pIC50 9.0) > Lys[Leu8]desArg9BK, pIC50<5.0). The second part describes species-related B1 receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK > desArg9BK and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1 receptors (desArg9BK = LysdesArg9BK and [Leus]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their N-terminus. The last part describes species-related B2 receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2 receptors. Various hypotheses are considered to interpret these findings.     

SV2B regulates synaptotagmin 1 by direct interaction

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.     

Lack of a direct nitrate-trifoliin A interaction in the Rhizobium-clover symbiosis

Summary Nitrate added at critical concentrations to plant growth medium inhibits the infection of legume roots by Rhizobium. The direct interaction, of nitrate and trifoliin A, a Rhizobium-recognition lection from white clover (Trifolium repens L.), was examined as a possible basis for this regulation. Selective molecular ultrafiltration studies to detect ligand-protein interactions showed that radioactive¹³NO₃ ⁻ did not bind directly to trifoliin A when incubated at two molar ratios. Immunoprecipitation of trifoliin A by its homologous antibody was unaffected by 15 mM NO₃ ⁻. In addition, there was no apparent reduction in attachment ofR. trifolii 0403 to root hairs of clover seedings during 1 h of incubation in the presence of 15 mM NO₃ ⁻. These results show that nitrate inhibition of these early steps of the infection process is not due to a direct interaction of nitrate with trifoliin A or its glycosylated receptors.     

Molecular identification and pharmacological profile of the bovine kinin B1 receptor

To support the study of kinin pharmacology in bovine models of cultured endothelial cells (ECs), the Bovine Genome Project was searched for a B1 receptor (B1R) gene ortholog (BDKRB1). A contig complementary to an intronless coding nucleotide sequence was found. The sequence was amplified from bovine EC DNA, further cloned into pcDNA3 and expressed in COS-1 cells. The bovine B1R sequence was confirmed and extended. A putative Zn2+-binding motif HEXXH is not present (replaced by HDAWP). The receptor binds [3H]Lys-des-Arg9-bradykinin in a saturable manner (K(d) 0.36 nM) and exhibits a pharmacological profile similar to that of human B(1)R.     

A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone

Background

Multimeric naphthoquinones are redox-active compounds that exhibit antineoplastic, antiprotozoal, and antiviral activities. Due to their multimodal effect on perturbation of cellular oxidative state, these compounds hold great potential as therapeutic agents against highly proliferative neoplastic cells. In our previous work, we developed a series of novel dimeric naphthoquinones and showed that they were selectively cytotoxic to human acute myeloid leukemia (AML), breast and prostate cancer cell lines. We subsequently identified the oxidoreductase NAD(P)H dehydrogenase, quinone 1 (NQO1) as the major target of dimeric naphthoquinones and proposed a mechanism of action that entailed induction of a futile redox cycling.

Results

Here, for the first time, we describe a direct physical interaction between the bromohydroxy dimeric naphthoquinone E6a and NQO1. Moreover, our studies reveal an extensive binding interface between E6a and the isoalloxazine ring of the flavin adenine dinucleotide (FAD) cofactor of NQO1 in addition to interactions with protein side chains in the active site. We also present biochemical evidence that dimeric naphthoquinones affect the redox state of the FAD cofactor of NQO1. Comparison of the mode of binding of E6a with those of other chemotherapeutics reveals unique characteristics of the interaction that can be leveraged in future drug optimization efforts.

Conclusion

The first structure of a dimeric naphthoquinone-NQO1 complex was reported, which can be used for design and synthesis of more potent next generation dimeric naphthoquinones to target NQO1 with higher affinity and specificity.

     

No direct association between Ia antigens and Fc receptors on the B cell membrane.

The capping of Ia antigens does not induce redistribution of Fc receptors (FcR) on B lymphocytes. This rules out the possibility of a unidirectional association between Ia and FcR such as has been reported to link Ig and FcR. Ia-capping was achieved with hapten-sandwich antibodies devoid of Fc regions: hapten-conjugated Fab anti-Ia followed by (Fab')2 anti-hapten antibody. Three different immune complex systems were used to label FcR. With fluorescent double labeling, Ia and FcR were readily distinguished. The independent labeling and surface mobility of Ia and FcR are considered in connection with reports of the inhibition of FcR by anti-Ia antibodies.     

The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1

The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.