Cross-talk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys(9)-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase.     

Molecular cloning and pharmacological characterization of the canine B1 and B2 bradykinin receptors

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the N-terminal Lys residue of [des-Arg10]Lys-bradykinin. Significantly, the B1 receptor antagonist [des-Arg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the 'classical' pharmacological properties of this receptor subtype.     

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca²⁺ mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg⁹-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.     

Site-specific cleavage of G protein-coupled receptor-engaged beta-arrestin. Influence of the AT1 receptor conformation on scissile site selection

The discovery of beta-arrestin-related approximately 46-kDa polypeptide in transfected cells and mouse hearts led us to examine angiotensin II type 1 receptor (AT(1)R)-dependent proteolytic cleavage of beta-arrestin(s). Receptor-ligand induced proteolysis of beta-arrestin(s) is novel, especially in the endocrine system, since proteolytic and/or splice variants of nonvisual arrestins are unknown. We used a strategy to retrieve AT(1)R-engaged isoforms of beta-arrestin 1 to confirm direct interaction of fragments with this G protein-coupled receptor and determine cleavage sites. Here we show that the angiotensin II-AT(1)R complex is associated with full-length and approximately 46-kDa beta-arrestin forms. Mass spectrometric analysis of the AT(1)R-associated short form suggested a scissile site located within the Arg(363)-Arg(393) region in the bovine beta-arrestin 1. Edman degradation analysis of a beta-arrestin 1 C-terminal fragment fused to enhanced green fluorescent protein confirmed the major cleavage to be after Phe(388) and a minor cleavage after Asn(375). Rather unexpectedly, the inverse agonist EXP3174-bound AT(1)R generated different fragmentation of bovine beta-arrestin 1, at Pro(276). The angiotensin II-induced cleavage is independent of inositol 1,4,5-trisphosphate- and Ca(2+)-mediated signaling pathways. The proteolysis of beta-arrestin 2 occurs, but the pattern is more complex. Our findings suggest that beta-arrestin cleavage upon AT(1)R stimulation is a part of the unraveling beta-arrestin-mediated G protein-coupled receptor signaling diversity.     

The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately connected and some of the cardioprotective effects of Losartan are abolished by blocking the bradykinin B2 receptor (B2R) signaling. In this study, we investigated the ability of six clinically available ARBs to specifically bind and activate the B2R. First, we investigated their ability to activate phosphoinositide (PI) hydrolysis in COS-7 cells transiently expressing the B2R. We found that only Losartan activated the B2R, working as a partial agonist compared to the endogenous ligand bradykinin. This effect was blocked by the B2R antagonist HOE 140. A competitive binding analysis revealed that Losartan does not significantly compete with bradykinin and does not change the binding affinity of bradykinin on the B2R. Furthermore, Losartan but not Candesartan mimicked the ability of bradykinin to increase the recovery of contractile force after metabolic stress in rat atrial tissue strips. In conclusion, Losartan is a partial agonist of the B2R through direct binding and activation, suggesting that B2R agonism could partly explain the beneficial effects of Losartan.     

Changes in amino-terminal portion of human B2 receptor selectively increase efficacy of synthetic ligand HOE 140 but not of cognate ligand bradykinin

Recently, we have shown that a widely used antagonist of the human bradykinin B(2) receptor (B(2)R) HOE 140 acts as a full agonist of the chicken ornithokinin receptor (B(o)R). To understand the molecular mechanisms underlying differential efficacy of HOE 140 for the various kinin receptors, we have constructed chimeric kinin receptors (CKR) in which the amino-terminal portion including the first two transmembrane regions and the first extracellular loop (CKR-2) or only the second transmembrane region and the first extracellular loop (CKR-1) of B(2)R were substituted with the corresponding segments of B(o)R. Ligand efficacy of synthetic ligand HOE 140 decreased in the order B(o)R > CKR-2 > CKR-1 > B(2)R, whereas the efficacy of the endogenous kinin ligand was unchanged. Enhanced HOE 140 efficacy was not due to a structural change in the ligand binding site or to an enhanced receptor expression level. Rather, heterologous binding competition studies indicated that structural change(s) introduced into the engineered receptors caused a selective reduction in apparent affinity of HOE 140 for the uncoupled inactive receptor state R but not for the active G protein-coupled state R*, thereby increasing the ratio of R* over R for a given ligand concentration. Our results may help explain the unusually broad efficacy spectrum of HOE 140, which varies from inverse to full agonism, depending on kinin receptor subtype, tissue origin, or species.     

Fever-like temperature modification differentially affects in vitro signaling of bradykinin B(1) and B(2) receptors

The bradykinin (BK) B(2) and B(1) receptors (B(2)R, B(1)R) belong to the rhodopsin-like G protein-coupled receptors (GPCRs) and are involved in (patho)physiological processes such as blood pressure regulation or inflammation. They mediate the effects of the pro-inflammatory peptides bradykinin/kallidin and desArg(9)-BK/desArg(10)-kallidin, respectively. Whereas the B(2)R is constitutively expressed and gets internalized upon activation, the B(1)R is especially induced by inflammatory mediators and responds to stimulation with increased surface receptor numbers. Stimulation of both receptors activates phospholipase Cβ (PLCβ) and mitogen activated protein kinase (MAPK) signaling. Because inflammatory processes are characterized by heat (fever), we analyzed the effect of increased temperature (41°C vs. 37°C) on B(1)R and B(2)R signaling in HEK 293 and IMR 90 cells. Our results show that signaling of both receptors is temperature-sensitive, however to a different extent and with regard to the investigated pathways. Comparing PLCβ activity and Ca(2+)-regulated signals, a temperature-dependent increase was only observed for B(1)R but not for B(2)R activation, whereas MAPK activities were doubled at 41°C for both receptors. Taken together, our findings suggest that the observed temperature sensitivity of B(1)R-induced PLCβ activation is B(1)R-specific. In contrast, the enhanced stimulation of MAPK activity under hyperthermic conditions appears to be a common phenomenon for GPCRs.     

The role of bradykinin B1 receptor on cardiac remodeling in stroke-prone spontaneously hypertensive rats (SHR-SP)

An angiotensin-converting enzyme inhibitor (ACE-I) reduces cardiac remodeling and a bradykinin B2 receptor (B2R) antagonist partially abolishes this ACE-I effect. However, bradykinin has two different types of receptor, the B1 receptor (B1R) and B2R. Although B1R is induced under several pathological conditions, including hypertension, the role of cardiac B1R in hypertension is not clear. We therefore investigated the role of cardiac B1R in stroke-prone spontaneously hypertensive rats (SHR-SP) and Wistar-Kyoto (WKY) rats. The B1R mRNA expression level in the heart was significantly higher in SHR-SP than in WKY rats. Chronic infusion of a B1R antagonist for 4 weeks significantly elevated blood pressure and left-ventricular weight of SHR-SP. Morphological analysis indicated that cardiomyocyte size and cardiac fibrosis significantly increased after administration of the B1R antagonist. The phosphorylation of mitogen-activated protein (MAP) kinases, including ERK, p38, and JNK, was significantly increased in the hearts of SHR-SP rats receiving the B1R antagonist. The TGF-beta1 expression level was significantly increased in SHR-SP rats treated with the B1R antagonist compared to that in WKY rats. The B1R antagonist significantly increased phosphorylation of Thr495 in endothelial nitric oxide synthase (eNOS), which is an inhibitory site of eNOS. These results suggest that the role of B1R in the heart may be attenuation of cardiac remodeling via inhibition of the expression of MAP kinases and TGF-beta1 through an increase in eNOS activity in a hypertensive condition.     

Downregulation of bradykinin B2 receptor in human fibroblasts during prolonged agonist exposure

Sustained activation of G protein-coupled receptors results in an attenuation of cellular responses, a phenomenon termed desensitization. Whereas mechanisms for rapid desensitization of ligand-receptor-G protein-effector systems are relatively well characterized, much less is known about long-term adaptation processes that occur in the continuous presence of an agonist. Here we have studied the fate of endogenously expressed bradykinin B(2) receptors on human fibroblasts during prolonged agonist treatment. Stimulation with bradykinin for up to 24 h resulted in a 50% reduction of surface binding sites that was paralleled by a similar decrease of total B(2) receptor protein followed by Western blotting using monoclonal antibodies to the B(2) receptor. Whereas B(2) receptor mRNA levels did not change during 24 h of agonist treatment, B(2) receptor de novo synthesis was attenuated by 35-50%, indicating translational control of B(2) receptor levels. Furthermore, the half-life of B(2) receptor protein was shortened by 20-40% as shown by (35)S-labeled pulse-chase and immunoprecipitation experiments. This study demonstrates that bradykinin B(2) receptor expression during long-term agonist treatment is primarily regulated on the (post)translational level, i.e., by attenuation of de novo synthesis and by reduction of receptor stability.     

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.     

Role of bradykinin B1 and B2 receptors in normal blood pressure regulation

With inhibition or absence of the bradykinin B2 receptor (B2R), B1R is upregulated and assumes some of the hemodynamic properties of B2R, indicating that both participate in the maintenance of normal vasoregulation or to development of hypertension. Herein we further evaluate the role of bradykinin in normal blood pressure (BP) regulation and its relationship with other vasoactive factors by selectively blocking its receptors. Six groups of Wistar rats were treated for 3 wk: one control group with vehicle alone, one with concurrent administration of B1R antagonist R-954 (70 microg x kg(-1) x day(-1)) and B2R antagonist HOE-140 (500 microg x kg(-1) x day(-1)), one with R-954 alone, one with HOE 140 alone, one with concurrent administration of both R-954 and HOE-140 plus the angiotensin antagonist losartan (5 mg x kg(-1) x day(-1)), and one with only losartan. BP was measured continuously by radiotelemetry. Only combined administration of B1R and B2R antagonists produced a significant BP increase from a baseline of 107-119 mmHg at end point, which could be partly prevented by losartan and was not associated with change in catecholamines, suggesting no involvement of the sympathoadrenal system. The impact of blockade of bradykinin on other vasoregulating systems was assessed by evaluating gene expression of different vasoactive factors. There was upregulation of the eNOS, AT1 receptor, PGE2 receptor, and tissue kallikrein genes in cardiac and renal tissues, more pronounced when both bradykinin receptors were blocked; significant downregulation of AT2 receptor gene in renal tissues only; and no consistent changes in B1R and B2R genes in either tissue. The results indicate that both B1R and B2R contribute to the maintenance of normal BP, but one can compensate for inhibition of the other, and the chronic inhibition of both leads to significant upregulation in the genes of related vasoactive systems.     

Diversity of B2 bradykinin receptors with nanomolar affinity expressed in passaged IMR90 human lung fibroblasts.

IMR90 human fetal lung fibroblasts express bradykinin receptors activating the pathway for biosynthesis of PGE2. A receptor of the B2 subtype stimulates half-maximal PGE2 production at 4.8 nM bradykinin, and maximal output takes place at 25 nM bradykinin. Radioligand binding studies reveal a population of [3H]bradykinin binding sites whose affinity correlates with this B2 receptor's biologic activity, with a KD of 2.5 nM. As IMR90 cells reach 60% of their defined life span in culture, they spontaneously induce expression of a second site of lower affinity, with half-maximal binding of [3H]bradykinin at 44 nM. This second site displays a characteristic primary B2 receptor recognition profile, but differs from the 2.5 nM site on a secondary level in recognition among different B2 ligands. Bradykinin is the most potent ligand at both sites; they each preferentially recognize an N-terminal extended bradykinin peptide construct having selectivity for the rat myometrial B2 receptor, suggesting that both sites have structural features in common. However, they display diversity in their order of preference for Met-Lys-bradykinin versus Lys-Lys-bradykinin; at the 44 nM site this order is completely reversed from the order of potency exhibited at the 2.5 nM site. Expression of the second site changes the manner in which these fibroblasts control their PGE2 production; it affords a graded response of PGE2 production at bradykinin levels beyond those which would normally saturate the 2.5 nM site. The inducibility of the 44 nM site in cultured fibroblasts addresses in vivo conditions in an inflammatory environment where continuing generation of bradykinin-related peptides takes place and presents a possible mechanism for overriding constraints that would otherwise limit the progression of inflammation.     

Coulombic and hydrophobic interactions in the first intracellular loop are vital for bradykinin B2 receptor ligand binding and consequent signal transduction

The role of the first intracellular loop (IC1) in the function of the rat bradykinin B2 receptor (BKB2R) was probed. On the basis of the bovine rhodopsin X-ray structure, the BKB2R IC1 consists of six residues: (60)HKTNCT. Exchange of this sequence with the bradykinin B1 receptor IC1 (PRRQLN) resulted in a chimera which bound bradykinin and signaled as wild-type (WT) BKB2R. In contrast, a chimera containing the IC1 of rat angiotensin II type Ia receptor (AT1aR) (YMKLKT) did not bind BK nor signal in response to BK at a concentration as high as 5 microM. ELISA illustrated that this receptor was still processed and inserted into the plasma membrane. Employing portions of the IC1, we observed that (60)HKT of BKB2R could be exchanged as a group with either the BKB1R (PRR) or AT1aR (YMK) with no change in receptor binding or signaling activities. When only the YM of AT1aR replaced the HK of BKB2R, leaving the N-terminal portion of IC1 without a positively charged residue, binding and signaling were reduced by more than 70%. When only N63 was replaced with the corresponding leucine of AT1aR, binding and signaling were ablated. In fact, replacement of the entire IC1 with the AT1aR except for N63 resulted in binding and signaling as WT BKB2R. However, N63 could be replaced by glutamine (in BKB1R) or aspartate and continued to function as WT BKB2R. NMR data indicated that the BKB2R IC1 extends beyond the bovine rhodopsin prototype to include HKTNCTVAEI. When E68 was exchanged with a serine (in AT1aR), ligand binding decreased by 60% and PI turnover decreased by 69%. Molecular modeling points to a strict requirement for a hydrophilic residue at position 63 (N) at the middle of the IC1 and a Coulombic charge interaction between the positive charges (H60 and K61) at the N-terminus and a negative charge (E68) at the C-terminus of the IC1.     

K317, R319, and E320 within the proximal C-terminus of the bradykinin B2 receptor form a motif important for phospholipase C and phospholipase A2 but not connective tissue growth factor related signaling

We showed previously that large domain exchanges between the bradykinin B2 (BKB2) and angiotensin II type 1a (AT1a) receptors can result in functional hybrids. However, when we proceeded to exchange the entire bradykinin B2 receptor (BKB2R) C-terminal tail with the AT1aR C-terminus, the hybrid, while continuing to bind BK and be endocytosed as wild type (WT) BKB2R, lost much of its ability to activate phosphatidylinositol (PI) turnover or the release of arachidonic acid (ARA). In this study, we constructed chimeric receptors within the proximal C-terminus between the BKB2R and AT1aR or bradykinin B1 receptor (BKB1R). The mutant and WT receptor cDNAs were stably transfected into Rat-1 cells. Also, point mutations were generated to evaluate the role of the individual residues within this region. These chimeric studies revealed that the proximal portion of the BKB2R C-tail is crucial for G protein-linked BKB2R functions. This region could not be swapped with the AT1aR to obtain a BK activated PI turnover or ARA release. Further studies demonstrated that the distal portion (325-330) of this region is exchangeable; however, the middle portion (317-324) is not. Small motif exchanges within this section identified the KSR and EVY motifs as crucial for G(alphaq), G(alphai) related signaling of the BKB2R. Point mutations then showed that the charged amino acids K317, R319, and E320 are the residues critical for linking to PI turnover and ARA release. However, these proximal chimeras showed normal receptor uptake. Interestingly, while apparently not activating G protein-linked signaling, the proximal tail AT1aR exchange mutant and the entire C-terminus exchange hybrid continued to cause a substantial bradykinin effected increase in connective tissue growth factor (CTGF) mRNA level, as WT BKB2R.     

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.     

Dual antagonists of the bradykinin B1 and B2 receptors based on a postulated common pharmacophore from existing non-peptide antagonists

We have recently drawn attention to the fact that most non-peptide antagonists of the kinin B1 receptor reported so far are structurally related, possessing the core motif phenyl-SO2-NR-(spacer(2-4))-CO-NRR. This is found in compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2- yl)phenyl]ethyl] - 2- [(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide), a very potent and selective B1 receptor antagonist. A subset of specific bradykinin B2 receptor antagonists (LF16-0687, bradyzide and derivatives) possesses a similar 'scaffold' (phenyl-SO2-NR-CRR-CO-NRR). We investigated whether simple molecules mimicking the postulated pharmacophores could be identified in two public chemical databases. Receptor binding to B1 and B2 receptors expressed by rabbit cultured smooth-muscle cells was confirmed for some of these newly identified agents, with a loss of receptor subtype selectivity. For instance, compound 3[2-(3-oxo-1-(toluene-4-sulfonyl)-1,2,3,4-4H-quinoxalin-2-yl)-N-phenyl-acetamide] exhibits IC50 values of 2.13 and 126 microM in the radioligand competition assays for B1 and B2 receptors, respectively, and a pA2 of 6.27 at the rabbit B1 receptor in a functional test (Lys-des-Arg9-bradykinin-induced contractility of the isolated aorta). Compound 5 (a close analog of compound 3) is a more balanced dual antagonist of low potency (IC50 values of 30 and 117 microM, respectively). As predicted, compounds modeled on a postulated pharmacophore common to some non-peptide B1 or B2 receptor antagonists exhibit measurable binding with decreased receptor subtype selectivity. Dual B1/B(2) receptor antagonists are of possible therapeutic interest and should be developed.     

Bradykinin B2 receptors mediate pulmonary sympathetic afferents induced reflexes in rabbits

Endogenous bradykinin (BK) is an established mediator of pulmonary inflammation, yet its role in lung disease is unclear. In the rabbit, injecting BK into the lung parenchyma elicits reflex hyperpnea, tachypnea, hypotension, and bradycardia by stimulating pulmonary sympathetic afferents. To further explore bradykinin effects, breathing pattern (phrenic nerve and abdominal muscle activities) and hemodynamics (blood pressure and heart rate) were examined in anesthetized, open-chest, and mechanically ventilated rabbits. Three receptor agonists [bradykinin, selective B(1) (des-Arg(9)-BK), and selective B(2) (Tyr(8)-BK)], as well as three B(2) receptor antagonists, B6029 (N alpha-Adamantaneacetyl)-Bradykinin, B(1)650 (D-Arg-[Hyp(3), Thi(5,8), D-Phe(7)]-Bradykinin, or Hoe-140 (D-Arg-[Hyp(3), Thi(5), D-Tic(7), Oic(8)] bradykinin), were used to identify the responsible receptor subtype. In both intact and vagotomized rabbits, injecting BK or a selective B(2) agonist into the lung elicited similar cardiopulmonary responses. These reflex responses were greatly attenuated or blocked by pre-injecting B(2) antagonists into the right atrium or into the lung parenchyma. In contrast, the B(1) agonist elicited fewer cardiopulmonary effects in intact rabbits and had no effect in vagotomized rabbits. We conclude that BK stimulates pulmonary sympathetic afferents [Soukhova, G., Wang, Y., Ahmed, M., Walker, J., Yu, J., 2003. Bradykinin stimulates respiratory drive by activating pulmonary sympathetic afferents in the rabbit. J. Appl. Physiol. 95, 241-249.; Wang, Y., Soukhova, G., Proctor, M., Walker, J., Yu, J., 2003. Bradykinin causes hypotension by activating pulmonary sympathetic afferents in the rabbit. J. Appl. Physiol. 95, 233-240.], eliciting a characteristic cardiopulmonary reflex via B(2) receptors.     

Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of direct interaction between enalaprilat and the kinin B1 receptors

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.     

Roles for heterodimerization of APJ and B2R in promoting cell proliferation via ERK1/2-eNOS signaling pathway

Apelin receptor (APJ) and bradykinin B2 receptor (B2R) play an important role in many physiological processes and share multiple similar characteristics in distribution and functions in the cardiovascular system. We first identified the endogenous expression of APJ and B2R in human umbilical vein endothelial cells (HUVECs) and their co-localization on human embryonic kidney (HEK) 293 cells membrane. A suite of bioluminescence and fluorescence resonance energy transfer (BRET and FRET), proximity ligation assay (PLA), and co-immunoprecipitation (Co-IP) was exploited to demonstrate formation of functional APJ and B2R heterodimer in HUVECs and transfected cells. Stimulation with apelin-13 and bradykinin (BK) increased the phosphorylation of the endothelial nitric oxide synthase (eNOS) in HUVECs, which could be inhibited by the silencing of APJ or B2R, indicating the APJ-B2R dimer is critical for eNOS phosphorylation in HUVECs. Furthermore, the increase of NOS and extracellular signal regulated kinases1/2 (ERK1/2) phosphorylation mediated by APJ/B2R dimer can be inhibited by U0126 and U73122, respectively, suggesting that the heterodimer might activate the PLC/ERK1/2/eNOS signaling pathway, and finally leading to a significant increase in cell proliferation. Thus, we uncovered for the first time the existence of APJ-B2R heterodimer and provided a promising new target in cardiovascular therapeutics.