Activation of B2 bradykinin receptors by neurotensin

Many previous reports suggested that relatively high concentrations of neurotensin were required to exert its effects on neurotransmitter secretion. The neurotensin binding sites, which recognize high concentrations of neurotensin, were characterized in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with neurotensin, [3H]norepinephrine secretion and elevation of cytosolic calcium were evoked at EC(50) values of 59+/-4 and 37+/-7 microM, respectively. Both calcium release and inositol 1,4,5-trisphosphate (IP(3)) production induced by neurotensin suggested involvement of phospholipase C. Experiments with simultaneous or sequential treatment with neurotensin and bradykinin suggested that neurotensin and bradykinin act on the same binding sites. Furthermore, both inhibition of bradykinin- and neurotensin-induced calcium rises by bradykinin receptor antagonists with similar IC(50) values and receptor binding analysis using [3H]bradykinin confirmed that neurotensin directly binds to B2 bradykinin receptors. The data suggest that neurotensin binds and activates the B2 bradykinin receptors.     

Kallikrein activates bradykinin B2 receptors in absence of kininogen

Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.     

Age-related changes of bradykinin B1 and B2 receptors in rat heart

Aging is a major risk factor for the development of vascular diseases, such as hypertension and atherosclerosis, that leads to end organ damage and especially heart failure. Bradykinin has been demonstrated to have a cardioprotective role by affecting metabolic processes and tissue perfusion under conditions of myocardial ischemia. Its actions are exerted via the bradykinin B1- and B2-type receptors (B1Rs and B2Rs), but the functional status of these receptors during the aging process is poorly understood. This study aims to investigate whether changes in B1R and B2R gene and protein expression in rat heart are associated with the age-related alterations of cardiac structure and function. Using real-time PCR, we found that B1R mRNA expression increased 2.9-fold in hearts of older rats (24 mo of age) compared with younger rats (3 mo of age), whereas B2R gene expression remained unchanged. Western blot analysis showed that expression of B2R at the protein level is approximately twofold higher in young rats compared with old rats, whereas the B1R protein is approximately twofold higher in old rats compared with young rats. The present results provide clear functional and molecular evidence that indicate age-related changes of bradykinin B1Rs and B2Rs in heart. Because the cardioprotective actions of bradykinin are physiologically mediated via the B2Rs, whereas the B1Rs become induced by tissue damage, these results suggest that age-related decreases in B2R protein levels may leave the heart vulnerable to ischemic damage, and increases in B1R expression and activity may represent a compensatory reaction in aging hearts.     

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.     

Gangliosides and chondroitin sulfate desensitize and internalize B2 bradykinin receptors

Prolonged or repeated agonist activation of G-protein-coupled receptors (GPCRs) initiates their desensitization and internalization, rendering them unresponsive to agonist activation. We analyzed how gangliosides and chondroitin sulfate affect B2 bradykinin (BK) receptors (B2Rs). Gangliosides and chondroitin sulfate did not stimulate intracellular Ca(2+) release from B2R-expressing CHO-K1 cells, but repeated exposure desensitized B2Rs to BK stimulation. Microscopic observation of DsRed-fused B2Rs revealed that several gangliosides and chondroitin sulfate C (CSC) effectively internalized B2Rs. Ganglioside-CSC treatment of B2R mutant-expressing cells failed to desensitize and internalize the mutant receptors. As this mutant lacks the first extracellular domain and cannot activate GPCR kinase (GRK), gangliosides and CSC likely initiate B2R desensitization and endocytosis through GRK-mediated B2R phosphorylation.     

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.     

Molecular features characterizing non-peptide selectivity to the human B1 and B2 bradykinin receptors

Bradykinin is produced in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases. Actions of this peptide are mediated through two different G-protein coupled receptors, named B1 and B2 that have different pharmacological characteristics. The former is up-regulated during inflammation episodes or tissue trauma whereas, the latter is constitutively expressed in a variety of cell types. In a previous work we have characterized the molecular features that explain the observed structure-activity results for both receptors by means of molecular modeling studies, using diverse ligands for both receptors. These results were summarized in the form of two different pharmacophores that provided new insights to be used for the design of novel molecules with antagonistic profile. In the present work, we compare these pharmacophores to understand the features that characterize ligand selectivity to the two bradykinin receptors. The study shows that most of the residues involved in the binding pocket are similar in both receptors and consequently are the pharmacophores obtained. The main difference between the two pharmacophores remains on point #5 that involves a polar moiety for the B1 receptor and an aromatic ring for the B2 receptor. Accordingly, analysis of the prospective bound conformation of several non-selective small molecule ligands of the bradykinin receptors permits to conclude that fulfilment of point#5 is a requirement to produce selective ligands. However, the study also shows that this is a necessary condition only, since ligands need also to be bulky enough to avoid binding to these receptors in diverse poses. These results provide new insights for a better understanding of the molecular features that ligands are required to exhibit to be selective bradykinin ligands.     

Solubilization and characterization of B2 bradykinin receptors from cultured human fibroblasts.

Active B2 bradykinin (BK) receptors were solubilized in high yields from intact monolayers or particulate fractions of cultured human foreskin fibroblasts using 4 mM of the non-denaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Other detergents showed only minor (digitonin) or no (Triton X-100, n-octyl glucopyranosid) efficacy at all. The stability of CHAPS-solubilized BK binding activity was temperature dependent being reduced to 30% of initial binding after 3 days of storage at 4 degrees C. CHAPS extracts, however, retained BK binding activity for at least several days when they were stored at -20 degrees C in the presence of 10% glycerol. The pharmacological characterization gave a rank order of potency for unlabeled BK, BK agonists, and antagonists to compete with [3H]BK for specific binding very similar to that observed in intact fibroblasts. Association and dissociation kinetics demonstrated that the binding of [3H]BK to the soluble CHAPS extracts was time dependent and reversible. Scatchard analysis of equilibrium binding data exhibited saturable binding of a single class of high affinity BK-binding sites with a Kd of 1.68 +/- 0.8 nM. Gel filtration revealed an apparent molecular weight of 250,000 for the solubilized BK receptor complex in CHAPS extracts. The ability to solubilize the B2 BK receptor in an active and stable form should allow for its future purification and for the characterization of its chemical properties.     

Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation

Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.     

Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells

Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B(2) kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 microM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [(3)H]BK internalization (internalized [(3)H]BK in percentage of total bound [(3)H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B(2) kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.     

Regulation of kinin B(2) receptors by bradykinin in human lung cells

Bradykinin is a potent mediator of inflammation that has been shown to participate in allergic airway inflammation. The biologic effects of bradykinin are mediated by binding and activation of its cognate receptor, the B(2) receptor (B(2)R). In the lung fibroblast cell line IMR-90, binding of bradykinin to B(2)R triggers down-regulation of receptor surface expression, suggesting that bradykinin-induced inflammation is transient and self-limited. Notably, subjects with chronic airway inflammation continue to respond to BK following a first challenge. B(2)Rs are expressed on many different lung cell types, including airway epithelial cells. We therefore compared IMR-90 cells with the human lung epithelial cell line BEAS2B and found that B(2)R expression in the two cell types is differently regulated by BK. Whereas BK induces down-regulation of B(2)R in IMR-90 cells, the same treatment leads to up-regulation of the receptor in BEAS2B cells. These results provide a possible explanation for the potency of bradykinin in inducing ongoing airway inflammation.     

Effects of a nonpeptide bradykinin B2 receptor antagonist, FR167344, on guinea-pig tracheal smooth muscle bradykinin receptors

It is speculated that bradykinin may play an important role in asthma. Thus, bradykinin receptor antagonists may have therapeutic potential against asthma. Orally active bradykinin antagonists would be more desirable for the treatment of the disease. In the present study, we examined the effects of a novel, potent, selective, and orally active nonpeptide bradykinin B2 receptor antagonist, FR167344 (N-[N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2 ,4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylamin ocarbonyl)cinnamylamide hydrochloride), on guinea-pig tracheal smooth muscle bradykinin receptors. FR167344 inhibited [3H]bradykinin binding to bradykinin receptors in epithelium-denuded guinea-pig tracheal membrane with an IC50 of 2.1 nM and a Ki of 0.44 nM. This compound also inhibited bradykinin-induced contraction of epithelium-denuded guinea-pig trachea with a pK(B) of 10.8, but had no effect on carbachol-induced contraction of the trachea even at 10(-6) M. These results indicate that FR167344 has the specific antagonistic activity against guinea-pig tracheal smooth muscle bradykinin receptors.     

Non-selectivity of new bradykinin antagonists for B1 receptors.

Two new B1 receptor antagonists, [Hyp3,Thi5,DTic7,Oic8]desArg9-BK and DArg[Hyp3,Thi5,DTic7,Oic8]desArg9-BK were tested in vitro on the rabbit jugular vein and the guinea pig ileum (preparations containing B2 receptors) and on the rabbit aorta (preparation containing B1 receptors) for pharmacological characterization. The results indicate that both compounds are antagonists on both B1 and B2 receptors, are competitive and discriminate between B2A and B2B receptor subtypes.     

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.     

Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release

Bradykinin (BK) or kallikreins activate B2 receptors (R) that couple Galpha(i) and Galpha(q) proteins to release arachidonic acid (AA) and elevate intracellular Ca2+ concentration ([Ca2+]i). Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples Galpha(i), Galpha(q), and Galpha(12/13) proteins. In Chinese hamster ovary cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of Galpha(i), Galpha(q), and Galpha(12/13) signaling pathways, and a protein kinase C (PKC)-alpha inhibitor, G?-6976, blocked potentiation, while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca2+]i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca2+]i by thrombin and blocked potentiation of AA release by B2R agonists. Because either a nonselective inhibitor (isotetrandrine) of phospholipase A2 (PLA2) or a Ca2+-dependent PLA2 inhibitor abolished potentiation of AA release by thrombin, while a Ca2+-independent PLA2 inhibitor did not, we concluded that the mechanism involves Ca2+-dependent PLA2 activation. Both thrombin and TRAP modified activation and phosphorylation of the B2R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the NH2-terminal Ser1-Phe2 bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus PAR1 activation enhances AA release by B2R agonists through signal transduction pathway.     

Mechanisms regulating the expression,self-maintenance,and signaling-function of the bradykinin B2 and B1 receptors

Bradykinin (BK) is a potent short-lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G-protein-coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Galphai and Galphaq G-protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg(10)-kallidin or desArg(9)-BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin-1 or tumor necrosis factor-alpha. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and self-maintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self-maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews (Regoli and Barabé, 1980, Pharmacol Rev 32:1-46; Regoli et al., 1990, J Cardiovasc Pharmacol 15(Suppl 6):S30-S38; Regoli et al., 1993, Can J Physiol Pharmacol 71:556-557; Marceau, 1995, Immunopharmacology 30:1-26; Regoli et al., 1998, Eur J Pharmacol 348:1-10).     

Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors

Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT(1) and bradykinin B(2) receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT(1) and B(2) receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT(1), and B(2) receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT(1), and B(2) receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.     

Receptor phosphorylation does not mediate cross talk between muscarinic M3 and bradykinin B2 receptors

This study examined cross talk between phospholipase C-coupledmuscarinic M₃ and bradykininB₂ receptors coexpressed inChinese hamster ovary (CHO) cells. Agonists of either receptor enhanced phosphoinositide signaling (which rapidly desensitized) and caused protein kinase C (PKC)-independent, homologous receptorphosphorylation. Muscarinic M₃ butnot bradykinin B₂ receptors werealso phosphorylated after phorbol ester activation of PKC. Consistentwith this, muscarinic M₃ receptorswere phosphorylated in a PKC-dependent fashion after bradykininB₂ receptor activation, butmuscarinic M₃ receptor activationdid not influence bradykinin B₂receptor phosphorylation. Despite heterologous phosphorylation ofmuscarinic M₃ receptors, phosphoinositide and Ca²⁺signaling were unaffected. In contrast, marked heterologousdesensitization of bradykinin-mediated responses occurred despite noreceptor phosphorylation. This desensitization was associated with asustained component of muscarinic receptor-mediated signaling, whereasbradykinin's inability to influence muscarinic receptor-mediatedresponses was associated with rapid and full desensitization ofbradykinin responses. Thus the mechanism of functional cross talk mostlikely involves depletion of a shared signaling component. These data demonstrate that receptor phosphorylation is not a prerequisite forheterologous desensitization and that such desensitization is notobligatory after heterologous receptor phosphorylation.