Development of a multigram synthesis of the bradykinin receptor 2 agonist FR-190997 and analogs thereof.

Using Fujisawa's B2R agonist FR-190997, we recently demonstrated for the first time that agonism at the bradykinin receptor type 2 (B2R) produces substantial antiproliferative effects. FR-190997 elicited an EC50 of 80 nM in the triple-negative breast cancer cell line MDA-MB-231, a much superior performance to that exhibited by most approved breast cancer drugs. Consequently, we initiated a program aiming primarily at synthesizing adequate quantities of FR-190997 to support further in vitro and in vivo studies toward its repurposing for various cancers and, in parallel, enable the generation of novel FR-190997 analogs for an SAR study. Prerequisite for this endeavor was to address the synthetic challenges associated with the FR-190997 scaffold, which the Fujisawa chemists had constructed in 20 steps, 13 of which required chromatographic purification. We succeeded in developing a 17-step synthesis amenable to late-stage diversification that eliminated all chromatography and enabled access to multigram quantities of FR-190997 and novel derivatives thereof, supporting further anticancer research based on B2R agonists.

Function and structure of bradykinin receptor 2 for drug discovery.

Type 2 bradykinin receptor (B2R) is an essential G protein-coupled receptor (GPCR) that regulates the cardiovascular system as a vasodepressor. Dysfunction of B2R is also closely related to cancers and hereditary angioedema (HAE). Although several B2R agonists and antagonists have been developed, icatibant is the only B2R antagonist clinically used for treating HAE. The recently determined structures of B2R have provided molecular insights into the functions and regulation of B2R, which shed light on structure-based drug design for the treatment of B2R-related diseases. In this review, we summarize the structure and function of B2R in relation to drug discovery and discuss future research directions to elucidate the remaining unknown functions of B2R dimerization.

Sex-specific Regulation of Prolactin Secretion by Pituitary Bradykinin Receptors.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

Potent antiproliferative activity of bradykinin B2 receptor selective agonist FR-190997 and analogue structures thereof: A paradox resolved?

Βradykinin stimulation of B2 receptor is known to activate the oncogenic ERK pathway and overexpression of bradykinin receptors B1 and B2 has been reported to occur in glioma, colorectal and cervical cancers. B1R and B2R antagonists have been shown to reverse tumor proliferation and invasion. Paradoxically, B1R and B2R agonism has also been reported to elicit antiproliferative benefits. In order to complement the data accumulated to date with the natural substrate bradykinin and peptidic B2R antagonists, we decided to examine for the first time the response elicited by B2R stimulation in breast cancer lines with a non-peptidic small molecule B2R agonist. We synthesized and assessed the highly selective and potent B2R partial agonist FR-190997 in MCF-7 and MDA-MBA-231 breast cancer lines and found it possessed significant antiproliferative activity (IC50 2.14 and 0.08 µΜ, respectively). The modular nature of FR-190997 allowed us to conduct a focused SAR study and discover compound 10 which exhibits subnanomolar antiproliferative activity (IC 50 0.06 nΜ) in the TNBC MDA-MBA-231 cell line. This performance surpasses, in most cases by several orders of magnitude, those of established anticancer agents and FDA-approved breast cancer drugs. In line with the established literature we suggest that this remarkable activity precipitates from a dual mode of action involving agonist-induced receptor internalization/degradation combined with sequestration of functional intracellular B2 receptors and inhibition of the associated endosomal signaling. The latter mode may be realized by appropriate ligands regardless of B2R agonist/antagonist designation which only relates to membrane residing GCPRs. Under this prism the controversy over the antiproliferative effects of B2 agonists and antagonists is potentially neutralized.

Bradykinin B2 receptor and dopamine D2 receptor cooperatively contribute to the regulation of neutrophil adhesion to endothelial cells.

Leukocyte adhesion to the vascular endothelium contributes to many immunological and inflammatory disorders. These processes have been shown to be mediated by bradykinin receptor type 2 (B2R) and dopamine receptor type 2 (D2R). In a previous study, we reported the formation of a B2R-D2R heterodimer, possibly altering cellular functions. Hence, in the present study, we examined the effect of co-activation of endothelial cells with B2R and D2R agonists on the interaction of these cells with neutrophils. Bradykinin, the main B2R agonist, significantly increased cell adhesion, and this effect was reversed when the endothelial cells were additionally co-treated with a selective D2R agonist, sumanirole. These results were dependent on the incubation time, showing an opposite tendency after prolonged stimulation. Significant changes in the expression of adhesion proteins, such as E-selectin and intercellular adhesion molecule 1 in endothelial cells were observed. Additionally, the cells preincubated with tumor necrosis factor-α showed decreased cell adhesion and IL-8 release after long incubation with both agonists. The modulation of cell adhesion by D2R and B2R seem to be mediated via STAT3 phosphorylation. In summary, this study demonstrated a protective role of D2R in neutrophil-endothelial cell adhesion induced by bradykinin, especially in cytokine-stimulated endothelial cells.

Modulation of Kinin B2 Receptor Signaling Controls Aortic Dilatation and Rupture in the Angiotensin II-Infused Apolipoprotein E-Deficient Mouse.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Activity of the local kallikrein-kinin system may be important in cardiovascular disease. The effect of kinin B2 receptor (B2R) agonist and antagonist peptides on experimental AAA was investigated. APPROACH AND RESULTS: AAA was induced in apolipoprotein E-deficient mice via infusion of angiotensin II (1.0 µg/kg per minute SC). B2R agonists or antagonists were given via injection (2 mg/kg IP) every other day. The B2R agonist (B9772) promoted aortic rupture in response to angiotensin II associated with an increase in neutrophil infiltration of the aorta in comparison to controls. Mice receiving a B2R/kinin B1 receptor antagonist (B9430) were relatively protected from aortic rupture. Neutrophil depletion abrogated the ability of the B2R agonist to promote aortic rupture. Progression of angiotensin II-induced aortic dilatation was inhibited in mice receiving a B2R antagonist (B9330). Secretion of metalloproteinase-2 and -9, osteoprotegerin, and osteopontin by human AAA explant was reduced in the presence of the B2R antagonist (B9330). B2R agonist and antagonist peptides enhanced and inhibited, respectively, angiotensin II-induced neutrophil activation and aortic smooth muscle cell inflammatory phenotype. The B2R antagonist (B9330; 5 µg) delivered directly to the aortic wall 1 week post-AAA induction with calcium phosphate in a rat model reduced aneurysm growth associated with downregulation of aortic metalloproteinase-9. CONCLUSIONS: B2R signaling promotes aortic rupture within a mouse model associated with the ability to stimulate inflammatory phenotypes of neutrophils and vascular smooth muscle cells. B2R antagonism could be a potential therapy for AAA.

Mechanisms of bradykinin-induced expression of connective tissue growth factor and nephrin in podocytes.

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies.

Bradykinin inhibits oxidative stress-induced senescence of endothelial progenitor cells through the B2R/AKT/RB and B2R/EGFR/RB signal pathways.

Circulating endothelial progenitor cells (EPCs) have multiple protective effects that facilitate repair of damage to tissues and organs. However, while various stressors are known to impair EPC function, the mechanisms of oxidative stress-induced EPC senescence remains unknown. We demonstrated that B2 receptor (B2R) expression on circulating CD34(+) cells was significantly reduced in patients with diabetes mellitus (DM) as compared to healthy controls. Furthermore, CD34(+) cell B2R expression in patients with DM was inversely correlated with plasma myeloperoxidase concentrations. Bradykinin (BK) treatment decreased human EPC (hEPC) senescence and intracellular oxygen radical production, resulting in reduced retinoblastoma 1 (RB) RNA expression in H2O2-induced senescent hEPCs and a reversal of the B2R downregulation that is normally observed in senescent cells. Furthermore, BK treatment of H2O2-exposed cells leads to elevated phosphorylation of RB, AKT, and cyclin D1 compared with H2O2-treatment alone. Antagonists of B2R, PI3K, and EGFR signaling pathways and B2R siRNA blocked BK protective effects. In summary, this study demonstrates that BK significantly inhibits oxidative stress-induced hEPC senescence though B2R-mediated activation of PI3K and EGFR signaling pathways.

Bradykinin-related peptides (BRPs) from skin secretions of three genera of phyllomedusine leaf frogs and their comparative pharmacological effects on mammalian smooth muscles.

While bradykinin has been identified in the skin secretions from several species of amphibian, bradykinin-related peptides (BRPs) are more common constituents. These peptides display a plethora of primary structural variations from the type peptide which include single or multiple amino acid substitutions, N- and/or C-terminal extensions and post-translational modifications such as proline hydroxylation and tyrosine sulfation. Such modified peptides have been reported in species from many families, including Bombinatoridae, Hylidae and Ranidae. The spectrum of these peptides in a given species is thought to be reflective of its predator profile from different vertebrate taxa. Here we report the isolation of BRPs and parallel molecular cloning of their respective biosynthetic precursor-encoding cDNAs from the skin secretions of the Mexican leaf frog (Pachymedusa dacnicolor), the Central American red-eyed leaf frog (Agalychnis callidryas) and the South American orange-legged leaf frog (Phyllomedusa hypochondrialis). Additionally, the eight different BRPs identified were chemically synthesized and screened for bioactivity using four different mammalian smooth muscle preparations and their effects and rank potencies were found to be radically different in these with some acting preferentially through bradykinin B1-type receptors and others through B(2)-type receptors.

Targeting the 'Janus face' of the B2-bradykinin receptor.

INTRODUCTION: Kinins are main active mediators of the kallikrein-kinin system (KKS) via bradykinin type 1 inducible (B1R) and type 2 constitutive (B2R) receptors. B2R mediates most physiological bradykinin (BK) responses, including vasodilation, natriuresis, NO, prostaglandins release. AREAS COVERED: The article summarizes knowledge on kinins, B2R signaling and biological functions; highlights crosstalks between B2R and renin-angiotensin system (RAS). The double role (Janus face) in physiopathology, namely the beneficial protection of the endothelium, which forms the basis for the therapeutical utilization of B2 receptor agonists, on the one side, and the involvement of B2R in inflammation or infection diseases and in pain mechanisms, which justifies the use of B2R antagonists, on the other side, is extensively analyzed. EXPERT OPINION: For decades, the B2R has been unconsciously activated during angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) treatments. Whether direct B2R targeting with stable agonists could bring additional therapeutic benefit to RAS inhibition should be investigated. Efficacy, established in experimental models, should be confirmed by translational studies in cardiovascular pathologies, glaucoma, Duchenne cardiopathy and during brain cancer therapy. The other face of B2R is targeted by antagonists already approved to treat hereditary angioedema. The use of antagonists could be extended to other angioedema and efficacy tested against acute pain and inflammatory diseases.

Dual kinin B1 and B2 receptor activation provides enhanced blood-brain barrier permeability and anticancer drug delivery into brain tumors.

The low permeability of the BBB is largely responsible for the lack of effective systemic chemotherapy against primary and metastatic brain tumors. Kinin B1R and B2R have been shown to mediate reversible tumor-selective BBB disruption in preclinical animal models. We investigated whether co-administration of two novel potent kinin B1R and B2R agonists offers an advantage over administering each agonist alone for enhancing BBB permeability and tumor targeting of drugs in the malignant F98 glioma rat model. A new covalent kinin heterodimer that equally stimulates B1R and B2R was also constructed for the purpose of our study. We found that co-administration of B1R and B2R agonists, or alternatively administration of the kinin heterodimer more effectively delivered the MRI contrast agent Gd-DTPA and the anticancer drug carboplatin to brain tumors and surrounding tissues than the agonists alone (determined by MRI and ICP-MS methods). Importantly, the efficient delivery of carboplatin by the dual kinin receptor targeting on the BBB translated into increased survival of glioma-bearing rats. Thus, this report describes a potential strategy for maximizing the brain bioavailability and therapeutic efficacy of chemotherapeutic drugs.

Protein expression, biochemical pharmacology of signal transduction, and relation to intraocular pressure modulation by bradykinin B2 receptors in ciliary muscle.

PURPOSE: To examine the bradykinin (BK) B2-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. METHODS: Previously published procedures were used throughout these studies. RESULTS: The human and monkey ciliary bodies expressed high levels of B2-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²âº](i) mobilization in primary h-CM cells (BK EC50=2.4±0.2 nM > Hyp³,ß-(2-thienyl)-Ala5,Tyr(Me)8-(®)-Arg9-BK (RMP-7) > Des-Arg9-BK EC50=4.2 µM [n=3-6]), and this was blocked by B2-selective antagonists, HOE-140 (IC50=1.4±0.1 nM) and WIN-63448 (IC50=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²âº](i) mobilization. Total prostaglandin (primarily PGE2) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B2-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1-1 µM; at 24 h) caused a 1.4-3.1-fold increase in promatrix metalloproteinases-1-3 release. Topical ocular BK (100 µg) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 µg of BK, but not Des-Arg9-BK, lowered IOP in rabbit eyes (22.9±7.3% and 37.0±5.6% at 5 h and 8 h post-injection; n=7-10). CONCLUSIONS: These studies have provided evidence of a functional endogenously expressed B2-receptor system in the CM that appears to be involved in modulating IOP.

Further pharmacological evaluation of a novel synthetic peptide bradykinin B2 receptor agonist.

We recently identified a novel human B2 receptor (B2R) agonist [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin (NG291) with greater in vitro and in vivo potency and duration of action than natural bradykinin (BK). Here, we further examined its stability and selectivity toward B2R. The hypotensive, antithrombotic, and profibrinolytic functions of NG291 relative to BK and its analogue ([Hyp(3),Thi(5),(4-Me)Tyr(8)(ΨCH(2)NH)Arg(9)]-BK) (RMP-7) were also tested. Contraction assays using isolated mouse stomachs (containing kinin B1R, B2R, and kininase I- and II-like activities) showed that NG291 is a more potent contractant than BK and is inhibited by HOE-140 (B2R antagonist) but unaffected by R954 (B1R antagonist), whereas both decreased the potency of BK. In stomach tissues from B2R knockout mice, BK maintained its activity via B1R, whereas NG291 had no contractile effect, indicating that it was selective for B2R. Unlike BK, NG291 was not degraded by rabbit lung ACE. Comparing intravenously administered BK and NG291 revealed that NG291 exhibited more potent and prolonged hypotensive action and greater antithrombotic and profibrinolytic activities. These effects were of comparable magnitude to RMP-7 and were absent in B2R knockout mice. We concluded that NG291 is a novel biostable B2R-selective agonist that may prove suitable for investigating the (pre)clinical cardioprotective efficacy of B2R activation.

Bifunctional epitope-agonist ligands of the bradykinin B(2) receptor.

Two bradykinin (BK) B(2) receptor agonists N-terminally extended with the myc epitope were synthesized and evaluated: myc-KPG-BK and myc-KGP-B-9972. The latter was modeled on the inactivation-resistant agonist B-9972 (D-Arg(0), Hyp(3), Igl(5), Oic(7), Igl(8)-BK) and is also resistant to endosomal inactivation. Despite a large loss of affinity relative to the parent peptide, the tagged analogs are conventional agonists in the umbilical vein contractility assay and compete for [(3)H]BK binding at the rabbit B(2) receptor. Endocytosed myc-KGP-B-9972 most effectively carried AlexaFluor-488-conjugated anti-myc monoclonal antibodies into intact cells expressing the B(2) receptor. Results support the prospects of functionally-active cargoes entering cells in a pharmacologically controlled manner.

Functional and molecular characterization of kinin B1 and B 2 receptors in human bladder cancer: implication of the PI3Kγ pathway.

Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.

Effects of kinin B(1) and B(2) receptor antagonists on overactive urinary bladder syndrome induced by spinal cord injury in rats.

BACKGROUND AND PURPOSE: Kinin B(1) and B(2) receptors have been implicated in physiological and pathological conditions of the urinary bladder. However, their role in overactive urinary bladder (OAB) syndrome following spinal cord injury (SCI) remains elusive. EXPERIMENTAL APPROACH: We investigated the role of kinin B(1) and B(2) receptors in OAB after SCI in rats. KEY RESULTS: SCI was associated with a marked inflammatory response and functional changes in the urinary bladder. SCI resulted in an up-regulation of B(1) receptor mRNA in the urinary bladder, dorsal root ganglion and spinal cord, as well as in B(1) protein in the urinary bladder and B(1) and B(2) receptor protein in spinal cord. Interestingly, both B(1) and B(2) protein expression were similarly distributed in detrusor muscle and urothelium of animals with SCI. In vitro stimulation of urinary bladder with the selective B(1) or B(2) agonist elicited a higher concentration-response curve in the SCI urinary bladder than in naive or sham urinary bladders. Cystometry revealed that treatment of SCI animals with the B(2) selective antagonist icatibant reduced the amplitude and number of non-voiding contractions (NVCs). The B(1) antagonist des-Arg(9) -[Leu(8) ]-bradykinin reduced the number of NVCs while the non-peptide B(1) antagonist SSR240612 reduced the number of NVCs, the urinary bladder capacity and increased the voiding efficiency and voided volume. CONCLUSIONS AND IMPLICATIONS: Taken together, these data show the important roles of B(1) and B(2) receptors in OAB following SCI in rats and suggest that blockade of these receptors could be a potential therapeutic target for controlling OAB.

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.

Bradykinin B2 receptor-mediated transport into intact cells: anti-receptor antibody-based cargoes.

Endocytosis of the bradykinin-stimulated B(2) receptors is parallel to the transport and subsequent degradation of the ligand. To implement biotechnological applications based on receptor-mediated transport, one strategy is to conjugate the agonist ligand to a cargo. Alternatively, we studied whether the B(2) receptor can transport large antibody-based cargoes into intact cells and characterized the ensuing endosomal routing. Myc-tagged B(2) receptors (coded by the vector myc-B(2)R) and a truncated construction devoid of the Ser-Thr phosphorylation domain (myc-B(2)R(trunc) vector) were coupled to anti-myc monoclonal antibodies that did not impair bradykinin binding or elicit calcium signaling in intact cells. Anti-myc antibodies, conjugated or not with secondary antibodies optionally coupled to Qdot nanomaterials, were transported into early endosome autoantigen 1-, and ß-arrestin-positive vesicles in bradykinin-stimulated intact cells expressing receptors encoded by myc-B(2)R. Antibody-conjugated cargoes progressed into late-endosomes-lysosomes within 3h without evidence of autophagy. Receptors encoded by myc-B(2)R(trunc) did not support the ligand-controlled endocytosis of anti-myc antibodies. Aside from small ligand-conjugated cargoes, very large antibody-based cargoes can be transported by agonist-stimulated B(2) receptors into intact cells. The latter type of cargo requires a receptor competent for interaction with ß-arrestins, enters the degradation pathway separately from the receptor as a function of time and has the potential to confer a qualitatively novel function to a receptor.

Functional role of M-type (KCNQ) K⁺ channels in adrenergic control of cardiomyocyte contraction rate by sympathetic neurons.

M-type (KCNQ) K⁺ channels are known to regulate excitability and firing properties of sympathetic neurons (SNs), but their role in regulating neurotransmitter release is unclear, requiring further study. We sought to use a physiological preparation in which SNs innervate primary cardiomyocytes to evaluate the direct role of M-channels in the release of noradrenaline (NA) from SNs. Co-cultures of rat SNs and mouse cardiomyocytes were prepared, and the contraction rate (CR) of the cardiomyocyte syncytium monitored by video microscopy. We excited the SNs with nicotine, acting on nicotinic acetylcholine receptors, and monitored the increase in CR in the presence or absence of the specific M-channel opener retigabine, or agonists of bradykinin B2 or purinergic P2Y receptors on the SNs. The maximal adrenergic effect on the CR was determined by application of isoproterenol (isoprenaline). To isolate the actions of B2 or P2Y receptor stimulation to the neurons, we prepared cardiomyocytes from B2 receptor or P2Y2 receptor knock-out mice, respectively. We found that co-application of retigabine strongly decreased the nicotine-induced increase in CR. Conversely, co-application of bradykinin or the P2Y-receptor agonist UTP augmented the nicotine-induced increase in CR to about half of the level produced by isoproterenol. All effects on the CR were wholly blocked by propranolol. Our data support the role of M-type K⁺ channels in the control of NA release by SNs at functional adrenergic synapses on cardiomyocytes.We conclude that physiological receptor agonists control the heart rate via the regulation of M-current in SNs.