Isoproterenol acts as a biased agonist of the alpha-1A-adrenoceptor that selectively activates the MAPK/ERK pathway.

The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e.

ATP and P2X purinoceptors in urinary tract disorders.

The pharmacological concept of specifically targeting purinoceptors (receptors for ATP and related nucleotides) has emerged over the last two decades in the quest for novel, differentiated therapeutics. Investigations from many laboratories have established a prominent role for ATP in the functional regulation of most tissue and organ systems, including the urinary tract, under normal and pathophysiological conditions. In the particular case of the urinary tract, ATP signaling via P2X1 receptors participates in the efferent control of detrusor smooth muscle excitability, and this function may be heightened in disease and aging.

Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats.

Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone.

AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist.

Background And Purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist.

Experimental Approach: The antagonistic potencies (pIC(50)) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology.

Endogenous purinergic control of bladder activity via presynaptic P2X3 and P2X2/3 receptors in the spinal cord.

P2X(3) and P2X(2/3) receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X(3) and P2X(2/3) antagonist, AF-792 [5-(5-ethynyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine, previously known as RO-5], in addition to less selective purinoceptor ligands, was applied intrathecally in vivo.

Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist.

Despite the extensive literature describing the role of the ATP-gated P2X(3) receptors in a variety of physiological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-like selective molecules. In this paper we report the discovery and optimization of RO-85, a novel drug-like, potent and selective P2X(3) antagonist. High-throughput screening of the Roche compound collection identified a small hit series of heterocyclic amides from a large parallel synthesis library.

Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function.

NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age.

Selective pharmacological blockade of the TRPV1 receptor suppresses sensory reflexes of the rodent bladder.

Purpose: We investigated the pharmacological effect of TRPV1 antagonists in anesthetized rodent models of bladder function.

Materials And Methods: The TRPV1 antagonists JNJ17203212 and JYL1421 were evaluated in the anesthetized rat volume induced micturition reflex model. JNJ17203212 was further evaluated in this model in capsaicin (Sigma) desensitized rats, and in rat capsaicin and mouse citric acid models of irritant induced detrusor overactivity.

Identification and SAR of novel diaminopyrimidines. Part 1: The discovery of RO-4, a dual P2X(3)/P2X(2/3) antagonist for the treatment of pain.

P2X purinoceptors are ligand-gated ion channels whose endogenous ligand is ATP. Both the P2X(3) and P2X(2/3) receptor subtypes have been shown to play an important role in the regulation of sensory function and dual P2X(3)/P2X(2/3) antagonists offer significant potential for the treatment of pain. A high-throughput screen of the Roche compound collection resulted in the identification of a novel series of diaminopyrimidines; subsequent optimization resulted in the discovery of RO-4, a potent, selective and drug-like dual P2X(3)/P2X(2/3) antagonist.

Identification and SAR of novel diaminopyrimidines. Part 2: The discovery of RO-51, a potent and selective, dual P2X(3)/P2X(2/3) antagonist for the treatment of pain.

The purinoceptor subtypes P2X(3) and P2X(2/3) have been shown to play a pivotal role in models of various pain conditions. Identification of a potent and selective dual P2X(3)/P2X(2/3) diaminopyrimidine antagonist RO-4 prompted subsequent optimization of the template. This paper describes the SAR and optimization of the diaminopyrimidine ring and particularly the substitution of the 2-amino group.

Facilitatory interplay in alpha 1a and beta 2 adrenoceptor function reveals a non-Gq signaling mode: implications for diversification of intracellular signal transduction.

Agonist occupied alpha(1)-adrenoceptors (alpha(1)-ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress alpha(1)- and beta-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state.

The role of metabotropic glutamate receptor mGlu5 in control of micturition and bladder nociception.

In micturition control, the roles of ionotropic glutamate (iGlu) receptors NMDA and AMPA are well established, whereas little is known about the function of metabotropic glutamate (mGlu) receptors. Since antagonists for mGlu5 receptors are efficacious in animal models of inflammatory and neuropathic pain, we examined whether mGlu5 receptors play a role in the voiding reflex and bladder nociception and, if so, via centrally or peripherally localized receptors. The mGlu5 receptor antagonist MPEP dose-dependently increased the micturition threshold (MT) volume in the volume-induced micturition reflex (VIMR) model in anesthetized rats.

Expression and function of rat urothelial P2Y receptors.

The control and regulation of the lower urinary tract are partly mediated by purinergic signaling. This study investigated the distribution and function of P2Y receptors in the rat urinary bladder. Application of P2Y agonists to rat urothelial cells evoked increases in intracellular calcium; the rank order of agonist potency (pEC(50) +/- SE) was ATP (5.

Effects of the selective prostacyclin receptor antagonist RO3244019 on the micturition reflex in rats.

Purpose: We investigated the role of prostacyclin on afferent modulation of the micturition reflex using the novel selective prostacyclin receptor antagonist RO3244019 in rat models of bladder function.

Materials And Methods: The effects of RO3244019 on urodynamic parameters were evaluated in 3 rat models. In the anesthetized isovolumetric bladder contraction and the volume induced micturition reflex (Refill) models the effects of RO3244019 and chronic capsaicin desensitization were compared.

Pharmacology of P2X channels.

Significant progress in understanding the pharmacological characteristics and physiological importance of homomeric and heteromeric P2X channels has been achieved in recent years. P2X channels, gated by ATP and most likely trimerically assembled from seven known P2X subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacological classification holds true in native cells and in vivo is less well-established.

Purinoceptors as therapeutic targets for lower urinary tract dysfunction.

Lower urinary tract symptoms (LUTS) are present in many common urological syndromes. However, their current suboptimal management by muscarinic and alpha(1)-adrenoceptor antagonists leaves a significant opportunity for the discovery and development of superior medicines. As potential targets for such therapeutics, purinoceptors have emerged over the last two decades from investigations that have established a prominent role for ATP in the regulation of urinary bladder function under normal and pathophysiological conditions.

RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists.

Prostacyclin (PGI2) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. Two structurally distinct series of IP receptor antagonists have been developed: 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) and R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO3244794).

P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP.

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X2 and P2X3 subunits. P2X2 and P2X3 subunits can form homomultimeric P2X2, homomultimeric P2X3, or heteromultimeric P2X2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood.

Identification of a 5-HT4 receptor antagonist clinical candidate through side-chain modification.

Replacement of the N-butyl side-chain of lead 5-HT4 receptor antagonist 2 with propanesulfonylpiperidinyl, morpholinyl, and piperazinyl groups led to higher affinity analogs 4-6. In vitro drug metabolism screens and cassette pharmacokinetic studies in the dog led to identification of the N-methylpiperazinyl analog (6b), which displayed pharmacokinetic, selectivity, and safety parameters sufficient for advancement to the clinic for the treatment of urinary incontinence.

Effect of neurokinins on canine prostate cell physiology.

Background: Sensory peptide neurotransmitters have been implicated as significant regulators of prostate growth. This study was designed to evaluate the role of neurokinins in proliferation, differentiation, and contraction of canine prostate cells in culture.

Methods: NK1, NK2, and NK3 receptor subtypes were localized in canine prostate tissue by immunocytochemistry and ligand binding studies.

Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium.

The bladder urothelium exhibits dynamic sensory properties that adapt to changes in the local environment. These studies investigated the localization and function of bradykinin receptor subtypes B1 and B2 in the normal and inflamed (cyclophosphamide (CYP)-induced cystitis) bladder urothelium and their contribution to lower urinary tract function in the rat. Our findings indicate that the bradykinin 2 receptor (B2R) but not the bradykinin 1 receptor (B1R) is expressed in control bladder urothelium.

Pharmacological and functional characterization of bradykinin B2 receptor in human prostate.

The objective of this study was to pharmacologically characterize bradykinin receptors, a component of the kallikrein-kinin system, in normal human prostate cells. In primary cultured human prostate stromal cells, bradykinin, but not [des-Arg9]bradykinin or [des-Arg10]kallidin, produced calcium mobilization or inositol phosphates accumulation with potencies (pEC50) of 8.8+/-0.

Uropathic observations in mice expressing a constitutively active point mutation in the 5-HT3A receptor subunit.

Mutant mice with a hypersensitive serotonin (5-HT)3A receptor were generated through targeted exon replacement. A valine to serine mutation (V13'S) in the channel-lining M2 domain of the 5-HT3A receptor subunit rendered the 5-HT3 receptor 70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT3B subunit. Mice homozygous for the mutant allele (5-HT3Avs/vs) had decreased levels of 5-HT3A mRNA.

Pharmacological characterization of canine bradykinin receptors in prostatic culture and in isolated prostate.

The objective of this study was to characterize pharmacologically bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, BK) receptors in the canine prostate. Primary cultures of canine prostate stromal (PS) and epithelial cells (PE) were established and then characterized using cell-specific antibodies (actin, vimentin and cytokeratin). Cultured cells were assayed for BK receptors using fluorometric imaging plate reader assays.

Identification of P2X receptors in cultured mouse and rat parasympathetic otic ganglion neurones including P2X knockout studies.

We have used patch-clamp recording from cultured neurones, immunohistochemistry and gene deletion techniques to characterize the P2X receptors present in mouse otic ganglion neurones, and demonstrated the presence of similar receptors in rat neurones. All neurones from wild-type (WT) mice responded to ATP (EC(50) 109 microM), but only 38% also responded to alpha beta-meATP (EC(50) 39 microM). The response to alpha beta-meATP was blocked by TNP-ATP with an IC(50) of 38.