Inhibition of prejunctional parasympathetic pathways by ß3-adrenoceptor agonists in the isolated pig detrusor: comparison with human detrusor studies.

Adrenergic receptors of the ß3-subtype (ß3-ADRs) seem to represent a new target for a more effective pharmacological treatment of overactive bladder (OAB), a wide spread urinary disorder. A promising opportunity for OAB therapy might rely on the development of selective ß3-ADR agonists, but an appropriate preclinical screening, as well as investigation of their pharmacological mechanism(s), is limited by poor availability of human bladder samples and of translational animal models. In this study, we used the porcine urinary bladder as experimental tool to ascertain the functions of ß3-ADRs in the control the parasympathetic motor drive. Tritiated acetylcholine ([3H]-ACh), mainly originated from neural stores, was released by electrical field stimulation (EFS) in epithelium-deprived detrusor strips from pigs bred without estrogens. EFS produced simultaneously [3H]-ACh release and smooth muscle contraction allowing to asses neural (pre-junctional) and myogenic (postjunctional) effects in the same experiment. Isoprenaline and mirabegron produced on the EFS-evoked effects a concentration-dependent inhibition antagonized by L-748,337, a high selective ß3-ADR antagonist. The analysis of the resultant pharmacodynamic parameters supports the notion that in pig detrusors, as well as in previously described human detrusors, the activation of inhibitory ß3-ADRs can modulate neural parasympathetic pathways. In such inhibitory control, the involvement of membrane K+ channels, mainly of the SK type, seems to play a pivotal role similarly to what previously described in humans. Therefore, the isolated porcine detrusor can provide a suitable experimental tool to study the mechanisms underlying the clinical efficacy of selective ß3-ADR compounds for human use.

Involvement of ß3-adrenoceptors in the inhibitory control of cholinergic activity in human bladder: Direct evidence by [(3)H]-acetylcholine release experiments in the isolated detrusor.

Bladder overactivity (OAB) is a multifactorial bladder disorder that requires therapeutics superior to the current pharmacological treatment with muscarinic antagonists. ß3-adrenoceptor (ß3-ADR) agonists represent a novel promising approach that differently addresses the parasympathetic pathway, but the clinical efficacy of these drugs has not been fully elucidated to date. Therefore, we aimed to study the pharmacological mechanisms activated by ß3-ADR agonists at muscular and neural sites in the isolated human bladder. Detrusor smooth muscle strips obtained from male patients undergoing total cystectomy were labelled with tritiated choline and stimulated with electrical field stimulation (EFS). EFS produced smooth muscle contraction and simultaneous acetylcholine ([(3)H]-ACh) release, which mostly reflects the neural origin of acetylcholine. Isoprenaline (INA), BRL37344 and mirabegron inhibited the EFS-evoked contraction and [(3)H]-ACh release in a concentration-dependent manner, yielding concentration-response curves (CRCs) that were shifted to the right by the selective ß3-ADR antagonists L-748,337 and SR59230A. Based on the agonist potency estimates (pEC50) and apparent affinities (pKb) of antagonists evaluated from the CRCs of agonists, our data confirm the occurrence of ß3-ADRs at muscle sites. Moreover, our data are consistent with the presence of inhibitory ß3-ADRs that are functionally expressed at the neural site. Taken together, these findings elucidate the mechanisms activated by ß3-ADR agonists because neural ß3-ADRs participate in the inhibition of detrusor motor drive by reducing the amount of acetylcholine involved in the cholinergic pathway.

Purinergic P2X3 heteroreceptors enhance parasympathetic motor drive in isolated porcine detrusor, a reliable model for development of P2X selective blockers for detrusor hyperactivity.

Various forms of low urinary tract symptoms (LUTS) seem dependant upon dysregulation of the purinergic pathway which produces sensory- or motor-activated incontinence. A body of evidence in human urinary bladders supports a link between up-regulation of purinergic activity and the pathogenesis of detrusor instability. This study investigated the potential role of adenosine 5'-triphosphate (ATP) in the control of detrusor motor drive in a model of porcine urinary bladder. The involvement of ATP on excitatory activity was assessed by measuring neurally-evoked [(3)H]-acetylcholine (ACh) release and smooth muscle contraction in detrusor strips. Epithelium-deprived preparations were used to minimize the influence of non-neural sources of ACh and ATP on parasympathetic neurotransmission. ACh release and smooth muscle contractility were not significantly affected by neural ATP in normal detrusor, but markedly enhanced when ATP hydrolysis was reduced by ectoATPase inhibitors, as well as by α,ß-methylene-ATP (ABMA), agonist resistant to ecto-enzymes degradation. Prejunctional P2X receptors located on cholinergic nerves are involved in such potentiating effect. These purinergic heteroreceptors were characterized as P2X(3) subunits by means of the putative antagonists: NF449 (P2X(1,3) selective), NF023 (P2X(1,3) selective), PPNDS (P2X(1) selective) and A-317491 (P2X(3) selective). In porcine detrusor, P2X(3) receptors are functionally expressed at neural site facilitating neurogenic ACh release. When purine breakdown is experimentally down-regulated to mimicking the impaired purinergic pathway observed in pathological human bladders, endogenous ATP can markedly enhance detrusor contractility through activation of these receptors. Since P2X(3) blockade represents a potential therapeutic approach for diseases of the urinary tract, isolated porcine detrusor represents a reliable model for development of novel selective P2X(3) antagonists beneficial in the treatment of detrusor hyperactivity.

Characterization of prejunctional serotonin receptors modulating [3H]acetylcholine release in the human detrusor.

Bladder overactivity (OAB) is a chronic and debilitating lower urinary tract (LUT) disorder that affects millions of individuals worldwide. LUT symptoms associated with OAB, such as urgency and urinary incontinence, cause a hygienic and social concern to patients, but their current pharmacological treatment is largely inadequate due to the lack of uroselectivity. Although OAB etiology remains multifactorial and poorly understood, increasing evidence indicates that serotonin [5-hydroxytryptamine (5-HT)] is an endogenous substance involved in the control of micturition at central and peripheral sites. In this study, we demonstrated the presence of three distinct 5-HT receptors localized at parasympathetic nerve terminals of the human bladder by measuring electrically evoked tritiated acetylcholine release in isolated detrusor strips. These prejunctional receptors, involved in both positive and negative feedback mechanisms regulating cholinergic transmission, have been characterized by means of three highly selective 5-HT antagonists for 5-HT(4), 5-HT(7), and 5-HT(1A) receptors, namely GR113808A ([1-[2-[(-methylsulphonyl) amino] ethyl]4-piperinidyl]methyl1-methyl-1H-indole-3-carboxylate succinate), SB269970 [(R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl)phenol hydrochloride], and WAY100635 [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)-cyclohexane-carboxamide trichloride]. Under these conditions, we confirmed the facilitatory role of 5-HT(4) heteroreceptors on acetylcholine release and revealed for the first time the occurrence of 5-HT(7) and 5-HT(1A) heteroreceptors with a facilitatory and an inhibitory action, respectively. Our findings strengthen the novel concept for the use of recently patented selective 5-HT agonists and antagonists for the control of OAB dysfunctions associated with inflammatory conditions, although their therapeutic efficacy needs to be explored in the clinical setting.

Facilitation of acetylcholine signaling by the dithiocarbamate fungicide propineb.

Dithiocarbamates (DTCs) are used mainly in agriculture as pesticides and as alcohol deterrent drugs. Neurological complications as well as movement disorders characterized by plastic rigidity, muscle twitch and paralysis are the prevailing symptoms in chronically exposed animals and humans. We investigated whether propineb interfered with peripheral cholinergic transmission in various isolated model systems. In electrically stimulated longitudinal muscle-myenteric plexus preparations (LMMPs), propineb (0.01-1000 nM) concentration-dependently enhanced the amplitude of both neurogenic twitch contractions and tritiated acetylcholine ([3H]ACh) release. The maximum percent increase was achieved by 10 nM propineb and was 19% and 14%, respectively. The effect on twitch contractions was partially antagonized by hexamethonium, a ganglionic nicotinic receptor blocker. In unstimulated LMMPs, propineb (10 pM, 10 nM, 10 microM) did not affect contractions to applied acetylcholine (ACh; 1 nM-10 microM), a finding indicating that propineb has no anticholinesterase activity. In human neuroblastoma cells (SH-SY5Y), propineb facilitated ACh release evoked by KCl depolarization. The increase in ACh release was not associated with detectable alterations of intracellular Ca2+([Ca2+]i) homeostasis. Binding studies carried out with alpha-bungarotoxin in striated muscle cells (L6) failed to demonstrate any influence of propineb on both affinity and capacity of skeletal muscle nicotinic receptors. In conclusion, propineb was found to interfere with cholinergic transmission in LMMPs and SH-SY5Y cells. In LMMPs, the potentiation of cholinergic transmission is partly dependent on the activation of ganglionic nicotinic receptors. Other targets relevant to cholinergic transmission seem not to be affected by propineb.