Sabatino Ventura

Cholinergic innervation and function in the prostate gland

The mammalian prostate is densely innervated by hypogastric and pelvic nerves that play an important role in regulating the growth and function of the gland. While there has been much interest in the role of the noradrenergic innervation and adrenoceptors in prostate function, the role of cholinergic neurones in prostate physiology and pathophysiology is not well understood. This review focuses on the role of acetylcholine and cholinoceptors in prostate function. Nitric oxide, vasoactive intestinal polypeptide, and/or neuropeptide Y are co-localised with cholinesterase and/or acetylcholine transporter in some of the nerve fibres supplying the prostate. Their roles are also briefly discussed in this review. A dense network of cholinesterase-staining fibres supplies both prostate epithelium and stroma, suggesting a role of acetylcholine and/or co-localised neuropeptides in the modulation of prostatic secretions, as well as smooth muscle tone. A predominantly epithelial location for prostate muscarinic receptors indicated a major secretomotor role for acetylcholine. The muscarinic receptor subtype mediating muscarinic agonist-induced smooth muscle contraction or enhancement of contractions evoked by nerve stimulation differs in different species. In the human, there is evidence for M(1) receptors on the epithelium, M(2) receptors on the stroma, and both M(1) and M(3) receptors in some prostate cancer cell lines. Several recent investigations indicate that muscarinic receptors may also mediate or modulate normal, benign, and malignant prostate growth. The role of muscarinic agonists and their receptors and the influences of age, testicular, and other steroids in regulating the effects are reviewed.

Novel drug targets for the pharmacotherapy of benign prostatic hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) is the major cause of lower urinary tract symptoms in men aged 50 or older. Symptoms are not normally life threatening, but often drastically affect the quality of life. The number of men seeking treatment for BPH is expected to grow in the next few years as a result of the ageing male population. Estimates of annual pharmaceutical sales of BPH therapies range from

Adenosine 5′-triphosphate (ATP) is an excitatory cotransmitter with noradrenaline to the smooth muscle of the rat prostate gland

US 3 to 10 billion, yet this market is dominated by two drug classes. Current drugs are only effective in treating mild to moderate symptoms, yet despite this, no emerging contenders appear to be on the horizon. This is remarkable given the increasing number of patients with severe symptoms who are required to undergo invasive and unpleasant surgery. This review provides a brief background on prostate function and the pathophysiology of BPH, followed by a brief description of BPH epidemiology, the burden it places on society, and the current surgical and pharmaceutical therapies. The recent literature on emerging contenders to current therapies and novel drug targets is then reviewed, focusing on drug targets which are able to relax prostatic smooth muscle in a similar way to the α1‐adrenoceptor antagonists, as this appears to be the most effective mechanism of action. Other mechanisms which may be of benefit are also discussed. It is concluded that recent basic research has revealed a number of novel drug targets such as muscarinic receptor or P2X‐purinoceptor antagonists, which have the potential to produce more effective and safer drug treatments.

Sympathetic co‐transmission to the cauda epididymis of the rat: characterization of postjunctional adrenoceptors and purinoceptors

This study investigated whether adenosine 5′‐triphosphate (ATP) is involved in neurotransmission to the rat prostate gland. Fluorescence immunohistochemistry carried out on formaldehyde‐fixed and frozen sections of rat prostate showed immunoreactivity for the P2X1‐receptor in the fibromuscular stroma surrounding the secretory acini but not in the glandular epithelium. P2X2‐, P2X3‐, P2X4‐ and P2X7‐receptors were immunonegative in the rat prostate stroma. Double‐staining procedures showed P2X1‐receptor immunoreactivity to be colocalized with α‐actin immunoreactivity. Isolated organ bath studies investigated whether drugs, which modify purinergic mechanisms, are able to affect contractility of the rat prostate gland. Suramin (100 μM) and αβmethylene ATP (10 μM) inhibited contractile responses to trains of electrical‐field stimulation (70 V, 0.5 ms, 0.1–2 Hz) in the absence and presence of prazosin (300 nM). Responses to 5–20 Hz were unaffected by suramin or αβmethylene ATP. Exogenous application of ATP analogues to unstimulated isolated preparations of rat prostate produced concentration‐dependent suramin (100 μM) sensitive transient contractions with a relative order of potency: αβmethylene ATP>βγmethylene ATP>ATP. Adenosine and adenosine 5′‐monophosphate (AMP) did not produce contractile responses. These results suggest that P2X1‐receptors for ATP, which mediate contractions are present in the fibromuscular stroma of the rat prostate. The relative order of potency of ATP analogues in producing contractions of the rat prostate is consistent with the activation of P2X1‐receptors. Inhibition by suramin and αβmethylene ATP of electrically evoked nerve‐mediated contractions of the rat prostate implies that ATP contributes to this contractile response and is therefore a cotransmitter with noradrenaline during low‐frequency stimulation.

Identification and Profiling of Novel α1A-Adrenoceptor-CXC Chemokine Receptor 2 Heteromer

1 Electrical field stimulation (10 Hz, 60 V, 1 ms, for 10 s) produced monophasic contractions of isolated preparations of rat cauda epididymis which could be abolished by guanethidine, and attenuated by prazosin and α,β‐methylene ATP. 2 The rank order of potency of adrenoceptor agonists in causing contraction of the preparation in the presence of the neuronal uptake blocker, nisoxetine (0.1 μm) was: adrenaline ≥ phenylephrine ≥ noradrenaline > clonidine > methoxamine > metaraminol > dopamine ≥ isoprenaline > xylazine. 3 Responses to the agonists were blocked by prazosin but not by propranolol or idazoxan. 4 The rank order of potency of purinoceptor agonists in causing contraction of the cauda epididymis was: α,β‐methylene ATP > β,γ‐methylene ATP ≥ 2‐methylthioATP > ATP > ADP. AMP and adenosine did not cause contractions. 5 Contractile responses to the purine nucleotide analogues were blocked by repeated application of α,β‐methylene ATP. 6 It is concluded that both ATP and noradrenaline may act as co‐transmitters in the sympathetic nerves supplying the smooth muscle of the rat cauda epididymis, and that α1‐adrenoceptors and P2X‐purinoceptors are present postjunctionally.

Calcitonin gene-related peptide (CGRP) inhibits contractions of the prostatic stroma of the rat but not the guinea-pig.

Background: Receptor heteromers are macromolecular complexes containing at least two different receptor subunits, resulting in distinct pharmacology. Results: The observed α1AAR-CXCR2 heteromer recruits β-arrestin strongly upon activation with norepinephrine, in contrast to α1AAR alone. Conclusion: Heteromerization with CXCR2 dramatically changes α1AAR pharmacology, revealing the potential for heteromer-specific biased agonism. Significance: Such heteromer-specific novel pharmacology has important implications for drug discovery. We have provided the first evidence for specific heteromerization between the α1A-adrenoceptor (α1AAR) and CXC chemokine receptor 2 (CXCR2) in live cells. α1AAR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α1AAR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α1AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α1AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α1AAR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α1AAR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.

α2-Adrenoceptor binding sites vary along the length of the male reproductive tract : a possible basis for the regional variation in response to field stimulation

This study investigated the presence and effects of calcitonin gene-related peptide (CGRP) within the rat and guinea-pig prostate glands. Immunohistochemical studies demonstrated that CGRP immunoreactive nerve fibres are sparsely distributed throughout the prostatic fibromuscular stroma in both species. These CGRP immunopositive nerve fibres shared a similar distribution profile but were not colocalized with tyrosine hydroxylase immunopositive nerve fibres which also innervate the prostatic stroma of these species. Nerve terminals within rat and guinea-pig prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). In guinea-pig preparations, application of human alpha-CGRP, rat adrenomedullin or rat amylin (0.1 nM-1 microM) had no effect on responses to field stimulation. In contrast, both rat and human alpha-CGRP (10 pM-300 nM), rat adrenomedullin (0.3 nM-1 microM) and rat amylin (3 nM-1 microM) concentration-dependently inhibited electrically evoked contractile responses in the rat prostate. The relative order of potency was rat alpha-CGRP=human alpha-CGRP>rat adrenomedullin>rat amylin. The inhibition by rat alpha-CGRP of field stimulation-induced contractions in the rat prostate was competitively antagonized by human CGRP((8-37)) (1, 3 and 10 microM) with a pA(2) of 6.20+/-0.13. Rat alpha-CGRP (10 nM) attenuated contractile responses of the rat prostate to exogenously added noradrenaline (1-100 microM). Inhibitory concentration-response curves to rat alpha-CGRP in rat prostates were unaffected by preincubation in either glibenclamide (10-100 microM), N-nitro-L-arginine methyl ester (L-NAME) (10 microM), bestatin (10 microM), captopril (10 microM) or phosphoramidon (3 microM). Our results indicate that CGRP-induced inhibition of electrically evoked contractions in the rat prostate occurs through activation of postjunctional CGRP(2) receptors which act independently of a K(ATP) channel or nitrergic mechanisms. Degradation of rat alpha-CGRP via peptidases does not appear to occur in the rat prostate.

Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice

Field stimulation (60V, 1 ms, 10 Hz, 10 s) produced monophasic contractions of cauda epididymides that were enhanced by the noradrenaline uptake inhibitor, nisoxetine, but unaffected by the alpha 2-adrenoceptor antagonist, idazoxan. Similar stimulation of vas deferens produced biphasic contractions that in the presence of idazoxan were enhanced and became monophasic. Nisoxetine accentuated the separation of the two phases. Radioligand binding studies yielded dissociation constants (KD) for [3H]prazosin binding that were similar (0.1-0.2 nM) in membrane preparations of both halves of the vas deferens and in the cauda epididymis; maximum binding density (Bmax) was slightly lower in cauda epididymis (20 fmol/mg protein) than in vas deferens (approximately 50 fmol/mg protein). KD values for [3H]rauwolscine were similar in the two halves of the vas deferens but Bmax values were higher in the prostatic half of the tissue (39 fmol/mg protein) than in the epididymal half (22 fmol/mg protein). We were unable to detect specific binding of [3H]rauwolscine in cauda epididymis. Absence or masking of alpha 2-adrenoceptors in this tissue would explain our findings that alpha 2-adrenoceptor agonists and antagonists do not modulate the responses of the cauda epididymis to trains of field stimulation.

Regional vascular responses to ATP and ATP analogues in the rabbit kidney in vivo: roles for adenosine receptors and prostanoids

Significance The search for a viable male contraceptive target has been a medical challenge for many years. Most strategies have focused on hormonal or germ-line strategies to produce dysfunctional sperm that are incapable of fertilization. The problem with such approaches is that they have intolerable side effects such as affecting male sexual activity or causing long-term irreversible effects on fertility. In addition, some strategies may transmit detrimental changes to future offspring. This manuscript describes a male contraceptive target within the autonomic nervous system, which would not affect the long-term viability of sperm nor the sexual or general health of males. In addition, due to the nature of the target, the contraceptive has the potential to be orally administered. Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive.

An immunohistochemical and pharmacological study of tachykinins in the rat and guinea-pig prostate glands.

Our knowledge of the effects of P2‐receptor activation on renal vascular tone comes mostly from in vitro models. We aimed to characterise the pharmacology of ATP in the renal circulation in vivo.