An investigation of the non-adrenergic contractile mechanisms in the mouse prostate gland
thesisposted on 07.02.2017 by White, Carl William
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The aims of this thesis were: 1) to characterise the muscarinic receptor subtype mediating contraction in the mouse prostate gland, 2) to investigate the signalling pathways involved in contraction mediated by the receptor determined in the first aim, 3) to investigate the effect of age on the contractile mechanisms in the mouse prostate gland, 4a) to confirm the role of muscarinic receptors in mediating contraction in the mouse prostate gland by generating and characterising dual α1A-adrenoceptor/P2X1-purinoceptor knockout mice and 4b) to investigate the effect of dual α1A-adrenoceptor/P2X1-purinoceptor deletion on male mouse fertility. 1. Using isolated organ bath studies the muscarinic receptor subtype mediating the cholinergic contractile response in the mouse prostate was characterised pharmacologically. The rank order of antagonist potency for seven muscarinic receptor antagonists in the mouse prostate was determined and correlated with known antagonist affinity values (pKi) for each of the 5 muscarinic receptor subtypes. With the best correlation (r = 0.98, p < 0.001, slope = 1.05 ± 0.08), the M3 muscarinic receptor subtype was determined to mediate contraction in the mouse prostate. Using genetically modified M3 muscarinic receptor knockout mice, the M3 muscarinic receptor subtype was subsequently confirmed to elicit nerve and agonist mediated contraction in the mouse prostate. 2. Smooth muscle contraction requires a rise in intracellular calcium (Ca2+) and subsequent binding of intracellular Ca2+ to calmodulin which activates myosin light chain kinase (MLCK) and in turn phosphorylates myosin light chain (MLC) filaments resulting in contraction. This thesis shows that in the mouse prostate the M3 muscarinic receptor subtype couples to the prototypical Gq/11 second messenger signalling pathway via activation of phospholipase C, suggesting that prostate contraction is mediated by IP3-mediated intracellular Ca+2 release, as well as influx of extracellular Ca2+ via voltage-gated L-type Ca2+ channels. Additionally, RhoA and Rho-kinase were found to be immunolocalised in the mouse prostatic smooth muscle, and both M3 muscarinic receptors and α1A-adrenoceptors activate the Rho kinase-dependent Ca2+ sensitisation signalling pathway, which is involved in contraction, indicating a convergent signalling pathway. These results further the understanding of muscarinic receptor mediated contraction in the prostate and suggest that the convergent Rho-kinase pathway may be a suitable target for the relaxation of prostatic smooth muscle in patients with lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH). 3. BPH is an age related disease of the prostate, however the effect of age on morphology and contractility of the mouse prostate model is unknown. With age the mouse prostate increased in size and weight, although histological BPH was not observed. Age did not affect the α1A-adrenoceptor mediated component of the contractile response in the mouse prostate. Interestingly, a decrease in the size of prostates taken from 12 month old α1A-adrenoceptor knockout mice was observed, indicating a role for α1A-adrenoceptors in growth of the mouse prostate. No change in the nerve mediated muscarinic contractile response was observed, however a decrease in the potency of acetylcholine but not carbachol was observed with age suggestive of an age related change in the activity of acetylcholinesterase. Furthermore, a nerve mediated non-adrenergic non-cholinergic contractile response developed with age and was shown to be mediated by P2X1-purinoceptors. This purinergic contractile response appeared to develop, in part, due to an increase in P2X1-purinoceptor number, however a large increase in the potency of ATP was also observed. The change in potency of ATP in the aged mouse prostate was subsequently shown to be caused by a decrease in the rate of ATP breakdown due to a loss of ATPase activity. These results indicate that α1A-adrenoceptor antagonists may in part relieve symptoms associated with BPH by decreasing prostate size. Furthermore, as a purinergic component of contraction develops with age in the prostate, purinoceptors may be a suitable additional target for the treatment of BPH. 4. This thesis is the first to generate dual α1A-adrenoceptor/P2X1-purinoceptor double knockout (DKO) mice and to observe the effects of this dual receptor deletion. DKO mice were generated by selective cross-breeding of single receptor knockout mice and are phenotypically normal with no observable changes to the cardiovascular system greater than that observed in single α1A-adrenoceptor knockout mice. However, significant changes were observed in the male reproductive tract. A marked decrease in nerve mediated vas deferens contractility was observed which physiologically prevents the transport of sperm from the epididymis to the urethra resulting in infertility in male DKO mice. However, a small residual nerve mediated contractile response was observed in the vas deferens of DKO mice and noradrenaline elicited a diminutive contractile response, both responses appear to be mediated by α1D-adrenoceptors, possibly due to compensatory up-regulation induced by the dual receptor deletion. Nerve mediated contraction of both the seminal vesicle and coagulating gland were inhibited in DKO mice which prevented the formation of a coagulum plug following copulation. In the prostate, the muscarinic receptor antagonist atropine abolished the nerve mediated contractile response confirming the role of the M3 muscarinic receptors in mediating the non-adrenergic contractile response in the young adult mouse prostate. In addition to confirming the role of M3 muscarinic receptors in mediating contraction of the mouse prostate, these results suggest that dual pharmacological blockade of α1A-adrenoceptors and P2X1-purinoceptors may be a safe and effective non-hormonal target for male contraception.
Awards: Winner of the Mollie Holman Doctoral Medal for Excellence, Faculty of Pharmacy and Pharmaceutical Sciences, 2013.