AbstractLower urinary tract symptoms (LUTS) affect a large proportion of the world’s population (~17%). The world population is also aging and the prevalence of LUTS will only increase as they are associated with ageing. The current treatments for LUTS, particularly those for the subgroup of patients with overactive bladder syndrome, exert adverse effects, such as dry mouth and constipation which can be severe and result in patient non-compliance. These drugs can also impair cognitive function and may put patients at an increased risk of developing Alzheimer’s disease. However, a common target for new drug treatments has been suggested, the vasculature of the lower urinary tract.
The main resistance artery supplying the bladder with blood is the superior vesical artery (SVA). The mediators regulating tone of this artery have not been investigated, although several publications have examined the microvasculature within the suburothelium of the bladder wall. The rat bladder microvasculature is innervated with sympathetic and nitrergic nerves, where nerve-evoked vasoconstriction is mediated by noradrenaline on α1- adrenoceptors and vasodilation by nitric oxide which is produced via both endothelial and neuronal nitric oxide synthase (eNOS and nNOS). It is not known whether the SVA is regulated by the same vasoactive mediators. The bladder vasculature has also been demonstrated to lack the typical myogenic response seen in other vessels.
There is mounting evidence that reductions in bladder perfusion are involved in the development of LUTS. Improving bladder perfusion may potentially be an effective treatment in ameliorating symptoms. Animal and clinical trials investigating lower urinary tracts symptoms have demonstrated that the clinically used phosphodiesterase-5 inhibitors (e.g. tadalafil) and α1-adrenoceptor antagonists (e.g. tamsulosin) improve lower urinary tract symptoms by improving blood flow to the bladder wall.
The aim of these studies was to investigate the pharmacology of the porcine superior vesical artery, due to its importance to bladder perfusion, and the potential for clinical treatments to exert action on this site. Specifically, the α1-adrenoceptors mediating contraction were classified, age-related changes to α1-adrenoceptor populations were identified, the neurotransmitters mediating responses to electrical field stimulation were examined, and the effects of phosphodiesterase-5 inhibition were investigated.
Classification of α1-adrenoceptors and age-related changes were investigated via in vitro functional organ bath studies using ring segments of isolated porcine SVA. The neurotransmitters mediating contractile and relaxatory responses were examined using electrically field stimulated intact and endothelium-denuded porcine SVA. Phosphodiesterase-5 inhibition was examined in both drug stimulated and electrically field stimulated porcine SVA. For age-related changes, isolated vessels from young (6-months old) and older (average 36-months old) pigs were utilised, but only young superior vesical arteries were used for the rest of the project.
Electrical field stimulation of the isolated SVA tissues induced an initial fast vasoconstriction followed by a slower, long vasodilation. Nerve-evoked vasoconstrictions were mediated by the release of two co-transmitters adenosine triphosphate (ATP) and noradrenaline acting on purinergic and α1-adrenergic receptors respectively. ATP was the predominant neurotransmitter contributing 60% to vasoconstrictor responses following nerve stimulation.
After removing the contractile components of the responses to nerve stimulation the main neurotransmitter mediating relaxation was identified as nitric oxide, with the nitric oxide synthase inhibitor L-NNA removing 80% of the nerve-evoked vasodilations in intact and endothelium-denuded vessels. The remaining neurogenic vasodilation (20%) was mediated by noradrenaline acting on β-adrenoceptors. In endothelium-denuded arterial rings, the selective nNOS inhibitor L-NPA, reduced vasodilations by 80%, with the remainder abolished by L-NNA. Therefore, nerve-evoked vasodilations of the porcine superior vesical
artery were mediated by nitric oxide (80%) via endothelial and neuronal nitric oxide synthase and noradrenaline acting on β-adrenoceptors.
In the presence of the phosphodiesterase-5 inhibitors tadalafil and sildenafil, nerve-evoked vasodilations were significantly enhanced. Noradrenaline was used to pre-contract the tissues before examining relaxations, and these inhibitors reduced the magnitude of the precontractions to noradrenaline. They also increased the potency of nitric oxide donors. As nitric oxide is the predominant endogenous vasodilator of the SVA, the results suggest that phosphodiesterase-5 inhibitors exert at least part of their therapeutic action via an action on this artery, thus improving bladder wall perfusion and bladder function.
Phenylephrine, noradrenaline and A-61603 all evoked concentration-dependent
vasoconstrictions of the porcine SVA. The α1A-adrenoceptor selective agonist A-61603 was the most potent agonist in this tissue. The low affinity of the selective α1D-adrenoceptor antagonist BMY-7378 indicated the lack of a role for α1D-adrenoceptors in the SVA. Prazosin which is selective for α1A-, α1B- and α1D-adrenoceptor subtypes also yielded a low affinity estimate and the slope of the Schild plot for this antagonist was less than one, suggesting phenylephrine was acting on two or more receptors. The affinity estimates of prazosin suggested that the α1L-adrenoceptor phenotype was mediating at least part of the vasoconstriction to phenylephrine. The α1A- and α1L-adrenoceptor selective antagonists tamsulosin and silodosin both yielded high affinity estimates, suggesting that α1A- and/or α1L-adrenoceptor were present. The slope of the Schild plot for tamsulosin was equal to unity, whereas for silodosin was significantly less than one. All of the other α1A-adrenoceptor selective antagonists examined, RS-100329, RS-17053 and SB-216469, produced nonuniform shifts of phenylephrine and noradrenaline concentration—response curves, with higher concentrations of the agonist being antagonized more than lower concentrations of the agonist. This unusual effect was not observed with these antagonists when using A- 61603 as the agonist. Parallel shifts of agonist curves were observed to prazosin when using A-61603 as the agonist, but affinity estimates for this antagonist and for RS-17053 were still low, suggesting the presence of the α1L-adrenoceptor. Silodosin, tamsulosin and RS-100329
yielded high affinity estimates with A-61603 as the agonist, suggesting that the α1A- and/or α1L-adrenoceptor was present. These data with selective agonists and antagonists suggest that vasoconstrictions of the young porcine superior vesical artery were mediated by noradrenaline acting via the α1L-adrenoceptor.
Similar non-uniform shifts of agonist curves when using these selective antagonists were also seen in SVA tissues from older pigs. Additionally, prazosin yielded an affinity estimate of 8.9, with a Schild plot slope of unity, suggesting that the α1A-adrenoceptor mediates vasoconstriction. This suggests that the pharmacology of the porcine superior vesical artery may change with age, from α1L- to α1A-adrenoceptors. However, this does not affect the potency of either tamsulosin or silodosin as a potential therapy in ischaemic lower urinary tract symptoms, since both drugs have a high affinity at α1A- and α1L- drenoceptors.
Thus, a novel pharmacology for the porcine SVA was observed in arterial rings from both young and older animals. Non-uniform shifts, where antagonists were more effective at high agonist concentrations could be explained by the existence of orthosteric and allosteric binding sites. The binding of a ligand could be causing an upregulation of 2nd messenger molecules or inactivating this upregulation leading to non-uniform shifts.
The porcine superior vesical artery has demonstrated novel pharmacology and presents as a potential target for new drug treatments, as both clinically used phosphodiesterase-5 inhibitors and α1-adrenoceptor antagonists exert potent actions at this site. Further research will be necessary to determine the role of allosteric binding or coupling in association with the non-uniform shifts by antagonists when using noradrenaline and phenylephrine. Additionally, these binding or coupling sites may provide new novel drug development targets for ischaemic lower urinary tract symptoms with potential to be selective for the
|Date of Award||2023|
|Supervisor||Russ Chess-Williams (Supervisor) & Donna Sellers (Supervisor)|