Ageing influences detrusor contractions to prostaglandin, angiotensin, histamine and 5-HT (serotonin), independent to the Rho kinase and extracellular calcium pathways

Ageing is associated with deteriorating urinary bladder function and an increasing prevalence of disorders such as underactive bladder. There are suggestions that G protein-coupled receptor (GPCR) second messenger pathways are altered during ageing, rather than the receptor proteins themselves. The aim of this study was to identify age-related variations in GPCR activation systems in urinary bladder smooth muscle (detrusor). Isolated porcine detrusor strips were mounted in organ baths and contractile responses induced by receptor agonists were assessed and compared between juvenile (6 months) and adult (2 years) animals. The effects of drugs disrupting intracellular calcium signalling were also studied. Adult tissue was far more sensitive to stimulation by 5-hydroxytryptamine (42% greater increase than juvenile), prostaglandin-E2 (26% greater increase), and angiotensin-II (39% greater increase), however less sensitive to histamine. Although nifedipine and Y-27632 impacted the contraction to all agonists, there were no significant differences between juvenile and adult detrusor. Impairment of IP3-mediated calcium release by 2-aminoethyl diphenylborinate had no effect on any contractile activity, except for neurokinin-A which inhibited both juvenile and adult detrusor, and prostaglandin-E2 which inhibited juvenile. Carbachol, histamine, 5-hydroxytryptamine, and angiotensin-II were not affected by the application of 2-aminoethyl diphenylborinate. In conclusion, the contractile responses to all the GPCR agonists involved extracellular calcium influx and calcium sensitisation, but for prostaglandin-E2 the dependence on calcium from intracellular sources was greater in the younger animals.


Intracellular Ca 2+ for GPCR activation of juvenile and adult tissues
After application of an inhibitor of IP3-induced Ca 2+ release, 2-APB (300 μM), there was no significant difference in the change in baseline tension for responses to carbachol (1 μM, n = 8), histamine (100 μM, n = 8), 5-HT (100 μM, n = 8), and ATII (100 nM, n = 8) for both juvenile and adult detrusor smooth muscle (Fig. 3).GPCRmediated contraction in the presence of 2-APB compared to controls was reduced in both juvenile and adult detrusor tissues in response to NKA (300 nM) by 38% (n = 8, p = 0.04) and 31% (n = 8, p = 0.004) respectively, with no significant difference between the two groups.However, after the application of 2-APB, a difference was recorded between juvenile and adult responses to PGE2 (10 μM, p = 0.03, unpaired Student's two-tailed t-test).While the response to PGE2 activation was inhibited in juvenile samples by 35% (n = 8, p = 0.02), there was no inhibition in aged samples (Fig. 3).

Rho kinase pathway for GPCR activation of juvenile and adult tissues
After activation of the six GPCRs with agonists, in the presence of the Rho kinase inhibitor, Y-27632 (1 μM), increases in the baseline tension of detrusor smooth muscle tissues for both juvenile and adult samples was significantly reduced compared to controls (p < 0.05 for all, paired Student's two-tailed t-test, Fig. 4).Inhibition of GPCR-mediated contractions by Y-27632 for juvenile and adult detrusor samples, respectively, was: 1 μM carbachol by 42% and 34% (n = 8); 100 μM histamine by 66% and 61% (n = 6); 100 μM 5-HT by 60% and 52% (n = 6); 300 nM NKA by 51% and 52% (n = 8); 10 μM PGE2 by 47% and 43% (n = 8); and 100 nM ATII by 78% and 54% (n = 8).When comparing the difference between juvenile and adult responses to the inhibition of Rho kinase,  www.nature.com/scientificreports/Y-27632 was equally as effective at inhibiting contractile activity between the two age groups, with no significant differences (unpaired Student's two-tailed t-test) between the magnitude of responses to any of the agonists.

Discussion
As the prevalence of underactive bladder increases with age, this study sought to investigate different time points to see how receptor-mediated contractile activity of the urinary bladder differs.Using porcine detrusor smooth muscle, various G protein-coupled receptor systems and their downstream signalling pathways that contribute to detrusor smooth muscle contractions were explored.The findings from this research highlight the varying responses to key mediators of contraction between juvenile and adult urinary bladder smooth muscle.Whilst the alterations do not appear to be related to G q/11 -coupled second messenger pathways, particularly extracellular Ca 2+ or Rho kinase, there were differences in the sensitivity to intracellular Ca 2+ .The activation of the muscarinic, histamine, 5-HT, NKA, PGE2, or ATII receptors increased the force of contraction in both juvenile and adult urinary bladder detrusor smooth muscle.Muscarinic receptors, in particular the M3 muscarinic receptor remain the most widely studied receptor in the detrusor 29 and urothelium/ lamina propria 30 , as they are primarily responsible for mediating contractions through cholinergic activation, and therefore remain the main target for treating bladder contractile disorders 31 .However, due to the side effects and lower-than-expected treatment outcomes associated with muscarinic receptor therapeutics 2 , there is a growing interest in additional receptor systems in the urinary bladder that may present novel targets for future pharmaceutical development.Of note, the H1 histamine 32 , 5-HT 2A 33 , neurokinin-2 34 , EP1 prostaglandin E2 35 , and AT 1 angiotensin II 15 receptors are known to cause contractions in the urinary bladder, and therapeutics that target these G q/11 -coupled receptors could be effective in the treatment of bladder contractile disorders, such as underactive bladder.
Across the GPCRs explored, developmental differences were identified in detrusor contractile activity in response to some of the agonists.Contractile responses to histamine in the adult detrusor were significantly smaller when compared to contractions observed in juveniles, which is consistent with findings by Stromberga et al. 20 .This could be explained by reduced compliance in the adult detrusor as a result of a greater deposition of collagen in the aged bladder 36,37 .In contrast, the urinary bladder of adult detrusor responded to a variety of agonists, including 5-HT, PGE2, and ATII, to a higher degree than the juvenile detrusor.It is known that these agonists are involved in bladder contraction, and the finding that the detrusor contractile response is more sensitive with age may indicate involvement in bladder dysfunction.Beyond receptor activation, intracellular signalling mechanisms contributing to contraction could also be accessible targets for pharmaceutical development 38 , and these may also be altered with age.Extracellular Ca 2+ , intracellular Ca 2+ , and Rho kinase are important components of the G q/11 -coupled contraction pathway 39 , and alterations to the availability of these molecules for contraction may contribute to the development of bladder contraction disorders.
The contribution of extracellular Ca 2+ and Rho kinase for contractions was consistent across both juvenile and adult detrusor, indicating that extracellular Ca 2+ entry into the tissue via voltage-gated Ca 2+ channels and Ca 2+ sensitisation via the Rho kinase pathway is unlikely to be impaired with age for various G q/11 receptors known to cause contraction.The influence of Ca 2+ from intracellular sources for agonist-induced contractions of tissues was minimal, and this was similar for the two age groups.This highlights that there is a stronger dependence on extracellular Ca 2+ and Rho kinase for mediating muscarinic, histamine, 5-HT, and ATII detrusor contractions.Impairment of IP3-mediated Ca 2+ release with 2-APB inhibited contractions induced by NKA for both juvenile and adult detrusor samples and juvenile samples inhibited in response to PGE2.These findings are unique to these receptors in the detrusor, as the influence of intracellular Ca 2+ release was found to be minimal for neurokinin and prostaglandin receptors in the urothelium and lamina propria 17 .The finding that there was decreasing sensitivity to intracellular Ca 2+ in response to PGE2-mediated contractions during ageing indicates an age-related difference in the G q/11 second messenger pathway.The EP1 prostaglandin E2 receptor may be of particular interest in future studies, as the magnitude of inhibition was significantly greater in juvenile detrusor than adult detrusor.This may be supported by findings that urinary bladder PGE2 levels are negatively correlated with age 40,41 , and as such, further investigations focussing on the receptor expressions could assist.One hypothesis for this age-related difference is that the juvenile detrusor may be more dependent on IP3-mediated Ca 2+ release for PGE2-induced contractions, whereas activation of the prostaglandin receptors in the adult detrusor relies on an increase in extracellular Ca 2+ , which may activate ryanodine receptors to induce intracellular Ca 2+ release 42 .

Limitations and future directions
Although a viable and well-validated model, porcine tissue may not directly correlate to human function, and experiments that replicate this study on human tissue would be of interest.It is also unclear which age would be equivalent to a 2-year-old sow.These animals are past their reproductive prime, and often referred to as the older pigs.However, it would likely not be correct to classify these animals as "elderly".As such, the terms juvenile and adult were used throughout, and future studies could investigate different age groups.Further, finding the molecular mechanisms involved in the differences observed would present an interesting direction for future investigations, as well as comparing different animal models.Lastly, future studies could explore age-related alterations in the signalling pathways associated with GPCR-mediated contractions across the various tissue layers in the urinary bladder, such as the urothelium and lamina propria.The increased contraction to 5-HT, PGE2, and ATII, and decreased sensitivity to histamine in older tissue, may provide insights into potential systems that could contribute to dysfunction in the contractility of the urinary bladder.In most cases, these observed alterations do not appear to be related to G q/11 -coupled second messenger pathways that involve extracellular Ca 2+ or Rho kinase, highlighting a potentially important age-related alteration.Alternatively, the decreased sensitivity to intracellular Ca 2+ in response to PGE2-mediated contractions in aged tissues, compared to juvenile samples may also warrant further investigation.Overall, juvenile and adult bladders demonstrate clear differences in their ability to contract in response to agonist stimulation, suggesting additional mechanisms that may contribute to bladder contractile dysfunction.

Tissue collection
Porcine urinary bladders from Large White-Landrace-Duroc cross-bred pigs (Suf scrofa domestica) were obtained from the local abattoir after slaughter for the routine commercial provision of food.Juvenile samples were taken from prepubescent pigs aged 6 months old at 80 kg live weight, and adult samples from sow pigs aged 2-3 years old at 200 kg live weight.After collection from the abattoir, tissues were transported in a portable cooler in cold Krebs-Henseleit bicarbonate solution (Krebs, composition in mM: NaCl 118.4;NaHCO 3 24.9;d-glucose 11.7;

Figure 1 .
Figure 1.Representative experimental trace comparing juvenile (left) and adult (right) detrusor tension response to 5-HT (100 μM).Note the significantly greater response in the adult detrusor compared to the juvenile detrusor.

Figure 2 .
Figure 2. The influence of 1 μM nifedipine on GPCR-mediated contractile responses in juvenile (upper chart) versus adult (lower chart) detrusor smooth muscle (n = 8 for all).*p < 0.05, **p < 0.01, ***p < 0.001 (paired Student's two-tailed t-test).Data reported as mean ± SEM.As carbachol induced a stronger contraction than the other receptors, the y-axis for this data set was expanded.

Table 1 .
Comparison of responses to GPCR activation between juvenile and adult detrusor tissues.Data reported as mean ± SEM.Bold font indicates statistical significance (p < 0.05, unpaired Student's two-tailed t-test).