Urinary bladders from Large White-Landrace-Duroc pigs (6 months old, 80 kg live weight) were used as the tissue in this study. All bladders were obtained from the local abattoir after slaughter for the routine commercial provision of food with no animals bred, harmed, culled, interfered, or interacted with as part of this research project. As such, animal ethics approval was not required (Business Queensland, 2015). Only female bladders were used in this study. After collection from the abattoir, tissues were transported in a portable cooler in cold Krebs-Henseleit bicarbonate solution (NaCl 118.4 mM, NaHCO₃ 24.9 mM, D-glucose 11.7 mM, KCl 4.6 mM, MgSO₄ 2.41 mM, CaCl₂ 1.9 mM, and KH₂PO₄ 1.18 mM) maintained at 4 °C to the University research facilities and used within 3 hours of the animal’s slaughter.

A single 4 cm strip was taken longitudinally from the bladder wall at the midpoint between the trigone and the bladder apex. Tissue strips were continually washed with a cold Krebs-Henseleit bicarbonate solution during the preparation and dissection stage. The white-coloured detrusor smooth muscle was dissected away from the pink-coloured urothelium and lamina propria using fine scissors. Histological assessment is known to effectively separate the two layers (Moro et al., 2012). After dissection and preparation, this single strip was cut in the middle, resulting in 2 × 2 cm strips which were mounted and suspended in 10 ml isolated tissue baths (Labglass, Brisbane, Australia) containing warmed Krebs solution at 37°C and perfused with carbogen gas (95% oxygen and 5% carbon dioxide). A maximum of two 4 cm strips were taken from each unique porcine bladder across the experiments. Throughout this manuscript, n values quoted are from paired tissue strips, and as such, the number of animals (N) used can be calculated using n ÷ 2 . After mounting, tissue tension was manually adjusted to 2 g using each transducer positioner’s fine adjustment knob. Each bath was washed with warmed Krebs a total of three times prior to the start of experimentation. At the conclusion of each experiment, tissue weight was measured on a scale to an accuracy of 0.001 g. The mean weight of porcine U&LP tissues was 0.22 ± 0.01 g (n = 208).

Krebs-Henseleit bicarbonate solution ingredients were obtained from Sigma-Aldrich (Missouri, U.S.). Carbamylcholine chloride (carbachol) and histamine dihydrochloride (histamine) were obtained from Sigma-Aldrich (Missouri, U.S.), nifedipine and NKA were from Tocris Bioscience (Bristol, U.K.), 5-HT was from Toronto Research Centre (Toronto, CA), and ATII and PGE2 were obtained from Cayman Chemicals (Michigan, U.S.). Nifedipine and PGE2 were dissolved in 100% ethanol, while all other pharmaceutical agents were soluble in distilled water. Concentrations chosen for the agonists and antagonists were selected based on their selectivity at each receptor and consistent with concentrations used in previous studies. In each case, the agonist concentration used induced a submaximal contraction (with an aim to achieve around 80% of peak receptor-induced contraction).

In the nifedipine studies, a vehicle control of 100% ethanol was added to control tissues or nifedipine (1 µM) was added to the experimental tissues and left to equilibrate for 30 min. Nifedipine was kept in darkness until the final application in the organ bath and experiments were concluded within 30 min to ensure no adverse light impacts. In the Ca²⁺-free studies, control tissues were washed with Krebs solution as normal, whereas the experimental tissues were washed three times with a nominally Ca²⁺-free Krebs solution. This also ensured that any excess Ca²⁺ on the tissue, or Ca²⁺ leaking out from intracellular sources, was cleared from the bath. Tissues were then left to equilibrate for 3 min in nominally zero Ca²⁺ solution before agonists were added. A single dose of a select GPCR agonist was added to both the control and experimental tissues after equilibration. Tension, frequency, and amplitude of spontaneous contractions were measured with an isometric force transducer (MCT050/D, ADInstruments, Castle Hill, Australia) and recorded on a Powerlab system using Labchart v7 software (ADInstruments). Although a threshold was not applied, in all cases each spontaneous contraction exceeded 0.4 g.

Changes in tension and amplitude were measured in grams (g), where amplitude was measured from the lowest point of spontaneous phasic contraction to peak. Frequency was expressed as the number of spontaneous phasic contractions per minute (cpm). All data was analysed using GraphPad Prism version 9 (San Diego, CA), and results were presented as mean ± standard error of the mean (SEM). A paired t-test was used to analyse tissue responses before and after the addition of agonist. A paired Student’s two-tailed t-test was used to analyse the significance of results when comparing tissues with direct controls, as per previous studies (Stromberga et al., 2020a). A one-way ANOVA with Tukey post-test was also undertaken to compare the means where more than two variables were assessed. For all statistical analysis, p < 0.05 was considered statistically significant.

There is variation in the literature surrounding methods to remove extracellular Ca²⁺ from an isolated tissue bath. Firstly, this may involve the omission of calcium chloride (CaCl) from Krebs-Henseleit bicarbonate solution (Poyser, 1984). Secondly, the addition of Ca²⁺ chelating agents ethylenediaminetetraacetic acid (EDTA, 1–5 mM) (Maggi et al., 1989) or glycoletherdiaminetetraacetic acid (EGTA, 0.5 mM) solution (Yoshimura and Yamaguchi, 1997) have been proposed. However, EGTA may have impacts on other ions (Wheeler and Weiss, 1979) or other mechanisms within the tissue (Poyser, 1984), as well as potentially destabilise cell membranes, leading to increased Ca²⁺ permeability (Guan et al., 1988). To assess this feasibility of using Ca²⁺ chelators, we observed that compared to the simple omission of CaCl, the addition of 1 mM EDTA or 0.5 mM EGTA had no effect on contractile responses to our agonists (e.g., carbachol 1 µM). Thirdly, in some studies, the removal of Ca²⁺ appeared to result in a hypovolemic solution, and in order to rectify this, the concentration of other ions was altered. In our pilot studies, when incorporating additional Mg²⁺ to substitute for omitted Ca²⁺, there was no significant effect on contractions (n = 8). As such, no additional magnesium was added to replace the omission of the Ca²⁺ divalent cation. Fourthly, there is the potential for Ca²⁺ to enter the Ca²⁺-free bath solution from the cytoplasm, or via the activation of other cellular pumps/exchangers. To minimise the impact of this, after mounting in the bath, tissues were washed three times over 5 min in warmed Ca²⁺-free Krebs. As a final check, before and after contractions, the extracellular buffer was collected and checked with spectrophotometric assessments (Pacific Laboratory Products, Victoria, Australia), with no Ca²⁺ detected on this assay. This provided confidence in a nominally zero Ca²⁺ solution. As there were no observable or significant impacts to any contractions from adjusting for the methods listed above, the decision was made to henceforth solely remove Ca²⁺ from the Krebs solution and not alter anything else, as this was the path of least manipulation.

In the absence of stimulation from any agonist, U&LP tissue strips developed spontaneous phasic contractions at a frequency of 3.65 ± 0.08 cpm with an amplitude of 0.73 ± 0.04 g (n = 104). When receptor agonists carbachol (1 µM), histamine (100 µM), 5-HT (100 µM), NKA (300 nM), PGE2 (10 µM), and ATII (100 nM) were added to the tissues, U&LP baseline tension increased significantly for all activated receptors (p < 0.001, Table 1). In addition, the frequency of spontaneous phasic contractions increased for carbachol (p < 0.001), histamine (p < 0.05), 5-HT (p < 0.01), and ATII (p < 0.05), but not NKA or PGE2. Amplitude was reduced by all the agonists, but the changes were statistically significant for only carbachol (p < 0.05), 5-HT (p < 0.001), and ATII (p < 0.01).

When nifedipine (1 µM) or vehicle control (totaling 0.035% ethanol) were added to tissues at the start of the 30 min equilibration period, there were no immediate changes to baseline tension, frequency, or amplitude for any of the agonists.

The frequencies and amplitudes of spontaneous phasic contractions produced by the U&LP tissues were investigated in the absence and presence of nifedipine for each of the receptor agonists (Table 2). No significant differences in the contractile frequency or amplitude were observed between the presence and absence of nifedipine (1 µM) for responses to carbachol, histamine, 5-HT, or NKA. Contractions for PGE2 (10µM, n = 8, p < 0.01) and ATII (100nM, n = 8, p < 0.05) exhibited a reduction in the amplitude of spontaneous phasic contractions in the presence of nifedipine, and when compared to the response in the absence of nifedipine, was a statistically significant difference.

In this study, two different methods were applied to assess the impact of extracellular Ca²⁺: the immersion of the tissue in nominally zero Ca²⁺ solution; or through Ca²⁺ channel antagonism with nifedipine (1 µM). Both methods significantly inhibited contractile activity changes in response to the assessed agonists. Overall, when looking at the impact of either nifedipine or nominally zero Ca²⁺ solution, there were no significant differences (Student’s two-tailed unpaired t-tests for each) between the effectiveness of inhibitions of tension, frequency, or amplitude after the addition of carbachol (1 µM), histamine (100 µM), 5-HT (100 µM), NKA (300 nM), PGE2 (10 µM), and ATII (100nM, p = NSD for all).

Antimuscarinics and parasympathomimetics have shown success for the management of bladder contractile disorders, and currently sit as first-line pharmaceutical treatments for overactive bladder and underactive bladder, respectively. However, most patients cease treatment regimens due to lower than expected benefits and adverse side effects. Recent success has been found with combination therapies and there is increasing interest in the identification of alternative receptors systems that may be involved in contraction, and hence future targets for therapeutic treatments. This study’s identification of similarities between receptors mediating and modulating contraction in the urinary bladder may present future therapeutic targets or provide insights into mechanisms that may be dysfunctional in overactive or underactive bladder. Of particular interest was this study’s finding that histamine, neurokinin-A and angiotensin II are highly dependent on extracellular Ca²⁺, warranting further investigation into their clinical use for the management of bladder dysfunction.

A limitation of this study was the use of single-dose applications of receptor agonists to examine changes in frequencies and amplitudes of phasic contractions over 30-min time periods. It should be noted that in detrusor studies of carbachol-induced bladder contractions, human tissue responded to inhibited extracellular Ca²⁺ to a greater extent than pig tissue (Wuest et al., 2007), however, this has not been demonstrated in urothelial and lamina propria studies. Future studies could investigate the potential role of ageing in influencing receptor responses to extracellular contractions, such as histamine (Stromberga et al., 2020a), as well as explore the influence of other GPCRs and the influence of Ca²⁺ on their responses.