Background

The involvement of prostaglandins in bladder physiology was first recognised from their release after urinary bladder distension or injury to the urothelium [1, 2]. An increase of prostaglandins in the urine of patients suffering from OAB has been well-reported previously [3,4,5,6], suggesting the prostaglandin system as a potential future therapeutic target in various bladder dysfunctions. The exact role and mechanisms of endogenous prostaglandins in the urinary bladder are not well understood. However, previous studies utilising exogenous prostaglandins have shown that these chemicals can alter contractility and micturition reflex in human bladders [7].

Prostaglandin production is generally low in healthy tissue but can increase immediately following acute inflammation [8]. They are synthesised in the bladder by cyclooxygenase (COX) and then subsequently converted into five primary prostanoids via their respective synthases: PGE2, PGD2, PGF2α, prostacyclin (PGI2) and thromboxane (TXA2) [9]. Prostaglandins are synthesised in both the bladder urothelium with lamina propria (U&LP) and in detrusor smooth muscle in response to stretch, nerve stimulation, U&LP damage or other inflammatory mediators [10, 11]. The production of prostaglandins is determined by the cells present at sites of inflammation capable of synthesising prostaglandins and the activity of the two cyclooxygenase isoenzymes, namely COX-1 and COX-2. For example, macrophages predominantly generate PGE2 and TXA2, whereas mast cells produce PGD2 [12]. COX-1 is present in most cells, whereas the expression of COX-2 is generally low in cells, but can increase dramatically upon stimulation by immune cells [13]. Prostaglandin I2 is the main prostaglandin synthesised in the human bladder, followed by PGE2, PGF2α and TXA2 [14, 15].

Although past studies have explored the effects of the different prostaglandins on the urinary bladder with a large focus on the actions of PGE2, a complete understanding of the contractile effects of the other four prostaglandins on the urinary bladder remain unclear. Specifically, of interest is to determine how the actions of the prostaglandins affect urothelium with lamina propria that is separated from the detrusor smooth muscle. Therefore, this study aimed to assess the influence of PGE2, PGF2α, PGD2, TXA2 and PGI2 on the urinary bladder urothelium with lamina propria and detrusor smooth muscle contractions and phasic activity.

Methods

Tissue preparation

Urinary bladders were obtained from Large White-Landrace pigs (approximately six months old, weighing between 80 and 100 kg) from the local abattoir after slaughter for the routine commercial provision of food. All methods were carried out in accordance with relevant Australian guidelines and regulations, and all experimental protocols were in accordance the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose [16]. As no animals were bred, harmed, culled, interfered, or interacted with as part of this research project, Animal Ethics Approval was not required for offal use [17]. Urothelium with lamina propria was dissected from the underlying detrusor layer, consistent with methods carried out in past studies [18,19,20,21], and cut in strips. Adjacent strips of U&LP and detrusor (10 mm × 5 mm) were tied vertically between an isometric force transducer (MCT050/D, ADInstruments, Castle Hill, Australia) and a fixed hook in 10 mL organ baths (Labglass, Brisbane, Australia), and superfused with Krebs-bicarbonate solution (NaCl 118.4 mM, NaHCO3 24.9 mM, CaCl2 1.9 mM, MgSO4 2.41 mM, KCl 4.6 mM, KH2PO4 1.18 mM and D-glucose 11.7 mM) and carbogen gas (95% oxygen and 5% carbon dioxide) at 37 °C. After tissue mounting, strips of U&LP and detrusor were washed three times, tension adjusted to 1.5–2.0 g and tissues left to equilibrate for 30 min. After the equilibration period, a single dose of a prostaglandin receptor agonist was added to the tissue strip.

Pharmaceutical agents

The following compounds were used in this study: prostaglandin E2, prostaglandin F2α, prostaglandin D2, prostaglandin I2 and thromboxane A2 (U-46619, Cayman Chemicals, Michigan, USA). Prostaglandin E2, prostaglandin F2α, prostaglandin D2, and prostaglandin I2 were dissolved in 100% ethanol and diluted with distilled H2O. U-46619 was supplied as a solution in methyl acetate, which was diluted with distilled H2O. Two concentrations of each prostaglandin receptor agonists were selected, 1 μM and 10 μM.

Data analysis

Data were graphed and analysed using GraphPad Prism version 8.3 for Windows (GraphPad Software, La Jolla, California, USA). Statistical analysis was conducted using a paired Student’s t-test, where p < 0.05 was considered as significant. All values were reported as mean change ± SEM. n equates to the number of individual bladders used in this study.

Results

Prostaglandin agonists for increasing U&LP spontaneous phasic activity

Strips of U&LP exhibited spontaneous phasic contractions in the absence of any stimulation at a mean frequency of 3.26 ± 0.07 cycles per minute (cpm, n = 146). Treatment with PGE2 caused the most prominent increases to U&LP spontaneous contractile activity. When PGE2 (1 μM) was added to isolated tissues, spontaneous activity increased by 39.2% ± 6.7% (n = 38, p < 0.001, Fig. 1). A greater concentration of PGE2 (10 μM) showed similar increases of 40.4% ± 9.6% to the U&LP spontaneous activity (n = 42, p < 0.001). Treatment with PGF2α showed smaller increases of 10.5% ± 4.6% to spontaneous activity when treated with 1 μM (n = 10, p < 0.05) and 13.3% ± 5.3% when treated with 10 μM (n = 14, p < 0.05). The addition of PGI2 (10 μM) increased spontaneous activity by 6.2% ± 1.6% (n = 8, p < 0.01) but had no effect at a lower concentration (1 μM, n = 8). The frequency was not significantly affected by PGD2 (1–10 μM, n = 12) or TXA2 (1–10 μM, n = 16).

Fig. 1
figure 1

U&LP changes in the frequency of spontaneous phasic contractions after the treatment with 1 μM and 10 μM of each specific prostaglandin agonists E2, F2α, TXA2, D2, and I2. There were no statistically significant differences in frequency changes between the 1 μM and 10 μM concentrations for any of the agonists (unpaired Student’s 2-tailed t-test)

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The average amplitude of these spontaneous phasic contractions exhibited in U&LP strips in the absence of any stimulation was 0.57 ± 0.02 g (n = 146). In response to treatment with 1 μM PGE2, amplitude decrease of 0.14 ± 0.04 g (n = 38, p < 0.001, Table 1) were observed. Similar decreases of 0.16 ± 0.03 g were also observed in response to a higher PGE2 concentration (10 μM, n = 42, p < 0.01). Treatment with TXA2 (1 μM) showed a significant decrease in the amplitude by 0.28 ± 0.06 g (n = 8, p < 0.01), which was not observed at a higher concentration (10 μM, n = 6). The addition of PGI2 (10 μM) decreased amplitude of spontaneous activity by 0.14 ± 0.05 (n = 8, p < 0.05) but had no effect at a lower concentration (1 μM, n = 8). The amplitude of spontaneous contractions was not altered by the addition of either PGF2α (1–10 μM, n = 24) or PGD2 (1–10 μM, n = 12, Table 1). None of the decreases in the amplitude of spontaneous phasic contractions of the U&LP were significantly affected by the two different prostaglandin receptor agonist concentrations (1 μM and 10 μM).

Table 1 U&LP changes in the amplitude of phasic contractions in response to the five primary prostaglandin agonists (mean ± SEM)
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Prostaglandin agonists in stimulating phasic contractions in detrusor

Total of 34% (n = 48) of the detrusor preparations that were set up in the organ baths exhibited spontaneous activity prior to the addition of any agonists. These contractions occurred at an average frequency of 2.03 ± 0.12 cpm (n = 48) with an average amplitude of 0.26 ± 0.02 g (n = 48). However, the majority of the detrusor preparations, that were otherwise quiescent developed spontaneous phasic contractions after the addition of the agonist.

Of those detrusor preparations that did not exhibit initial phasic activity during baseline: PGE2 (1 μM) sparked contractions in 68% of preparations (n = 19) and PGE2 (10 μM) in 69% (n = 22); PGF2α (1 μM) initiated contractions in in 56% (n = 5) and PGF2α (10 μM) in 88% (n = 7); TXA2 (1 μM) initiated contractions in 63% (n = 5) and TXA2 (10 μM) in 80% (n = 4); PGD2 (1 μM) initiated phasic activity in 50% (n = 2) and PGD2 (10 μM) in 75% (n = 6); and lastly PGI2 (10 μM) initiated contractions in 40% (n = 2) of preparations. This demonstrates the ability of prostaglandin agonists to induce spontaneous activity in otherwise quiescent detrusor tissue strips.

Prostaglandin agonists in stimulating tonic contractions in U&LP

All assessed prostaglandin agonists contracted the U&LP with the rank order of contractile response effectiveness as: PGE2 > PGF2α > TXA2 > PGD2 > PGI2. The addition of PGE2 (1 μM) to isolated U&LP induced tissue contractions, with increases of 1.01 ± 0.08 g (n = 38, p < 0.001) to the tonic contractions. When a greater concentration of PGE2 (10 μM) was selected, increases of 1.36 ± 0.09 g (n = 42, p < 0.001, Fig. 2) were observed. Treatment with 1 μM PGF2α showed a small increase to tonic contractions of 0.15 ± 0.04 g (n = 10, p < 0.01) when compared to a higher concentration of 10 μM, which exhibited increases of 0.79 ± 0.06 g (n = 14, p < 0.001). The addition of two concentrations of TXA2 induced similar contractions, where tonic contraction increased by 0.70 ± 0.07 g when treated with 1 μM (n = 8, p < 0.001), and by 0.65 ± 0.12 g after treatment with 10 μM (n = 6, p < 0.001).

Fig. 2
figure 2

U&LP changes in tonic contractions in response to the treatment with 1 μM and 10 μM of prostaglandin E2 (top row), F2α (middle row) and TXA2 (bottom row). Sample traces of the responses observed to two concentrations of prostaglandin agonist (left & middle columns). Increases in tonic contractions after treatment with each agonist are represented as mean change ± SEM (right column). Significant changes in the tonic contractions between 1 μM and 10 μM were evaluated using an unpaired Student’s two-tailed t-test, where *p < 0.05, **p < 0.01, ***p < 0.001

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When PGD2 (1 μM) was added to the U&LP tissue preparations, tonic contractions increased by 0.19 ± 0.04 g (n = 4, p < 0.05, Fig. 3). Treatment with a higher concentration of PGD2 (10 μM) exhibited increases of 0.63 ± 0.09 g (n = 8, p < 0.001). The addition of PGI2 showed small increases in tonic contractions of 0.11 ± 0.02 g in response to 1 μM PGI2 (n = 8, p < 0.001), and 0.22 ± 0.03 g in response to 10 μM PGI2 (n = 8, p < 0.001, Fig. 3).

Fig. 3
figure 3

U&LP changes in tonic contractions in response to the treatment with 1 μM and 10 μM of prostaglandin agonists D2 (top row) and I2 (bottom row). Sample traces of the responses observed to two concentrations of prostaglandin agonist (left & middle columns). Increases in tonic contractions after treatment with each agonist are represented as mean change ± SEM (right column). Significant changes in the tonic contractions between 1 μM and 10 μM were evaluated using an unpaired Student’s two-tailed t-test, where *p < 0.05, **p < 0.01, ***p < 0.001

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Prostaglandin agonists in stimulating tonic contractions in detrusor

All assessed prostaglandin agonists contracted the detrusor smooth muscle preparations with the rank order of contractile response effectiveness as: PGE2 > PGF2α > TXA2 > PGD2 > PGI2. In detrusor preparations, PGE2 (1 μM) increased the tonic contractions by 0.73 ± 0.09 g (n = 34, p < 0.001), whereas PGE2 (10 μM) nearly doubled the response, producing an average increase of 1.32 ± 0.13 g (n = 38, p < 0.001, Fig. 4). Treatment with 1 μM PGF2α showed a small increase of 0.20 ± 0.05 g (n = 10, p < 0.01), whereas 10 μM of PGF2α increased the tonic contractions by 0.97 ± 0.14 g (n = 12, p < 0.001). When TXA2 was added, tonic contractions increased by 0.47 ± 0.12 g when treated with 1 μM (n = 8, p < 0.001), and by 1.03 ± 0.14 g (n = 6, p < 0.001, Fig. 4) when treated with 1 μM TXA2.

Fig. 4
figure 4

Detrusor changes in tonic contractions in response to the treatment with 1 μM and 10 μM of prostaglandin E2 (top row), F2α (middle row) and TXA2 (bottom row). Sample traces of the responses observed to two concentrations of prostaglandin agonist (left & middle columns). Increases in tonic contractions after treatment with each agonist are represented as mean change ± SEM (right column). Significant changes in the tonic contractions between 1 μM and 10 μM were evaluated using an unpaired Student’s two-tailed t-test, where *p < 0.05, **p < 0.01, ***p < 0.001

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PGD2 showed a small increase in the tonic contractions of 0.12 ± 0.04 g when 1 μM was added (n = 4, p < 0.05), and an increase of 0.36 ± 0.06 g when 10 μM PGD2 was added (n = 6, p < 0.01, Fig. 5). PGI2 showed small increases in tonic contractions at both concentrations, showing an increase of 0.16 ± 0.02 g when treated with 1 μM (n = 8, p < 0.001), and 0.13 ± 0.03 g when treated with 10 μM PGI2 (n = 8, p < 0.001, Fig. 5). The effects of prostaglandin agonists on tonic contractions of the detrusor smooth muscle were significantly different between the two concentrations (1 μM and 10 μM) for PGE2 (p < 0.001), PGF2α (p < 0.001) and PGD2 (p < 0.05).

Fig. 5
figure 5

Detrusor changes in tonic contractions in response to the treatment with 1 μM and 10 μM of prostaglandin agonists D2 (top row) and I2 (bottom row). Sample traces of responses observed to two concentrations of a prostaglandin agonist (left & middle columns). Increases in the tonic contractions after treatment with an agonist are represented as mean change ± SEM (right column). Significant changes in the tonic contractions between 1 μM and 10 μM were evaluated using an unpaired Student’s t-test, where *p < 0.05, ***p < 0.001

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Discussion

Previous research has shown that stimulation of the M3 muscarinic receptor in U&LP causes immediate contractions, as well as increases in the frequency of spontaneous phasic contractions, and reduction in their amplitude [22, 23]. In our study, the prostaglandin agonists have shown similar contractile responses to both tonic contractions and spontaneous activity, thereby associating the actions of prostaglandins with many of the bladder contractile dysfunctions, such as OAB and IC/BPS.

The ability to contract the tissue was varied between the different prostaglandin agonists. The rank order of agonist response in stimulating contractions in U&LP and detrusor was: PGE2 > PGF2α > TXA2 > PGD2 > PGI2. This furthers previous research which reported the involvement of PGE2 in the initiation of micturition in both humans and animals [24], suggesting a contribution to bladder overactivity. Treatment with PGF2α showed minimal increases at a concentration of 1 μM, yet responses were significantly enhanced in both U&LP and detrusor when increased to 10 μM. At the smaller concentration of 1 μM, treatment with TXA2 reached maximal contractile responses, and as such, was not enhanced at the higher agonist concentration of 10 μM. This was not the case with detrusor preparations, wherein the higher concentration of TXA2 (10 μM) resulted in significantly enhanced contractions. The responses observed in porcine tissue in response to PGF2α, and TXA2 are consistent with the Palea [25] findings. In addition, our study has established that U&LP isolated tissue is also capable of responding and producing definite increases in tonic contractions in response to these prostaglandin agonists.

Of the five prostaglandins, PGD2 and PGI2 had the smallest effect on both tonic contractions and spontaneous activity. This lack of increases to the tonic contractions or spontaneous contractile frequency may be explained by PGD2 having potential inhibitory actions via the stimulation of DP receptor [26]. An explanation for the small contractile effects observed in our study in response to PGI2, the main prostaglandin synthesised in the human bladder [14, 27], is that the aqueous solutions of PGI2 are extremely chemically unstable with a relatively short half-life, depending on the buffer concentration [28]. As such, future studies utilising more chemically stable PGI2 agonist analogous might provide further insights into the actions of this inflammatory mediator on the urinary bladder.

Conclusions

The urinary bladder is capable of responding to all five major prostaglandins produced in the urinary bladder. Out of these prostaglandins, PGE2 and PGF2α had the most significant impact on both contraction and increases to the spontaneous contractile frequency in the U&LP. All five prostaglandin receptor agonists were also capable of inducing spontaneous phasic contractions in otherwise quiescent detrusor tissue strips.