The five primary prostaglandins stimulate contractions and phasic activity of the urinary bladder urothelium, lamina propria and detrusor

Background Inflammation is often associated with several bladder dysfunctions, including overactive bladder (OAB) and interstitial cystitis/bladder pain syndrome (IC/PBS). As such, inflammation of the bladder and the actions of inflammatory mediators may contribute to the development of urinary symptoms. This study assessed the actions of PGE2, PGF2, PGD2, TXA2, and PGI2 on urinary bladder urothelium with lamina propria (U&LP), and detrusor smooth muscle. Methods Studies were carried out using isolated tissue baths, where strips of porcine bladder U&LP or detrusor were exposed to varying concentrations of prostaglandin agonists (1 μM and 10 μM). Results All assessed prostaglandin agonists contracted both the U&LP and detrusor smooth muscle, with the rank order of contractile response effectiveness as: PGE2 > PGF2α > TXA2 > PGD2 > PGI2. In U&LP, treatment with PGE2 (10 μM) increased tonic contractions by 1.36 ± 0.09 g (n = 42, p < 0.001) and phasic contractions by 40.4 ± 9.6% (n = 42, p < 0.001). In response to PGF2α (10 μM), U&LP tonic contractions increased by 0.79 ± 0.06 g (n = 14, p < 0.001) and phasic activity by 13.3% ± 5.3% (n = 15, p < 0.05). In detrusor preparations, PGE2 (10 μM) increased tonic contractions by 1.32 ± 0.13 g (n = 38, p < 0.001) and PGF2α (10 μM) by 0.97 ± 0.14 g (n = 12, p < 0.001). Only 34% (n = 48) of all detrusor preparations exhibited spontaneous activity prior to the addition of any agonist at a frequency of 2.03 ± 0.12 cpm. In preparations that did not exhibit initial phasic activity, all of the prostaglandin agonists were capable of commencing phasic activity. Conclusions The urinary bladder U&LP and detrusor respond to a variety of prostaglandin agonists, with their activation resulting in direct contractions, as well as increases to spontaneous contractile activity. This study presents the prostaglandin receptor system as a potential therapeutic target for lower urinary tract dysfunction.


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: PGE 2 , PGD 2 , PGF 2α , prostacyclin (PGI 2 ) and thromboxane (TXA 2 ) [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 PGE 2 and TXA 2 , whereas mast cells produce PGD 2 [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 I 2 is the main prostaglandin synthesised in the human bladder, followed by PGE 2, PGF 2α and TXA 2 [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 PGE 2 , 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 PGE 2 , PGF 2α , PGD 2 , TXA 2 and PGI 2 on the urinary bladder urothelium with lamina propria and detrusor smooth muscle contractions and phasic activity.

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, NaHCO 3 24.9 mM, CaCl 2 1.9 mM, MgSO 4 2.41 mM, KCl 4.6 mM, KH 2 PO 4 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 E 2 , prostaglandin F 2α , prostaglandin D 2 , prostaglandin I 2 and thromboxane A 2 (U-46619, Cayman Chemicals, Michigan, USA). Prostaglandin E 2 , prostaglandin F 2α , prostaglandin D 2, and prostaglandin I 2 were dissolved in 100% ethanol and diluted with distilled H 2 O. U-46619 was supplied as a solution in methyl acetate, which was diluted with distilled H 2 O. 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.

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.

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: PGE 2 > PGF 2α > TXA 2 > PGD 2 > PGI 2 . This furthers previous research which reported the involvement of PGE 2 in the initiation of micturition in both humans and animals [24], suggesting a contribution . 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 to bladder overactivity. Treatment with PGF 2α 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 TXA 2 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 TXA 2 (10 μM) resulted in significantly enhanced contractions. The responses observed in porcine tissue in response to PGF 2α , and TXA 2 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, PGD 2 and PGI 2 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 PGD 2 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 PGI 2 , the main prostaglandin synthesised in the human bladder [14,27], is that the aqueous solutions of PGI 2 are extremely chemically unstable with a relatively short half-life, depending on the buffer concentration [28]. As such, future studies utilising more chemically stable PGI 2 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, PGE 2 and PGF 2α had the Fig. 4 Detrusor changes in tonic contractions in response to the treatment with 1 μM and 10 μM of prostaglandin E 2 (top row), F 2α (middle row) and TXA 2 (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 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. . 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