Local or systemic inflammation can severely impair urinary bladder functions and contribute to the development of voiding disorders in millions of people worldwide. Isoprostanes are inflammatory lipid mediators that are upregulated in the blood and urine by oxidative stress and may potentially induce detrusor overactivity. The aim of the study was to investigate the effects and signal transduction of isoprostanes in human and murine urinary bladders to provide potential pharmacological targets in detrusor overactivity. Contraction force was measured with myograph in murine and human urinary bladder smooth muscle (UBSM) ex vivo. Isoprostane 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in murine UBSM, which was abolished in mice deficient for the thromboxane prostanoid receptor (TP-KO). The responses remained unaltered after removal of the mucosa or incubation with tetrodotoxin. Smooth muscle specific deletion of Gα12/13-protein or inhibition of Rho-kinase (ROCK) by Y-27632 decreased the contractions. In Gαq/11‐KO mice, responses were reduced and in the presence of Y‐27632 abolished completely. In human UBSM the TP agonist U-46619 evoked dose‐dependent contractions. Neither atropine, nor the purinergic receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS) decreased the effect, indicating that TP receptors directly mediate detrusor muscle contraction. 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in human UBSM, and these responses were abolished by the TP antagonist SQ-29548 and were decreased by Y-27632. Our results indicate that isoprostanes evoke contraction in murine and human UBs, an effect mediated by the TP receptor. The G12/13-Rho-ROCK pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target.

Bradykinin (BK) has been proposed to modulate urinary bladder functions and implicated in the pathophysiology of detrusor overactivity. The present study aims to elucidate the signaling pathways of BK-induced detrusor muscle contraction, with the goal of better understanding the molecular regulation of micturition and identifying potential novel therapeutic targets of its disorders. Experiments have been carried out on bladders isolated from wild-type or genetically modified [smooth muscle-specific knockout (KO): Gαq/11-KO, Gα12/13-KO and constitutive KO: thromboxane prostanoid (TP) receptor-KO, cyclooxygenase-1 (COX-1)-KO] mice and on human bladder samples. Contractions of detrusor strips were measured by myography. Bradykinin induced concentration-dependent contractions in both murine and human bladders, which were independent of secondary release of acetylcholine, ATP, or prostanoid mediators. B2 receptor antagonist HOE-140 markedly diminished contractile responses in both species, whereas B1 receptor antagonist R-715 did not alter BK's effect. Consistently with these findings, pharmacological stimulation of B2 but not B1 receptors resembled the effect of BK. Interestingly, both Gαq/11- and Gα12/13-KO murine bladders showed reduced response to BK, indicating that simultaneous activation of both pathways is required for the contraction. Furthermore, the Rho-kinase (ROCK) inhibitor Y-27632 markedly decreased contractions in both murine and human bladders. Our results indicate that BK evokes contractions in murine and human bladders, acting primarily on B2 receptors. Gαq/11-coupled and Gα12/13-RhoA-ROCK signaling appear to mediate these contractions simultaneously. Inhibition of ROCK enzyme reduces the contractions in both species, identifying this enzyme, together with B2 receptor, as potential targets for treating voiding disorders.

The overactive bladder (OAB) is a clinical condition characterized by symptoms of frequency, urgency with/without incontinence, with a prevalence of 16% in the developed countries. The current pharmacological treatment mainly consists of anticholinergic drugs with several side effects. According to prior experimental results the arachidonic acid (AA) derivate prostanoids and isoprotanes, the latter produced non‐enzymatically during oxidative stress, are not only biomarkers of systemic oxidative stress, but are excreting in the urine and might act directly on the detrusor muscle leading to detrusor overactivity. Our aim was to examine the effects and the signal transduction pathways of prostanoids and isoprotanes in the urinary bladder smooth muscle, and potentially provide theoretical basis for the development of more specific medication of OAB with less adverse effects. Detrusor muscle strips were prepared from wild type (C57BL/6) and knockout mice, deficient for the thromboxane receptor (TP) or the α‐subunits of heterotrimeric G proteins (Gαq/11‐KO, Gα12/13‐KO) without urothelium under dissection microscope. Contraction force was measured by myograph under isometric conditions and normalized to the reference contractions evoked by 124 mM K+. The prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), as well as the isoprostane 8‐epi‐PGE2 and 8‐iso‐PGF2α evoked contraction in the urinary bladder strips. The effect of the prostanoids was decreased, and the effect of the isoprostanes was abolished in the strips of TP KO mice, suggesting that the effect of the prostanoids is mediated partially, whereas that of the isoprostanes mainly by the TP. The TP receptor selective agonist U‐46619 evoked dose‐dependent contraction in the bladder strips, and neither the cholinerg antagonist atropine, nor the purinerg P2X‐antagonist pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS) decreased the responses, indicating that the TP agonist has a direct effect on the detrusor muscle. The responses evoked by U‐46619 were comparable to the effect evoked by cholinergic agonist, carbachol. The contraction responses were decreased in the strips of the Gα12/13‐KO mice. Correspondingly, the responses evoked by the prostanoids and isoprostanes were reduced by the Rho‐kinase (ROCK) inhibitor Y‐27632. In the strips of the Gαq/11‐KO mice, the responses were also decreased and in the presence of Y‐27632 abolished completely. In conclusion, the examined prostanoids and isoprostanes evokes contraction acting directly on the detrusor muscle. These responses are mediated mainly by the TP receptor and are linked to the Gαq/11 and to the Gα12/13‐Rho‐ROCK intracellular signaling pathways in the murine urinary bladder. The Gα12/13‐Rho‐ROCK signaling pathway may provide a novel, more specific pharmacological target with less adverse effects in the treatment of OAB. Support or Funding Information K‐112964, K‐125174 and NVKP‐16‐1‐2016‐0042 from the Hungarian National Research, Development and Innovation Office and EFOP‐3.6.3‐VEKOP‐16‐2017‐00009.

Urinary bladder smooth muscle (UBSM) dysfunctions (overactive bladder, detrusor overactivity) affect millions of people worldwide, however their etiology is still poorly understood. Nevertheless, inflammatory mediators including isoprostanes (isoP) and bradykinin (BK) have been proposed as potential pathogenic factors in detrusor overactivity. Our preliminary data gained from mouse bladders proved that these mediators are potent constrictors of murine UBSM and the RhoA – Rho-kinase (ROCK) pathway plays a prominent role in mediating their effects.The present study aimed to investigate the signal transducion of isoP- and BK-induced contractions focusing on the role of RhoA – ROCK signaling in human UBSM. Our goal is to better understand the molecular mechanism of micturition and identifying potential novel therapeutic targets of its disorders.Experiments were performed on human bladders which were surgically removed due to bladder malignancy. UBSM strips were dissected from a macroscopically tumor-free area, approved by a pathologist, then they were mounted on a myograph system. The force and time course of their contractile responses were registered under isometric conditions BK and isoPs (8-iso-PGF2α and 8-iso-PGE2) induced concentration-dependent contractions in human UBSM. BK’s effect was significantly inhibited by the B2 receptor antagonist HOE-140, whereas it remained unaltered following incubation with B1 receptor antagonist R-715. Consistently with these findings, pharmacological stimulation of B2 but not B1 receptors resembled the effect of BK.Similarly, isoPs also induced concentration-dependent potent contractions in human bladder strips, which were inhibited by the thromboxane prostanoid (TP) receptor antagonist SQ29548. For further investigating the downstream signaling of B2 and TP receptors, we focused on the RhoA – ROCK pathway. Thus, we applied the specific ROCK-inhibitor, Y27632, which substantially reduced human UBSM contractions induced by BK or isoPs.Our results clearly demonstrate that both isoPs and BK induce potent contractions in human detrusor muscle. BK’s effect is mediated mainly via B2 receptors, while isoP-induced contractions are dependent on TP receptor activation in human bladders. Inhibition of ROCK enzyme reduced the contractions induced by these inflammatory mediators, identifying this enzyme, together with B2 or TP receptors as potential targets for treating bladder smooth muscle dysfunctions assocated with local or systemic inflammation. Hungarian NRDIO K-125174, K-135683, K-139230 and PD-132851, Ministry for Innovation and Technology from the Hungarian NRDI fund (2020-1.1.6-JÖVŐ-2021-00010, TKP2021-EGA-25), STIA-KFI-2021 and EFOP-3.6.3-VEKOP-16-2017-00009 grants. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Overactive bladder (OAB) is a clinical condition characterized by symptoms of frequency, urgency with/without incontinence. Despite of the fact that OAB affects millions of patients’ quality of life significantly, its etiology is still poorly understood. Currently, antimuscarinics are the first-line medical therapy for the management of OAB. However, their application is limited due to the decrease of patients’ compliance because of several adverse effects (e.g. dry mouth, obstipation).Our aim was to further analyze the signaling pathways of muscarinic receptors in detrusor muscle contraction with the goal of better understanding the intracellular regulation of micturition and identifying potential novel therapeutic targets for OAB.Experiments were performed on adult, male, wild-type (WT), M2, M3, M2/M3, Gαq/11 knockout (KO) and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM).Contractile responses induced by the muscarinic receptor agonist carbamoylcholine (CCh) in UBSM associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632. The CCh-induced contractions were markedly reduced in detrusor strips lacking either M2 or M3 receptors and were abolished in UBSM from M2/M3 KO mice. The RhoA activation was decreased in both M2 KO and M3 KO bladders and it was completely reduced in M2/M3 KO UBSM. Inhibition of Gαi-coupled signaling by PTX-treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gαq/11 KO mice, however, RhoA activation was unaffected.In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M2 and M3 receptors. Furthermore, CCh-induced contractions involve simultaneously the classical Gαq/11- and the Gαi-coupled signaling pathways, but the RhoA activation appears to be mediated exclusively by the Gαi proteins. These findings may aid the identification of more specific therapeutic targets for OAB. Hungarian NRDIO K-125174, K-135683, K-139230 and PD-132851, Ministry for Innovation and Technology from the Hungarian NRDI fund (2020-1.1.6-JÖVÖ-2021-00010, TKP2021-EGA-25), STIA-KFI-2021, EFOP-3.6.3-VEKOP-16-2017-00009 grants and Gedeon Richter Plc Talent Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.