Aims: Previous studies have shown increased density of M₂receptors in hypertrophied rat bladders that possess an M₂contractile phenotype. The aim of the current study is to determine whether human bladders with an M₂contractile phenotype also have a greater density of bladder M₂receptors.Materials and Methods: Human bladders were obtained from 24 different organ transplant donors. Darifenacin and methoctramine affinity was determined by the rightward shift of cumulative carbachol concentration contractile response curves for each bladder. Radioligand binding and immunoprecipitation was used to quantify M₂and M₃subtypes in isolated detrusor muscle and urothelium. In addition, pig bladder muscle and urothelial receptors were quantified for comparison.Results: In the human urothelium total, M₂and M₃muscarinic receptor density is significantly negatively correlated with the affinity of darifenacin for inhibition of contraction of the detrusor muscle. In the detrusor muscle there is no correlation between receptor density and darifenacin affinity for inhibition of contraction. Muscarinic receptor density is greater in the muscle than in the urothelium in human bladders whereas in the pig bladder the density is greater in the urothelium than in the muscle.Conclusions: The greater density of urothelial muscarinic receptors in human bladders with lower darifenacin affinity, indicative of a greater contribution of M₂receptors to the contractile response, points towards a possible role of the urothelium in controlling M₂mediated contractile phenotype. In comparison between human and pig bladders, the distribution of muscarinic receptor subtypes in the muscle and urothelium are quite different. Neurourol. Urodynam.

Localized provoked vulvodynia is characterized by chronic vulvar pain that disrupts every aspect of the patient's life. Pain is localized to the vulvar vestibule, a specialized ring of tissue immediately surrounding the vaginal opening involved in immune defense. In this article, we show inflammation is the critical first step necessary for the generation of pain signals in the vulva. Inflammatory stimuli alone or combined with the transient receptor potential cation channel subfamily V member 4 (TRPV4) agonist 4α-phorbol 12,13-didecanoate stimulate calcium flux into vulvar fibroblast cells. Activity is blocked by the TRPV4 antagonist HC067047, denoting specificity to TRPV4. Using lipidomics, we found pro-resolving lipids in the vulvar vestibule were dysregulated, characterized by a reduction in pro-resolving mediators and heightened production of inflammatory mediators. We demonstrate specialized pro-resolving mediators represent a potential new therapy for vulvar pain, acting on 2 key parts of the disease mechanism by limiting inflammation and acutely inhibiting TRPV4 signaling.

Background/Objectives: Localized provoked vulvodynia (LPV) is characterized by chronic vulvar pain upon light touch to the vestibule, a specialized ring of tissue immediately surrounding the vaginal opening. LPV affects about 14 million people in the US, yet the etiopathology of the disease is unknown. In LPV, the vestibule expresses elevated levels of the pro-nociceptive pro-inflammatory mediators prostaglandin E₂ (PGE2) and interleukin-6 (IL-6), which corresponds to lower pain thresholds. Previous studies have shown reduced amounts of arachidonic acid (AA)-derived pro-resolving lipid mediators in tissue biopsies from LPV patients that might impede the resolution of inflammation. AA is obtained from dietary linoleic acid, pointing to a defect in the metabolism of dietary polyunsaturated fatty acids in LPV. We aimed to further explore the involvement of AA metabolism in LPV, which appears dysregulated in the vestibule of LPV patients and culminates in chronic inflammation and chronic pain. Methods: Vestibular and vulvar tissue biopsies obtained from LPV and non-LPV patients were used to generate fibroblast strains and assessed for COX/LOX expression using qRT-PCR. Fibroblast strains were treated with inflammatory stimuli, and then COX-1 and COX-2 expression was assessed using Western blot analysis. Pro-inflammatory mediator production was assessed using enzyme-linked immunosorbent assays (ELISAs). ALOX5 and ALOX12 expression was assessed using qRT-PCR. Finally, lipidomic analysis was carried out to screen for 143 lipid metabolites following inflammatory challenge. Results: Tissue and fibroblasts from LPV patients exhibited altered expression of COX/LOX enzymes and production of AA-derived lipid mediators compared to non-LPV patients. Conclusions: Lipid profiles of tissue and vestibular fibroblasts from LPV patients differed from non-LPV patients, and this difference was attributed to differential COX/LOX expression and activity, which metabolizes AA derived from dietary linoleic acid. This dysregulation fosters chronic inflammation and reduced resolution capacity in LPV patients, causing chronic pain. While further work is needed, these findings suggest that dietary modifications could impact the LPV mechanism.

Localized provoked vulvodynia (LPV) is characterized by chronic vulvar pain upon light touch to the vulvar vestibule, a specialized ring of tissue immediately surrounding the vaginal opening. LPV affects ∼14 million people in the US. Current treatments for LPV include nonsteroidal anti-inflammatory drugs (NSAIDs), but clinical evidence has demonstrated that they are ineffective for vulvar pain. Here, the effects of NSAID treatment on inflammation and resolution in an in vitro model of LPV were explored using enzyme-linked immunosorbent assays (ELISAs), calcium imaging, and metabololipidomics. Additionally, the effectiveness of NSAID treatment on mitigating chronic vulvar pain was assessed using a validated LPV mouse model that mimics key features of vulvodynia. These findings indicate that although NSAID treatment reduced pro-inflammatory eicosanoid production in vulvar fibroblasts, lipidomic analysis revealed that COX inhibition also downregulated levels of pro-resolving lipids, namely epoxyeicosatrienoic acids (EETs), lipoxins (LXs), and resolvin E-series (RvEs). In vivo, NSAID treatment did not restore vulvar pain thresholds back to baseline levels in mice. Overall, this study offers one possible explanation for previous reports of the ineffectiveness of NSAIDs on managing LPV-associated vulvar pain. PERSPECTIVE: Taking NSAIDs to manage chronic vulvar pain may downregulate levels of specialized pro-resolving lipid mediators, thereby resulting in prolonged pain and delayed inflammation resolution. This study provides one possible explanation for clinical reports of the ineffectiveness of NSAIDs in the mitigation of vulvodynia-associated pain.

Localized provoked vulvodynia (LPV) causes chronic pain upon light touch to the vulvar vestibule of anatomical females, the ring of tissue surrounding the vaginal opening, afflicting ~14 million US women. Inflammation is accepted as a driver of LPV with NF-кB, the “master regulator” of inflammation, activating downstream calcium channels involved in the pain signaling mechanism. LPV-affected vestibular tissue and fibroblasts produce elevated levels of pro-nociceptive pro-inflammatory mediators prostaglandin E2 (PGE2) and interleukin-6 (IL-6), which correlate with pain thresholds. Thus, we explored the effect of NF-кB inhibition on calcium signaling through transient receptor potential vanilloid (TRPV) 4 and 1 channels. We observed decreased calcium influx (measured by Fmax/F0) when NF-кB was inhibited 24 or 1 h prior to imaging in absence of inflammatory mediators. However, we did not observe a decrease in calcium flux when NF-кB was inhibited in presence of inflammatory mediators. Paradoxically, acute inhibition of NF-кB resulted in an elevation in calcium influx in vulvar fibroblasts from both LPV and non-LPV patients. We then determined that NF-кB inhibitors, regardless of their mechanism of inhibition, have this effect, suggesting that their use during active inflammation may have unintended side effects. Caution is necessary when targeting NF-кB due to its role in immune defense and the potential for unregulated calcium entry that can result in cell death. Elevations in calcium flux in response to NF-кB inhibition do not appear to occur through pain channels, rather general calcium channels, such that the death of these cells would not confer any analgesic benefits.