Substance P (SP) is may play an important role in the physiological and pathophysiological functions of the urinary bladder, including bladder sensory disorders like interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder (OAB) [1]. However, the impact of SP on sensory function remains unclear. Moreover, the specific interaction between neurokinin-1 receptors (NK1R), a SP receptor, and transient receptor potential vanilloid 1 (TRPV1), a capsaicin (CAP) receptor, has also been reported, but it is not yet fully understood [2]. The aim of this study was to determine the effects of SP on bladder function in relation to CAP, specifically on bladder afferent nerve activity.

We used female Sprague-Dawley rats. The in vitro organ bath study assessed SP-induced detrusor contraction in the intact and inside-out preparations of isolated whole bladders [3]. To verify the desensitization of CAP, CAP was added prior to the SP administrations. The in vivo cystometry and multi-unit bladder afferent nerve activity were measured before and after intravesical SP-instillation. Finally, immunohistochemistry was performed to locate TRPV1 and NK1R in the urinary bladder.

The organ bath study revealed that the intravesical pressure significantly increased in the intact preparation (N=5), but not in the inside-out preparation (N=5), by administering SP after CAP-desensitization (Figure 1). Cystometry showed that during intravesical SP-instillation, cystometric parameters remained unchanged. In contrast, in the multi-unit bladder afferent nerve activity, the SP group’s firing rates gradually increased over time, whereas the control group showed no change (Figure 2AB). Spike2 was used to divide multi-unit recordings into individual single-units. As a result, roughly 15 single-unit per rat were extracted, with 83 single-units in the control group and 89 single-units in the SP group examined. 
Extracted single-units activities were classified into five groups based on the changes of firing ratio relative to baseline (score as 1): highly-decreased (ratio < 0.4), decreased (0.4 ≤ ratio < 0.8), unchanged (0.8 ≤ ratio ≤ 1.2), increased (1.2 < ratio ≤ 1.6), and highly-increased (ratio > 1.6). The SP group had a higher proportion of “highly-increased” and a lower proportion of “highly-decreased” than the control group; this proportional distribution between groups was significantly different  (Figure 2C). 
Immunohistochemistry revealed individual expression of TRPV1 and NK1R was observed throughout the urinary bladder, including the urothelium, lamina propria, and detrusor smooth muscle. Furthermore, the co-localization of TRPV1 and NK1R was primarily observed in the lamina propria.

The organ bath study revealed that SP does not penetrate the urothelium or act as an individual mechanism on the urothelium to change the contractile tone of the detrusor smooth muscle. During intravesical SP-instillation, bladder afferent nerve activity significantly increased, whereas the inter-contraction interval (ICI: time between micturition), which is indirectly reflected in sensory function, remained unchanged. This finding suggests that SP administered through the urothelium can enhance bladder sensory function while not altering the micturition interval threshold. Immunohistochemistry revealed TRPV1 and NK1R were individually expressed, but some expressions were colocalized. In particular, NK1R was expressed on the urothelium and lamina propria layers rather than the smooth muscle layer, which could explain the increased afferent activity during SP-instillation.

The present study revealed that SP caused detrusor contraction via the smooth muscle. In addition, this is the first direct report using in vivo measurement of bladder afferent nerve activity that SP via urothelium can enhance bladder sensory function without changing the threshold in the micturition interval. SP may cause such bladder function on its own rather than as a result of CAP exposure. These findings highlight SP’s critical role in bladder sensory signaling, which may contribute to the development of pain and urgency in conditions like IC/BPS and OAB.

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