Fig 2: The effects of NTs and levocabastine on the spontaneous contraction of the detrusor muscle strips.Tension recordings revealed the effects of NTs on the contractile activities of the rat detrusor strips (A). Compared with negative control, NTs (1 µM) can increase the amplitude and decrease the frequency (B), aP < 0.001, bP < 0.01, independent-sample t-test, 10 muscle strips) of detrusor strip contraction, which recovered after washing. The dose-response effects of NTs (10–11 to 10–6 M) and levocabastine (10–11 to 10–6 M) were also measured (C). Gradual increases in amplitude were found in the NTs group (D), cP < 0.001 indicates that the concentration 10–6 M had more significant effects than other concentrations, one-way ANOVA, seven muscle strips) but not for levocabastine (P = 0.115, one-way ANOVA, six muscle strips).

Fig 3: The effects of NTs on intracellular calcium concentration in SMCs.Every six pictures were chosen to represent the process of instantaneous [Ca2+]i changes in the SMCs after treatment with pharmacological agents (A,B). The real-time relative fluorescence intensities (F1/F0) are shown as continuous plots (C,D). In a calcium solution, application with NTs increased [Ca2+]i (E, aP < 0.001), which was drastically abolished by pretreatment with SR142948A (P < 0.001) and partly inhibited by FLA (bP < 0.001). In a calcium-free solution, NT administration also increased [Ca2+]i (eP < 0.001), and the increasing effects of NTs on calcium concentration with calcium solution was higher than that in the calcium-free solution (cP < 0.001). Pretreatment with SR142948A and U73122 can also completely abolish or partially inhibit the effects (dP < 0.001). In the whole cell patch clamp experiment, NTs triggered an inward current, from 40 pA to 180 pA, but the vehicle had no influence on the cell current.

Fig 4: NTs and NTSR mRNA and protein expression in the rat bladder.The mRNAs for NTs and the two main subtypes of NTSR, NTSR1 and NTSR2, were detected in the rat bladder. Brain tissue and water were used as the positive and negative controls respectively (A). The identities of all products were confirmed by sequencing and reverse PubMed BLAST analyses. Western blots of the tissue lysates were probed with anti-NTSR1, and anti-NTSR2 and anti-GAPDH served as an endogenous control. The molecular weight of the NTSR was 46 kDa (B). Semiquantitative analysis of the NTSR subtypes. The expression level of NTSR1 was higher than that of NTSR2 ((C), aP < 0.001, paired-sample t-test, four rats).

Fig 5: NTs-induced changes in bladder voiding.NTs-induced changes in bladder micturition were detected with rat cystometry. Infusion of NTs in rat bladder lumen can influence the MI but not the MBP (A). Compared with saline vehicle, NTs significantly decreased the MI but had no influence on MBP (B, aP = 0.001, bP = 0.100, independent-sample t-test, 6 rats).