Increased succinate synthesis and secretion are observed in metabolic syndrome (Mets) and diabetes mellitus and have been linked to Lower Urinary Tract Symptoms (LUTS). We have demonstrated previously that succinate modulates bladder function and contractility by relaxing detrusor muscle. Furthermore, we found that the knockout (KO) of succinate receptor (GPR91) caused thickening of the bladder mucosa and reduced bladder capacity in mouse model. However, the mechanisms involved in the relaxing effect of succinate/GPR91 in the bladder are still elusive. Our aim was to identify the mechanisms by which succinate/GPR91 produces its relaxing effect on the bladder.

Bladder strips from C57BL/6 and GPR91/crumb KO mice were used for contractility studies or fixed for microscopy. Urothelial and smooth muscle cells from Sprague Dawley (SD) rats and C57BL/6 and GPR91 KO mice were isolated from bladder using collagenase IV method. Cells responses to incubation with succinate was studied for nitric oxide (NO) by colorimetric method and ATP using ELISA (Cayman Chemicals).

Contractility of GPR91 KO bladder strips did not show differences in responses to different stimuli compared to wild type mice bladder strips. (Figure 1.A-D) The bladder strips from C57BL/6 showed more relaxation to succinate compared to the bladder strips of the GPR91 KO. (Figure 1.E) The contraction phase noticed at high concentrations of succinate is mainly related to the NaCl concentration build up in the organ bath medium (sodium succinate salt used to prepare succinate solution) observed in both wild type and KO strips (Figure 1.F). 
Neuronal marker PGP9.5 showed more uniform expression of the marker in all urothelial layers of WT compared to lower expression in the basal urothelial layers of the GPR91 KO. However, the PGP9.5 expression in the detrusor muscle looks more extensive in GPR91 KO compared to WT. (Figure 2)
Incubation with increasing concentrations of succinate (30 min) dose-dependently stimulated the release of NO in culture medium of SD SMCs while levels of cGMP were not significantly affected (n=6). NO release in response to succinate was equivalent if urothelial cells of C57BL/J and GPR91 KO. (Figure 3) Succinate reduced the levels of ATP in urothelial cells culture after 24-hour incubation (Figure 4.A). Intracellular changes were not seen after shorter incubation times (10-30 miuntes), but lower ATP levels were observed only in the supernatant (Figure 4.Band C).

Figure 1: Contractile characteristics of detrusor strips from C57BL/6 and GPR91 KO mice. Response to 60 mM KCl stimulation (A), Dose-response to carbachol 3 nM – 100 µM (B), Carbachol Emax (C57BL/6 n = 18; GPR91 KO n = 26)(C) and Electrical field stimulation 1 Hz – 32 Hz (C57BL/6 n = 12; GPR91 KO n = 22)(D). In response to succinate (1 mM – 100 mM) after stimulation with 1 µM carbachol (C57BL/6 n = 18; GPR91 KO n = 26), fC57BL/6 strips displayed relaxation, while KO mice strips remained unaffected (E). stimulation with NaCl (2 mM – 200 mM) after stimulation with 1 µM carbachol (C57BL/6 n = 6; GPR91 KO n = 6) explains the contraction observed at higher concentrations of sodium succinate (F). Responses are represented as percentage change from stimulation with 1 µM carbachol. Two-way ANOVA with Bonferroni post-hoc test for different succinate concentrations. *P < 0.05. 
Figure 2: Neuronal marker PGP9.5 in the bladder wall of C57BL/6 and GPR91 KO mice. (A) C57BL/6: top images: mucosa, bottom images: detrusor layer. (B) GPR91 KO; top images: mucosa, bottom: detrusor layer. Images on the right shows the distribution of PGP9.5 (Green florescence) and on the left images are superimposed to show nuclei (stained blue by DAPI), UT: urothelium, LP: lamina propria, SM: smooth muscle layer, scale bar 50 µm. Neuronal marker PGP9.5. 
Figure 3: Measure of nitric oxide secretion in cell culture. (A) SM Cells from SD rats were incubated with succinate at 10 mM for 10 or 30 minutes and nitric oxide measured in the supernatant. Preincubation of cells with L-NAME (1 mM) for 30 minutes completely abolished succinate (10 mM)-stimulated increase in NO. (B) Urothelial cells from C57BL/6 and GPR91KO mice displayed a similar increase in NO when incubated for 10 or 30 minutes with succinate (10 mM).
Figure 4: Intracellular and secreted ATP levels in SD urothelial cells. (A) Intracellular ATP levels decreased incubated for 24 hrs with succinate (10 mM) (n=5). Student T-test *P<0.05. (B) Intracellular and supernatant levels of ATP from cells incubated for 10 min with succinate 1, 10 or 30 mM displayed no differences (n=5). (C) In cells pre-incubated for 30 min with or without succinate (10 mM) then with or without carbachol (50 M), intracellular ATP content was unaffected while ATP levels in supernatant were decreased by succinate. Carbachol had no effect on ATP release. (n=8) (ANOVA One-way, **P<0.01, *P<0.05).

The absence of the GPR91 receptor in the KO animal did not affect the bladder contractile response to different stimuli in vitro, except for a loss of the relaxing effect of succinate. This suggests the involvement of the receptor in the bladder relaxation by succinate. Higher expression of PGP9.5 in the detrusor could explain the lower bladder capacity observed previously in GPR91 KO bladders. NO stimulation by high concentrations of succinate is not GPR91-dependent in urothelial cells. NO secretion from urothelial and smooth muscle cells is not related  to succinate/GPR91 activation. The differences in supernatant levels of ATP following a short incubation of succinate suggests that the relaxation effect of the acute administration of succinate relies on decrease of ATP secretion by the urothelium.

Despite the structural and molecular changes caused by the lack of GPR91 in the bladder, it does not influence bladder contractile response in vitro. However, bladder relaxation by acute administration of succinate appears to rely on decreased ATP secretion by urothelial cells.