Login to Access Video or Poster Abstract: MP94-18
Sources of Funding: FRQS

Introduction

Overactive bladder is associated with the metabolic syndrome. Increased succinate production is detected in the presence of hyperglycemia and hypoxemia, as with diabetes mellitus and metabolic syndrome. Succinate was recently identified as a major metabolic switch controlling metabolic functions in the body through its receptor GPR91 (SUCNR1). The aim of our study is to determine how succinate interacts with urothelial cells through its receptor.

Methods

Urothelial cells were isolated from female Sprague-Dawley rat bladder using a collagenase IV method. After confluency, cells were exposed to succinate then treated for microscopy and immunoblotting. Cyclic AMP and PGE₂ were measured using an Elisa kit from Cayman Chemical Company. Nitric oxide was assessed by an colorimetric method. Retroviruses were generated for shRNA-mediated knockdown of GPR91.

Results

Urothelial cells were characterized using Cytokeratin 17 and the AE1/AE3 antibody. RT-PCR confirmed expression of GPR91. Short-term incubation of cells with succinate (200 µM) results in phosphorylation of Erk and c-Jun amino-terminal kinases (JNKs). Inhibition of the MAPK pathway by PD98059 (10 µM) inhibited increases of Erk-P elicited by succinic acid. On the other hand, pre-incubation of cells with succinate dose-dependently decreased the concentrations of intracellular cyclic AMP stimulated by forskolin. Succinate triggers entry of calcium inside urothelial cells as visualized by confocal microscopy. Long-term incubation of cells with succinate increased secretion of nitric oxide and decreased PGE₂ release.Cells infected with shRNA retrovirus targeting GPR91 displayed a strong decrease in GPR91 expression. This was associated with a loss of succinate-stimulated Erk phosphorylation. Moreover, inhibition of cyclic AMP synthesis, increases in intracellular calcium and release of nitric oxide were all dramatically prevented.

Conclusions

GPR91 is expressed in urothelial cells. Binding of succinate to its receptor triggers phosphorylation of Erk and JNK, a process that requires the MAPK pathway. Inhibition of cyclic AMP production suggests the receptor is bound to protein G_i. Release of nitric oxide and decrease of PGE₂ are under succinic acid control suggesting a potential cross-talk between urothelium and detrusor muscle.

Funding

FRQS