Normal human urothelial cell lines express onabotulinumtoxinA SV2 high affinity receptors

INTRODUCTION & OBJECTIVES: Botulinum A toxin (BoNT/A) internalization within the target cells requires the binding with high affinity receptors (Synaptic Vesicle Proteins type 2 -SV2). To date, 3 different isoforms of SV2 have been identified in synaptic and endocrine tissues. Bladder SV2 immunoreactivity has been previously identified in parasympathetic, sympathetic and sensory fibers, but not into bladder urothelial cells. The aim of this study was to evaluate the expression of SV2 in human non neoplastic urothelial cells at genic and protein levels. MATERIAL & METHODS: The study was performed in normal human urothelial cell lines (NHUCs). Expression of the 3 SV2 isoforms (SV2A, -B –C) was investigated by quantitative Real Time PCR (qPCR) and Western blot analysis. In order to identify SV2 subcellular localization, NHUCs were fractionated and proteins from different cellular compartments including membrane, cytoplasm, soluble nuclear and cytoskeleton were separated by SDS-PAGE and probed with anti SV2-A, -B and -C Abs. Mouse brain tissue was used as positive control and ß actin was used as loading control. RESULTS: qPCR demonstrated the presence of SV2-A, -B and -C mRNAs in NHUCs. Higher levels of SV2C (about 1.8 folds) were expressed as compared to SV2A isoform mRNA levels. In addition, SV2B exhibited a 20- and 30-fold reduction in the amount of mRNA levels in comparison with SV2A and SV2C, respectively (Fig. 1). Western blot analysis showed bands with proper molecular weights, likely corresponding to SV2-A, -B and –C, in lysates from NHUCs and mouse brain tissue. All 3 isoforms were found to be accumulated primarily in the cytoplasmic extract, weakly expressed in the membrane fraction and were not detected in soluble nuclear and cytoskeletal compartments. CONCLUSIONS: We demonstrated for the first time the presence of SV2 BoNT/A high affinity receptors on the plasmatic membranes of NHUCs. Recent observations suggested a crucial role for the urothelium in modulating underlying sensory fibers. This may be due not only to the urothelial release of Ach, but also of other neurotransmitters, as ATP, CGRP and Substance P. BoNT/A could act through SV2-mediated internalization on human urothelial cells thus blocking neurotransmitters' exocytosis and controlling afferent transmission and pain. The higher level of SV2C, as compared to the other isoforms, is consistent with the previously described most robust BoNT/A binding activity with SV2C