INTRODUCTION AND OBJECTIVES: The clinical use of Botulinum A toxin (BoNT/A) is based on its ability to block the vesicular release of Acetilcholine (Ach) and other neurotransmitters at the level of the neuronal plasmatic membrane. This process requires the internalization of the neurotoxin within the target cell, what it happens by means of the binding with high affinity receptors. These receptors are the synaptic vesicle proteins type 2 (SV2). To date three different types of SV2 receptors have been identified in synaptic structures and endocrine tissues. SV2 immunoreactivity into the bladder has been previously identified at the level of parasympathetic, sympathetic and sensory fibers, but there are no consistent data about their presence in the membranes of urothelial cells. The aim of the present study was to evaluate the genic expression of SV2 receptors in human non neoplastic urothelial cells. METHODS: The analysis was performed in normal human urothelial cell lines. Total RNA was extracted from normal human urothelial cells, quantified with the spectrophotometer and transcript using the High Capacity Archive Kit (Applied Biosystems). 1 L of cDNA was used to perform Polymerase Chain Reaction (PCR) using specific primers built from sequences of SV2-A and – B receptors present in the GeneBank database with the Beacon Designer software (Bio Rad). PCR products were separated in agarose gel at 2%, colored with ethidium bromide and acquired with ChemiDoc (Bio Rad). RESULTS: PCR analysis showed the presence of two specific fragments for SV2-A and -B high affinity receptors for BoNT/A in human urothelial cells (Figure). CONCLUSIONS: To the best of our knowledge the present results show for the first time the presence of SV2 high affinity receptors for BoNT/A on the plasmatic membranes of human urothelial cells. Recent observations suggested a crucial role for the urothelium in the modulation of the underlying afferent sensory fibers. This may be due not only to the urothelial release of Ach, but also to the release of other neurotransmitters, as ATP, CGRP and Substance P, all involved in neurogenic inflammation. BoNT/A could act through SV2-mediated internalization on human urothelial cells thus blocking neurotransmitters’ exocytosis and controlling afferent nervous transmission and bladder pain.

Assessment of botulinum A toxin high affinity SV2 receptors on normal human urothelial cells.

GIANNANTONI, Antonella;
2011

Abstract

INTRODUCTION AND OBJECTIVES: The clinical use of Botulinum A toxin (BoNT/A) is based on its ability to block the vesicular release of Acetilcholine (Ach) and other neurotransmitters at the level of the neuronal plasmatic membrane. This process requires the internalization of the neurotoxin within the target cell, what it happens by means of the binding with high affinity receptors. These receptors are the synaptic vesicle proteins type 2 (SV2). To date three different types of SV2 receptors have been identified in synaptic structures and endocrine tissues. SV2 immunoreactivity into the bladder has been previously identified at the level of parasympathetic, sympathetic and sensory fibers, but there are no consistent data about their presence in the membranes of urothelial cells. The aim of the present study was to evaluate the genic expression of SV2 receptors in human non neoplastic urothelial cells. METHODS: The analysis was performed in normal human urothelial cell lines. Total RNA was extracted from normal human urothelial cells, quantified with the spectrophotometer and transcript using the High Capacity Archive Kit (Applied Biosystems). 1 L of cDNA was used to perform Polymerase Chain Reaction (PCR) using specific primers built from sequences of SV2-A and – B receptors present in the GeneBank database with the Beacon Designer software (Bio Rad). PCR products were separated in agarose gel at 2%, colored with ethidium bromide and acquired with ChemiDoc (Bio Rad). RESULTS: PCR analysis showed the presence of two specific fragments for SV2-A and -B high affinity receptors for BoNT/A in human urothelial cells (Figure). CONCLUSIONS: To the best of our knowledge the present results show for the first time the presence of SV2 high affinity receptors for BoNT/A on the plasmatic membranes of human urothelial cells. Recent observations suggested a crucial role for the urothelium in the modulation of the underlying afferent sensory fibers. This may be due not only to the urothelial release of Ach, but also to the release of other neurotransmitters, as ATP, CGRP and Substance P, all involved in neurogenic inflammation. BoNT/A could act through SV2-mediated internalization on human urothelial cells thus blocking neurotransmitters’ exocytosis and controlling afferent nervous transmission and bladder pain.
2011
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11391/971185
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