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695 Botulinum toxin A enters normal human urothelial cells and modulates the sensory activity of bladder urothelium
695
Botulinum toxin A enters normal human urothelial cells and modulates the sensory activity of bladder urothelium Eur Urol Suppl 2014;13;e695
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Amantini C.1 , Farfariello V. 1 , Proietti S. 2 , Vianello A. 3 , Gubbiotti M. 3 , Rossi De Vermandois J. 3 , Santoni G.1 , Giannantoni A. 3 1 University
of Camerino, School of Pharmacy, Camerino, Italy, 2 Humanitas Clinical and Research Center,, Dept. of Urology, Rozzano, Italy,
3 University
of Perugia, Dept. of Urology and Andrology, Perugia, Italy
INTRODUCTION & OBJECTIVES: Recent studies demonstrated that Botulinum A toxin (onabot/A) is able to inhibit neurotransmitters release from bladder urothelium in different experimental conditions. Until today, presence and distribution of onabot/A specific receptors which allow neurotoxin entering cells, namely Synaptic Vescicle Proteins type 2 (SV2) have never been demonstrated in bladder urothelium. We investigated the expression of the three SV2 isoforms (SV2-A, SV2-B and SV2-C) and the ability of onabot/A to enter primary Normal Human Urothelial Cells (NHUCs) and modulate expression and levels of NGF and ATP, respectively. MATERIAL & METHODS: NHUCs were isolated from cancer free bladder tissue specimens obtained from informed patients undergoing radical cystectomy for urothelial cancer. Presence and distribution of SV2 receptors in NHUCs were assessed by Western blot. Onabot/A entry was assessed by flow cytometry and fluorescence microscopy. NGF mRNA expression was evaluated by Real-Time PCR and ATP levels were detected by bioluminescence assay. Statistical analysis was performed by Student's t-test and ANOVA test. RESULTS:
Western blot analysis indicated the presence of SV2A, B and C in NHUCs. Interestingly, we found that NHUCs expressed higher level of SV2C as compared to SV2A isoform. All the three isoforms were found to be localized primarily in the cytoplasmic extract, weakly expressed in the membrane fraction and were not detected in soluble nuclear and cytoskeletal compartments. Flow cytometric analysis and fluorescence microscopy revealed a strong increase of red fluorescence in the majority of BoNT/A-treated cells. In addition, we found that onabot/A treatment induced a significant up-regulation of NGF mRNA expression and markedly reduced the release and the intracellular content of ATP, as compared to the untreated controls. (Fig. 1) CONCLUSIONS: NHUCs express all the three SV2 receptor isoforms. Onabot/A enters urothelial cells and directly modulates NGF mRNA expression and ATP production and release.
Botulinum toxin A enters normal human urothelial cells and modulates the sensory activity of bladder urothelium Eur Urol Suppl 2014;13;e695
Print! Print!
Amantini C.1 , Farfariello V. 1 , Proietti S. 2 , Vianello A. 3 , Gubbiotti M. 3 , Rossi De Vermandois J. 3 , Santoni G.1 , Giannantoni A. 3 1 University
of Camerino, School of Pharmacy, Camerino, Italy, 2 Humanitas Clinical and Research Center,, Dept. of Urology, Rozzano, Italy,
3 University
of Perugia, Dept. of Urology and Andrology, Perugia, Italy
INTRODUCTION & OBJECTIVES: Recent studies demonstrated that Botulinum A toxin (onabot/A) is able to inhibit neurotransmitters release from bladder urothelium in different experimental conditions. Until today, presence and distribution of onabot/A specific receptors which allow neurotoxin entering cells, namely Synaptic Vescicle Proteins type 2 (SV2) have never been demonstrated in bladder urothelium. We investigated the expression of the three SV2 isoforms (SV2-A, SV2-B and SV2-C) and the ability of onabot/A to enter primary Normal Human Urothelial Cells (NHUCs) and modulate expression and levels of NGF and ATP, respectively. MATERIAL & METHODS: NHUCs were isolated from cancer free bladder tissue specimens obtained from informed patients undergoing radical cystectomy for urothelial cancer. Presence and distribution of SV2 receptors in NHUCs were assessed by Western blot. Onabot/A entry was assessed by flow cytometry and fluorescence microscopy. NGF mRNA expression was evaluated by Real-Time PCR and ATP levels were detected by bioluminescence assay. Statistical analysis was performed by Student's t-test and ANOVA test. RESULTS:
Western blot analysis indicated the presence of SV2A, B and C in NHUCs. Interestingly, we found that NHUCs expressed higher level of SV2C as compared to SV2A isoform. All the three isoforms were found to be localized primarily in the cytoplasmic extract, weakly expressed in the membrane fraction and were not detected in soluble nuclear and cytoskeletal compartments. Flow cytometric analysis and fluorescence microscopy revealed a strong increase of red fluorescence in the majority of BoNT/A-treated cells. In addition, we found that onabot/A treatment induced a significant up-regulation of NGF mRNA expression and markedly reduced the release and the intracellular content of ATP, as compared to the untreated controls. (Fig. 1) CONCLUSIONS: NHUCs express all the three SV2 receptor isoforms. Onabot/A enters urothelial cells and directly modulates NGF mRNA expression and ATP production and release.