886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO

Abstract

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO Guillermo Villegas, Moses Tar, Arnold Melman, and Michael DiSanto Guillermo VillegasGuillermo Villegas More articles by this author , Moses TarMoses Tar More articles by this author , Arnold MelmanArnold Melman More articles by this author , and Michael DiSantoMichael DiSanto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1642AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that is mainly formed by phosphorylation of sphingosine by sphingosine kinase 1 (SPHK1) and can be reverted to sphingosine by sphingosine-1-phosphate phosphatase 1 (SPP1). Our recent studies show that S1P plays a mayor role in the Ca+2 sensitization pathway and contractility of corpus cavernosum smooth muscle (CCSM). Furthermore we demonstrated that SPHK1 expression is elevated in Type 1 diabetic patients with impotence while SPHK1 overexpression in vivo induces erectile dysfunction (ED) in normal rats and upregulates the expression of Rho-kinase (ROCK). Our goals were a) to asses if knocking down expression of SPHK1 in CCSM of diabetic (DM) rats improves erectile function and therefore confirm the determinant role of S1P in the etiology of DM-induced ED b) To examine if SPHK1 expression modulates smooth muscle calcium sensitization by increasing or decreasing ROCK activity and c) to determine whether ROCK is modulated directly by SPHK1 or by S1P. METHODS 1) SPHK1 mRNA was silenced to prevent the synthesis of S1P by using small hairpin RNA (sh) cloned into a shRNA expression vector (pSiren/SPHK1) 2) Human SPP1 and SPHK1 were cloned into the pVax vector to overexpress these proteins 3) For in vivo studies, 2 month strepotozotocin induced diabetic rats (STZ) with ED were injected in the CC with 100 μg pSiren/SPHK1 or pSiren/Luciferase (MOC) and erectile function assessed via cavernous nerve stimulation-induced increase in intracavernous pressure (ICP) one week post-injection 4) For the in vitro studies rat and human primary CCSM cells were transfected with either pSiren/SPHK1, pVax/SPP1 or pVax/SPHK1. In addition S1P receptors were blocked with suramin or N-ethylmaleimide. Cells were harvested at 48 hours, lysed for protein and RNA extraction and analyzed by real time PCR and Western blot. RESULTS a) pSiren/SPHK1 injected STZ rats showed an improved erectile function especially at lower stimulations compared to MOC b) Transfection of CCSM cells with either SPHK1 shRNA or SPP1 significant decreased ROCK activity and c) Blocking of the S1P receptors also downregulated ROCK activity. CONCLUSIONS We showed that in STZ rats, treatment of CC with SPHK1 shRNA improves erectile function confirming the direct role S1P plays in DM-induced ED. Our results further suggest that S1P mediates Ca+2 sensitization of CCSM cells by modulating ROCK activity and that the mechanism involves S1P and not SPHK1. Bronx, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e346-e347 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guillermo Villegas More articles by this author Moses Tar More articles by this author Arnold Melman More articles by this author Michael DiSanto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...