Purpose Several reports have suggested the rat Vcsa1 gene is down-regulated
Purpose Several reports have suggested the rat Vcsa1 gene is down-regulated in models of erectile dysfunction (ED). contrast, treatment of CSM cells that lowered NEP activity resulted in decreases in GPCR expression. These results suggest that the peptide product of Vcsa1, sialorphin, can effect GPCR expression by acting on NEP. In animals with bilaterally transected cavernous nerves the reduced expression of Vcsa1 is accompanied by increased GPCR expression in cavernosal tissue. Conclusions These experiments suggest that the mechanism by which Vcsa1 modulates erectile function is partly mediated through changes in GPCR AT-101 IC50 expression. expression of Vcsa1 in corporal smooth muscle (CSM) cells and microarray and RT-PCR to determine the effect on GPCR gene expression. In addition, we confirmed these results by using a rat model. ED was induced by bilateral transection of the cavernous nerve, and the tissues were then analyzed for Vcsa1 and GPCR gene expression. Bilateral transaction of the cavernosal nerve has previously been shown to cause decreased Vcsa1 expression in corporal tissue 1, 2 in rats. 2.0 Materials and Methods 2.1 Animals Rat CSM cells were isolated from freshly excised corporal tissues from Fischer F-344 AT-101 IC50 rats as AT-101 IC50 previously described 12 and grown in DMEM containing 10% FBS media. In addition, six 120-day old Fischer F-344 rats underwent surgical bilateral transection of the cavernous nerve (CN) as previously described1, 2. As controls, an additional six animals underwent sham operations without CN transection. After 9 days animals were killed by placing them in a CO2 chamber, and the corpora were harvested and flash frozen in liquid nitrogen and stored at ?70C until RNA preparation. These protocols are approved by the Animal Use Committee at the Albert Einstein Sirt6 College of Medicine. 2.2 Vcsa1-siRNA siRNA that would target Vcsa1 (Vcsa1-siRNA) was constructed. We found potential Vcsa1-siRNA sequences using the Ambion on-line resource (siRNA target finder: http://www.ambion.com/techlib/misc/siRNA_finder.html). The siRNA construct, Vcsa1-siRNA, was synthesized using the Silencer siRNA Construction Kit (Ambion, Foster City, CA) following the manufacturers instructions. The following template oligonucleotides for the siRNAs were used: antisense, 5-AATGGTGGACAAATAGGAGTACCTGTCTC-3; sense, 5-AATACTCCTATTTGTCCACCACCTGTCTC-3. 2.3 NEP-siRNA NEP-siRNA (smartpool) was obtained from Dharmacon Research, Inc. (Lafayette, CO). It was composed of four Smartselection designed siRNAs targeting the NEP gene with the following sequences: (1) sense, 5-GCAGAAAUCAGAUCGUCUUUU-3; antisense, 5-AAGACGAUCCUGAUUUCUGCUU-3; sense, (2) 5-GAACAAACAUAUGGUACUUUU-3; antisense, 5-AAGUACCAUAUGUUUCUUCUU-3; sense, (3) 5-UAACCAAACUUAAGCCUAUUU-3; antisense, 5-AUAGGCUUAAGUUUGGUUAUU-3; (4) sense, 5-GUACGGACUUCUUCAAAUAUU-3; antisense, 5-UAUUUGAAGAAGUCCGUACUU-3. 2.4 Transfection of corporal smooth muscle cells The Vcsa1-siRNA or NEP-siRNA was used to knock-down expression of the Vcsa1 or the NEP gene, respectively, in rat CSM cells following transfection of 10 nM Vcsa1-siRNA for 60 hours in DMEM containing 10% FBS. Expression of the various genes were determined by quantitative PCR, normalized to GAPDH, and the untreated cells used … We hypothesized that inhibiting NEP using phosphoramidon 15 would result in the down-regulation of GPCR expression as opposed to their up-regulation following treatment of cells with Vcsa1-siRNA. This is because NEP inhibition by phosphoramidon would result in longer binding times of agonist to GPCR, and would result in compensatory changes in cells that would down-regulate GPCR gene expression to prevent over activity of the G-protein downstream signaling pathways. As seen in Figure 2, the majority of GPCR are down-regulated by the presence of 1M phosphoramidon in the culture media. The exception was Celsr3, a cadherin EGF LAG seven-pass G-type receptor, which was up-regulated. This may be a result of over-lapping, but not identical, specificities in inhibiting cell surface expressed peptidases by sialorphin compared to phosphoramidon. Fig. 2 The expression of GPCR in AT-101 IC50 corporal cells following treatment with 1 M phosphoramidon. The experiment was repeated three times, and the expression of the GPCR determined in triplicate (significance is determined by the students AT-101 IC50 t-test, … We hypothesize that the effect on expression of GPCR expression by sialorphin and phosphoramidon is mediated through their affect on the NEP. In order to directly demonstrate.