This conclusion was strengthened with data obtained in functional experiments. For this, TIC cells have been stimu lated by 2 IU hCG or 1 ng ml FSH, and CREB phosphory lation was evaluated. It’s effectively established that gonadotropin receptors exert their actions by coupling to G proteins, expanding cAMP synthesis that, in conse quence, promotes CREB phosphorylation, It had been identified that in TIC cultures, CREB protein phosphoryla tion was elevated six fold by hCG stimula tion, whereas FSH did not induce any modify, as a result supporting the concept that these cultures contained mainly theca interstitial cells. To review the expression of p2y2r, p2y4r, and p2y6r tran scripts, RNA from TIC was reverse transcribed, then PCR was carried out with unique oligonucleotides for each receptor subtype. RNA samples from ovary, brain, and heart have been also analyzed as controls.
As proven in Figure 1A, a p2y2r fragment of 1032 bp selleck chemical Oligomycin A and a p2y6r frag ment of 257 bp had been amplified through the cDNA of all tis sues examined. However, the p2y4r fragment of 575 bp was only amplified through the whole ovary and brain cDNA. In the many assays, manage amplifications without the need of RT or with out cDNA template did not generate any PCR product, The amplified fragments have been cloned into the pCR4 TOPO vector, sequenced, and analyzed in BLAST, and the fragments have been identical to the reported sequences from mouse, These RT PCR final results indicated that TIC might express P2Y2 and P2Y6 receptors. In order to detect the protein, Western blot was carried out from homogenates to detect P2Y2, to detect P2Y6 receptor it had been essential to per type immunoprecipitation followed by Western blot, which advised a reduced expression degree of this receptor, P2Y2 was detected as a band of 58 kDa, a serious band near 70 kDa, along with a fainter band of 45 kDa.
P2Y6 was detected as three bands with molecular weights of somewhere around 45, forty, and 37 kDa. Within the latter case, the IgG hefty chain interfered using the immunoreactive bands corresponding for the receptor. Even so, all bands observed match the molecular weights reported previ ously for each receptor forms. UTP and UDP induced increase of intracellular Ca2 concentration in TIC Functional responses of P2Y receptors Camptothecine had been studied by applying ATP, UTP, or UDP to TIC and monitoring the adjustments in intracellular calcium concentration working with fluorescence microscopy of Fluo 4 AM loaded cells. In all scenarios, 25 to 40 cells from three independent cul tures were analyzed, Figure 2A exhibits a normal response elicited by a hundred uM ATP. With the highest concentration tested, ATP elicited a i improve of 458 18% compared using the basal level, this increase was mono tonic, dose dependent, and had an EC50 of six.