Anisodamine The mRNA expression of asEP studied by RT PCR sh
The mRNA expression of asEP4 studied by RT-PCR shows that EP4 is expressed in many different tissues in Atlantic salmon which is comparable to other species (Sugimoto and Narumiya, 2007) although some distinct differences are present. For example, in mice the expression of EP4 receptor was found to be more abundant in the thymus, part of the digestive (ileum) and reproductive (uterus) organs while it was least abundant in spleen and kidney (Sugimoto and Narumiya, 2007). However, in this study we found that it is strongly expressed in the immune organs (spleen and head kidney). The difference in tissue expression suggests that differences in functionality may also exist. Direct comparison between zebrafish and asEP4 is difficult due to the lack of quantitative expression data and the presence of multiple isoforms in zebrafish compared to only one identified so far in Atlantic salmon. It is however not unlikely that Atlantic salmon has other isoforms that are yet to be identified. Among the three identified zebrafish EP4 receptors, EP4b has a similar wide tissue distribution as asEP4 while the other two are exclusively expressed in few tissues (Tsuge et al., 2013). One of the main differences between the expression patterns of zebrafish EP4b and asEP4 is that zebrafish EP4b mRNA was found to be expressed in the Anisodamine but could not be detected in the heart by PCR while the opposite was found for asEP4. Another observation was that the level of expression observed by RT-PCR correlated with the intensity of staining observed by immunohistochemistry. The only exception was heart and muscle where no expression was detected by immunohistochemistry. This might be due to differences in detection limit between the two methods or simply due to impurities in the templates used for RT-PCR. In mammals, PGE2 is known to modulate the functions of different populations of immune cells (Harizi et al, 2003, Ikegami et al, 2001, Luschnig-Schratl et al, 2011, Minakuchi et al, 1990). The importance of EP4 in mediating these responses has been investigated. Using RT-PCR and western blot, it was shown that EP4 is expressed by B cells, T cells, eosinophils, dendritic cells and macrophages (Harizi et al, 2003, Ikegami et al, 2001, Mita et al, 2002, Nataraj et al, 2001). It was further shown that EP4 mediates the inhibitory effect induced by PGE2 on cytokine production by macrophages (Nataraj et al., 2001). In the present study, asEP4 was found to be strongly expressed in the spleen and head kidney by RT-PCR. Using immunohistochemistry, asEP4 was found expressed in leukocytic cells in both organs, and interestingly, strongly associated with the splenic melanomacrophages. Moreover, it was also found in liver monocyte-like cells and in the intra-epithelial leukocytes present in the intestine. These findings suggest that asEP4 might play a role in local immune functions as demonstrated in mammals. Further studies are required, however, to demonstrate this. Expression of the EP4 receptor in Atlantic salmon liver was limited to the bile ducts and intrahepatic monocytes-like cells. In rat liver, the expression of EP4 mRNA was detected strongly in the endothelial cells while weak or no expression was detected in Kupffer and liver stellate cells (Fennekohl et al., 1999). However, we did not find any study that describes the expression of EP4 receptor in hepatic tissues by immunohistochemistry in other species. While the expression of EP4 in the immune cells is well documented, the expression in the bile ducts has not been previously reported although EP4 mRNA expression was detected in gall bladder carcinoma (Asano et al., 2002). It is known that PGE2 plays an important role in protecting the epithelial lining of the gallbladder from the bile salts by stimulating the secretion of mucin in a cAMP dependent manner (Behar, 2013, Kuver et al, 1994). The expression of asEP4 in the epithelium of the bile ducts suggests that it may play a role in maintaining the integrity of the bile duct epithelium. Other roles cannot be excluded given that EP2 has been suggested to play a novel role in PGE2 mediated regulation of glucose metabolism in other fish species (Busby et al., 2002).