br Materials and methods br Results br
Materials and methods
Discussion In the present study, we found that SSTR2, SSTR5, and DA2R proteins are expressed in ACTH-positive AVE-1625 in healthy canine pituitary glands, with particularly high positive cell ratios in the intermediate lobes. Somatostatin has been reported to inhibit the production of growth hormone (GH), prolactin (PRL), thyroid stimulating hormone (TSH) and ACTH. This has been shown in studies on rats (GH, PRL), humans (TSH), and in the mouse AtT20 cell line (ACTH) (Cuevas-Ramos and Fleseriu 2014). Furthermore, SSTR2 knockout mice reportedly showed increased ACTH secretion. An association between SSTR2 and ACTH secretion has also been described (Cuevas-Ramos and Fleseriu 2014). However, SSTR5-mediated inhibition of ACTH secretion has been reported to be much stronger than SSTR2 (Cuevas-Ramos and Fleseriu 2014). Also, an in vitro study using cells derived from canine ACTH-secreting pituitary adenoma tissue, pasireotide and octoreotide reduced CRH-induced ACTH secretion (de Bruin et al. 2008). In contrast, it has not been clarified yet whether ACTH release is directly regulated by DA2R in human normal corticotrophic cells (de Bruin et al. 2009). In this study, SSTR2, SSTR5, and DA2R proteins were found to be expressed in ACTH-positive cells of normal canine pituitary glands. This result corresponds to a previous report that SSTR2, SSTR5 and DA2R mRNA expression can be detected in normal canine anterior pituitary lobe (de Bruin et al. 2008). Furthermore, SSTR2 protein expression in the intermediate lobe of the normal canine pituitary is very similar (de Bruin et al. 2008). Based on our results, it may be hypothesized that in dogs, these receptors might be associated with ACTH secretion and cell division in the pituitary gland. However, it is not known whether the distribution of the expression of these receptors in ACTH-positive cells will change or not, because the normal canine pituitary glands in this study were obtained from very young dogs. In addition, we found that in twelve of fourteen cases (86%), the ACTH-secreting pituitary adenomas expressed SSTR2 and/or SSTR5. In humans, hSSTR2- and hSSTR5-positive cell ratios are both reportedly 75% in patients with Cushing's disease (Cuevas-Ramos and Fleseriu 2014). The membrane expression of hSSTR5 is much higher than that of hSSTR2 (de Bruin et al. 2009). In an in vitro study using the AtT20 cell line, SSTR2 has been found to be more susceptible to hypercortisolism than SSTR5. SSTR2 expression is downregulated by hypercortisolism, whereas, SSTR5 expression is unchanged (de Bruin et al. 2009). However, in an in vitro study using canine ACTH-secreting pituitary adenoma cells, it has been reported that the addition of dexamethasone upregulates SSTR2 mRNA expression (de Bruin et al. 2008). In this study, fewer cases expressed SSTR2 than SSTR5. These data might be reflective of the difference between acute and chronic hypercortisolism, as the in vitro study with dexamethasone added to the cultured cells can be thought as an acute hypercortisolism. Additionally, of the 14 Cushing's disease cases, seven cases showed strong SSTR5-positive staining. This indicates that pasireotide, a somatostatin analog that has high affinity for SSTR5, might be effective. This result confirmed the results of in vitro and in vivo studies of the effectiveness of pasireotide in dogs with Cushing's disease (Colao et al. 2012). Our results indicate that somatostatin analogs might be effective in treating ACTH-secreting pituitary adenomas positive for α-MSH (and thus likely to be derived from intermediate lobes), because strong positive staining for SSTR2 and SSTR5 was observed in the intermediate lobes of normal canine pituitary glands. However, there were only two cases in which ACTH-secreting pituitary adenomas were positively stained by α-MSH. Hence, this suggests that somatostatin analogs would be effective in treating ACTH-secreting pituitary adenomas derived from both the anterior and the intermediate lobes. Depending on the immunohistological results for SSTR2 and SSTR5, somatostatin analogs could be chosen as a therapeutic option in cases of incomplete resection or recurrence, in veterinary medicine.