Vasopressin Receptor V ₂ , V ₃ Expression On ACTH Secretory Tumors

Objectives1. Investigating the expression and function of vasopressin receptor V2and V3in human pituitary corticotrophin adenoma and the ectopic ACTH syndrome.2. Observing the expression of vasopressin receptor V2and V3in mouse pituitary corticotrophin tumor cell line AtT-20.3. Studying the effects of both vasopressin receptor V2agonist and antagonist on ACTH secretion in AtT-20cells, and exploring the potential therapeutic effect of vasopressin receptor V2antagonist on ACTH secreting tumors.Methods1. By enrolling50pituitary corticotrophin adenoma and2ectopic ACTH syndrome patients in our study, whose pathological specimens were stained by immunohistochemistry technique after surgery, it was able to observe the expression of vasopressin receptor V2and V3in ACTH secreting tumors.2. Using the statistical methods to analyse the relationship between patients’ clinical data and the expression of vasopressin receptor V2and V3.3. The multispectral slides imaging flow cytometry system was used to make quantitative analysis on the expression of vasopressin receptor V2and V3in those12patients who had underwent BIPSS+DDAVP stimulation test before surgery. By combining the results of BIPSS+DDAVP test and the quantitative analysis, their correlations were investigated.4. The mouse pituitary corticotrophin tumor cell line AtT-20was successfully cultured in vitro, and Real-Time PCR was used to detect the mRNA expression of vasopressin receptor V2and V3in AtT-20cells.5. Three different concentrations of desmopressin,2nM、20nM and200nM, were used to observe their impact on ACTH secretion in AtT-20cells.6. Exploring the influence of vasopressin receptor V2antagonist Tolvaptan on ACTH secretion, three different concentrations of Tolvaptan,100nM、1μM、10μM, were used to deal with AtT-20cells.Results1. The expression of vasopressin receptor V2and V3was detected in50pituitary corticotrophin adenomas, and their positive rates were62%and76%respectively. The positive rate of V3receptor was higher than V2receptor. 2. The vasopressin receptor V3was positive in both two ectopic ACTH tumors, but V2receptor was positive in one and negative in the other one.3. Clinical manifestation of edema and purple striae were different in positive group and negative group of vasopressin receptor V2and V3respectively. There was no significant difference in other clinical data.4. In BIPSS+DDAVP stimulation test, the increase of ACTH level in central and tumor side was correlated only with the staining intensity of vasopressin receptor V2, but not with receptor V3. The correlation coefficients of ACTH level in central and tumor side with receptor V2were0.686and0.675respectively. On the other hand, the peripheral increase of ACTH level had nothing to do with the expression of vasopressin receptor V2and V3.5. The expression of vasopressin receptor V2and V3in mRNA level was detected by Real-time PCR in AtT-20cells.6. It was showed in desmopressin drug test that2nM and20nM desmopressin could apparently stimulate ACTH secretion in AtT-20cells after2h; whereas these effects became weak gradually after4h.7. Results showed that1μM and10μM Tolvaptan had different effects on inhibiting ACTH secretion stimulated by desmopressin. These effects became much stronger as the increasing in concentration of Tolvaptan. But100nM Tolvaptan hadn’t got this effect.Conclusions1. The vasopressin receptor V2and V3were truly expressed in human pituitary corticotrophin adenoma and ectopic ACTH syndrome, and the V3receptor had a higher positive rate than the V2receptor.2. Desmopressin regulated ACTH secretion through the vasopressin receptor V2and V3in tumor cells, and V2receptor possibly played a more important role than the V3receptor during the regulation.3. AtT-20cells also expressed the vasopressin receptor V2and V3through which desmopressin could effectively stimulate ACTH secretion.4. Tolvaptan, the selective V2receptor antagonist, could successfully inhibit ACTH secretion stimulated by desmopressin.5. In the future, Tolvaptan may have a potential worth and probably a bright prospect in the medical treatment of Cushing’s syndrome.