Figure 6

Calnuc-GFP or Calnuc(ΔSS)-CFP increases ACTH secretion triggered by mastoparan or GTPγS and the effect is G protein-dependent. (A) The amount of total ACTH secreted after stimulation with mastoparan increases from 30% in parental (NT) cells or those expressing GFP alone to 60% and 50% in AtT-20 cells overexpressing calnuc-GFP and calnuc(ΔSS)-CFP (CNGΔSS), respetively. Similar effects are observed after stimulation with GTPγS where 70–75% of the total ACTH is secreted in cells overexpressing calnuc-GFP (CNG) or calnuc(ΔSS)-CFP cells vs 40% in parental cells (NT) or those expressing GFP alone. Results (mean ± SD) represent the average of values obtained in 3 separate experiments performed in triplicate. (B). Introduction of anti-calnuc or anti-G protein antibodies decreases ACTH secretion triggered by mastoparan. In control cells (no-treatment) or those treated with preimmune or anti-Gα12 IgG, 55–60% of the total ACTH was secreted. After pre-treatment with anti-calnuc, anti-Gαi1/2, anti-Gαi3 and anti-GβIgG, ACTH secretion triggered by mastoparan was decreased to 37%, 43%, 32% and 37%, respectively. Results (mean ± SD) represent the average of values obtained in two separate experiments performed in duplicate.