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  • Title: Enhanced production of pituitary adenylate-cyclase-activating polypeptide by 1, N6-dibutyryladenosine 3',5'-monophosphate, phorbol 12-myristate 13-acetate and by the polypeptide itself in human neuroblastoma cells, IMR-32.
    Author: Suzuki N, Harada M, Hosoya M, Fujino M.
    Journal: Eur J Biochem; 1994 Jul 01; 223(1):147-53. PubMed ID: 7913440.
    Abstract:
    We investigated a regulatory mechanism for the production of pituitary adenylate-cyclase-activating polypeptide (PACAP) using the human neuroblastoma cell line, IMR-32. Although immunoreactive (ir-) PACAP38 was produced by these cells, it was not accumulated in the culture medium. To overcome this problem, we cultured the cells in the presence of a biotinylated monoclonal antibody to PACAP38 in order to trap ir-PACAP38 in the medium. Then, the trapped ir-PACAP38 was measured with a two-site enzyme immunoassay based on the biotin-avidin interaction. These studies showed that confluent IMR-32 cells constitutively secreted at least 130 pg/10(6) cells per day of ir-PACAP38. Treatment of the cells with 1 mM 1, N6-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) or 0.1 microM phorbol 12-myristate 13-acetate stimulated ir-PACAP38 production at almost twice the basal level in IMR-32 cells, and these two reagents additively stimulated ir-PACAP38 production. Northern blot analysis indicated that treatment of the cells with both Bt2cAMP and the phorbol ester increased the intensity of several mRNA bands that hybridized with a cDNA probe for human PACAP precursor. We also found that IMR-32 cells possessed high-affinity type-I PACAP receptors (Kd = 230 pM, binding sites: 8.6 x 10(4) sites/cell), which had a 1000-fold higher affinity for PACAP38 and PACAP27 than for vasoactive intestinal polypeptide. The treatment of IMR-32 cells with PACAP and vasoactive intestinal polypeptide increased intracellular cAMP levels, and also increased ir-PACAP production. Northern blot analysis revealed that PACAP at 10 nM markedly stimulated the expression of a 3.7-kb mRNA band that hybridized with PACAP precursor cDNA. These results indicate that IMR-32 cells produce ir-PACAP38, and that PACAP stimulates the synthesis of PACAP mRNA and peptides by increasing intracellular cAMP. Although it remains to be investigated whether PACAP secreted by IMR-32 cells really acts in an autocrine fashion, the present study provides a useful model for further studies of the biological role of PACAP in neuronal cells that produce PACAP and also respond to PACAP.
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