Phorbol ester-mediated neurotensin secretion is dependent on the PKC-alpha and -delta isoforms

Neurotensin (NT) plays an important role in gastrointestinal secretion, motility, and growth. The mechanisms regulating NT secretion are not entirely known. Our purpose was to define the role of the PKC signaling pathway in secretion of NT from BON cells, a human pancreatic carcinoid cell line that produces and secretes NT peptide. We demonstrated expression of all 11 PKC isoforms at varying levels in untreated BON cells. Expression of PKC-alpha, -beta2, -delta, and -mu isoforms was most pronounced. Immunofluorescent staining showed PKC-alpha and -mu expression throughout the cytoplasm and in the membrane. Also, significant fluorescence of PKC-delta was noted in the nucleus and cytoplasm. Treatment with PMA induced translocation of PKC-alpha, -delta, and -mu from cytosol to membrane. Activation of PKC-alpha, -delta, and -mu was further confirmed by kinase assays. Addition of PKC-alpha inhibitor G?-6976 at a nanomolar concentration, other PKC inhibitors G?-6983 and GF-109203X, or PKC-delta-specific inhibitor rottlerin significantly inhibited PMA-mediated NT release. Overexpression of either PKC-alpha or -delta increased PMA-mediated NT secretion compared with control cells. We demonstrated that PMA-mediated NT secretion in BON cells is associated with translocation and activation of PKC-alpha, -delta, and -mu. Furthermore, inhibition of PKC-alpha and -delta blocked PMA-stimulated NT secretion, suggesting a critical role for these isoforms in NT release.