Desensitization of δ-opioid-induced mobilization of Ca²⁺ stores in NG108-15 cells

Activation of δ-opioid receptors in NG108-15 cells induces the release of calcium from an inositol 1,4,5-trisphosphate-sensitive intracellular store. We used fura-2-based digital imaging to study the effects of prolonged exposure to agonist on opioid-induced increases in [Ca²⁺](i). Exposure to D-Ala²-D-Leu⁵ enkephalin (DADLE) (1 μM) for 30 min completely desensitized NG108-15 cells to a second DADLE-induced response. The cells recovered gradually over 25 min following washout of DADLE. The desensitization was not due to depletion of intracellular calcium stores and bradykinin failed to cross-desensitize the DADLE-evoked response, although both agonists mobilized the same Ca²⁺ store. Desensitization induced by 100 nM DADLE was overcome by a higher concentration of DADLE (100 μM). Treatment with 8-cpt-cAMP (0.1 mM) for 30 min did not influence the DADLE-induced increases in [Ca²⁺ ](i). Phorbol dibutyrate (PdBu) (1 μM) blocked the response completely. Treatment with the inhibitor of cyclic nucleotide-dependent kinases H8 (1 μM) for 45 min did not prevent DADLE-induced desensitization. Treatment with the protein kinase C (PKC) inhibitors staurosporin (10 nM) and GF-109203X (200 nM) for 45 min reduced desensitization. However, down-regulation of PKC by 24 h exposure to PdBu (1 μM) failed to prevent the DADLE-induced desensitization in NG108- 15 cells. Thus, we conclude that multiple pathways participated in desensitization of δ-receptor-mediated Ca²⁺ mobilization, one of which includes PKC.