Phosphorylation promotes the desensitization of the opioid-induced Ca 2+ increase in NG108-15 cells

Abstract

Using the fluorescent Ca 2+ indicator fura-2, we demonstrated that, in a single NG108-15 cell, acute repetitive challenge with leucine-enkephalin (EK) results in a gradual reduction of the increase of the cytosolic Ca 2+ concentration ([Ca 2+] i) at agonist exposure times of 90 s or less; increasing the EK exposure time of each challenge from 30 to 90 s results in greater desensitization, with complete desensitization occurring at 90 s exposure. Similar results are seen with ATP. In opioid-desensitized cells, bradykinin can still induce a marked [Ca 2+] i increase, while exposure of desensitized cell to 50 mM K + restores the response EK-induced, suggesting a role of intracellular Ca 2+ stores in the desensitization process. Pretreatment of cells with certain protein kinase inhibitors, including N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) and staurosporine, prevented desensitization, while others, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and {1-[ N, O-bis-(5-isoquinolinesulfonyl)- N-methyl- l-tyrosyl]-4-phenyl-piperazine (KN-62), had no effect. In contrast, activation of protein kinase C by phorbol 12-myristate 13-acetate promoted desensitization. Thus, the desensitization is dependent on protein phosphorylation. HA1004 alone did not alter EK- or bradykinin-induced inositol 1,4,5-trisphosphate (IP 3) generation; however, the inhibitory effect of calyculin A on EK- or bradykinin-induced IP 3 generation was reversed by HA1004. In addition, in the presence of HA1004, the blockade of Ca 2+ influx by either verapamil or removal of extracellular Ca 2+ or the depletion of Ca 2+ pools by thapsigargin still led to desensitization, suggesting that phosphorylation does not alter the activity of the Ca 2+ transporters involved in Ca 2+ influx and Ca 2+ release. Our results imply that emptying of intracellular Ca 2+ stores and protein phosphorylation in the phospholipase C signaling pathway play roles in the process of desensitization.