Cyclic AMP regulates the calcium transients released from IP3-sensitive stores by activation of rat κ-opioid receptors expressed in CHO cells

Abstract

We analyzed intracellular Ca2+and cAMP levels in Chinese hamster ovary cells expressing a cloned rat kappa opioid receptor (CHO-κ cells). Although expression of kappa (κ)-opioid receptors was confirmed with a fluorescent dynorphin analog in almost all CHO-κ cells, the κ-specific agonists, U50488H or U69593, induced a Ca2+transient only in 35% of the cells. The Ca2+response occurred in all-or-none fashion and the half-maximal dosage of U50488H (812.1nM) was higher than that (3.2nM) to inhibit forskolin-stimulated cAMP. The κ-receptors coupled to Gi/oproteins since pertussis toxin significantly reduced the U50488H actions on intracellular Ca2+and cAMP. The Ca2+transient originates from IP3-sensitive internal stores since the Ca2+response was blocked by a PLC inhibitor (U73122) or by thapsigargin depletion of internal stores while removal of extracellular Ca2+had no effect. Interestingly, application of dibutyryl cAMP (+ 56.2%) or 8-bromo-cAMP (+ 174.7%) significantly increased the occurrence of U50488H-induced Ca2+mobilization while protein kinase A (PKA) inhibitors, Rp-cAMP (−32.3%) or myr-ψ PKA (−73.9%) significantly reduced the response. Therefore, it was concluded that cAMP and PKA activity can regulate the Ca2+mobilization. These results suggest that the κ receptor-linked cAMP cascade regulates the occurrence of κ-opioid-mediated Ca2+mobilization.