μ-Opioid Inhibition of Ca2+ Currents and Secretion in Isolated Terminals of the Neurohypophysis Occurs via Ryanodine-Sensitive Ca2+ Stores

mu-Opioid agonists have no effect on calcium currents (I-Ca) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, mu-opioid receptor (MOR)-mediated inhibition of I-Ca is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca2+](i)) in response to application of [D-Ala(2)-N-Me-Phe(4), Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca2+](i) with BAPTA-AM completely blocked the inhibition of both I-Ca and high-K+-induced rises in [Ca2+](i) due to MOR activation,but had no effect on mu-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I-Ca and completely relieves MOR-mediated inhibition of high-K+-induced and DAMGO-induced rises in [Ca2+](i). Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca2+](i) and inhibition of I-Ca and high-K+-induced rises in [Ca2+](i) while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca2+](i) released by cADPr from ryanodine-sensitive stores.