[mu]-Opioid Inhibition of Ca super(2+) Currents and Secretion in Isolated Terminals of the Neurohypophysis Occurs via Ryanodine-Sensitive Ca super(2+) Stores

• Published in: The Journal of neuroscience 2014-03, Vol.34 (10), p.3733-3742
• Main Authors: Velazquez-Marrero, Cristina, Ortiz-Miranda, Sonia, Marrero, Hector G, Custer, Edward E, Treistman, Steven N, Lemos, Jose R
• Format: Article
• Language: English
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/JNEUROSCI.2505-13.2014

[mu]-Opioid agonists have no effect on calcium currents (I sub(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, [mu]-opioid receptor (MOR)-mediated inhibition of I sub(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 ([Ca super(2+)] sub(i)) in response to application of [d-Ala super(2)-N-Me-Phe super(4),Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH sub(2)(CTOP ), a MOR antagonist. Buffering DAMGO-induced changes in [Ca super(2+)] sub(i) with BAPTA-AM completely blocked the inhibition of both ICa and high-K super(+)-induced rises in [Ca super(2+)] sub(i) due to MOR activation, but had no effect on [kappa]-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 sub(Ca) and completely relieves MOR-mediated inhibition of high-K super(+)-induced and DAMGO-induced rises in [Ca super(2+)] sub(i). Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca super(2+)] sub(i) and inhibition of I sub(Ca) and high-K super(+)-induced rises in [Ca super(2+)] sub(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 [Ca super(2+)] sub(i) released by cADPr from ryanodine-sensitive stores.