RT Journal Article
SR Electronic
T1 μ-Opioid Inhibition of Ca<sup>2+</sup> Currents and Secretion in Isolated Terminals of the Neurohypophysis Occurs via Ryanodine-Sensitive Ca<sup>2+</sup> Stores
JF The Journal of Neuroscience
JO J. Neurosci.
FD Society for Neuroscience
SP 3733
OP 3742
DO 10.1523/JNEUROSCI.2505-13.2014
VO 34
IS 10
A1 Cristina Velázquez-Marrero
A1 Sonia Ortiz-Miranda
A1 Héctor G. Marrero
A1 Edward E. Custer
A1 Steven N. Treistman
A1 José R. Lemos
YR 2014
UL http://www.jneurosci.org/content/34/10/3733.abstract
AB μ-Opioid agonists have no effect on calcium currents (ICa) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, μ-opioid receptor (MOR)-mediated inhibition of ICa 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-Ala2-N-Me-Phe4,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 ICa and high-K+-induced rises in [Ca2+]i due to MOR activation, but had no effect on κ-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 ICa 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 ICa 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.