title: μ-Opioid inhibition of Ca2+ currents and secretion in isolated terminals of the neurohypophysis occurs via ryanodine-sensitive Ca2+ stores.
abstract: μ-Opioid agonists have no effect on calcium currents (I(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, μ-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 ([Ca(2+)]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 [Ca(2+)]i with BAPTA-AM completely blocked the inhibition of both I(Ca) and high-K(+)-induced rises in [Ca(2+)]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 I(Ca) and completely relieves MOR-mediated inhibition of high-K(+)-induced and DAMGO-induced rises in [Ca(2+)]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca(2+)]i and inhibition of I(Ca) and high-K(+)-induced rises in [Ca(2+)]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(2+)]i released by cADPr from ryanodine-sensitive stores.
authors.affiliation: Department of Microbiology and Physiological Systems and Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Cristina
authors.initials: C
authors.lastname: Velázquez-Marrero
authors.forename: Sonia
authors.initials: S
authors.lastname: Ortiz-Miranda
authors.forename: Héctor G
authors.initials: HG
authors.lastname: Marrero
authors.forename: Edward E
authors.initials: EE
authors.lastname: Custer
authors.forename: Steven N
authors.initials: SN
authors.lastname: Treistman
authors.forename: José R
authors.initials: JR
authors.lastname: Lemos
mesh_terms: D000701:Analgesics, Opioid / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D010904:Pituitary Gland, Posterior / Q000187:drug effects / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors* / Q000502:physiology; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000701:Analgesics, Opioid; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D012433:Ryanodine; D002118:Calcium
keywords: ICa; MOR modulation; NH terminals; RyR; exocytosis