Coupling Specificity of NOP Opioid Receptors to Pertussis-Toxin-Sensitive Gα Proteins in Adult Rat Stellate Ganglion Neurons Using Small Interference RNA

Abstract

The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Galphai/o subunits. However, the identification of the specific Galpha subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Galpha subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Galpha subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Galpha protein was examined by RT-PCR experiments. Fluorescence imaging showed that Galphai1, Galphai3, and Galphao were expressed in SG neurons. The transfection of Galphai1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Galphai3 or Galphao was without effect. Taken together, these results suggest that in SG neurons Galphai1 subunits selectively couple NOP receptors to N-type Ca2+ channels.

Modulation of Silent and Constitutively Active Nociceptin/Orphanin FQ Receptors by Potent Receptor Antagonists and Na+ Ions in Rat Sympathetic Neurons

“…Previous studies have shown that NOP receptors are natively expressed in SG neurons (Ruiz-Velasco et al, 2005;Margas et al, 2008). Thus, this model system was ideal to compare the effects of antagonists or Na ϩ ions under conditions in which NOP receptors are silent (i.e., physiological expression levels) or constitutively active (i.e., high expression levels) within a neuronal environment.…”

“…SG neurons were isolated from adult rats using the method described previously (Margas et al, 2008). In brief, male Wistar rats (125-175 g) were first anesthetized with CO 2 and then decapitated with a laboratory guillotine.…”

Pharmacogenomic study of the role of the nociceptin/orphanin FQ receptor and opioid receptors in diabetic hyperalgesia

“…Similar to opioid receptors, NOP receptors have been shown to couple to pertussis toxin-sensitive G i/o proteins to inhibit adenylate cyclase, activate inwardly rectifying potassium channels and inhibit calcium channels (Ma et al, 1997;Margas et al, 2008). However, unlike opioid receptors, NOP receptors can also couple to pertussis toxin-insensitive G proteins, including G z , G 16 and G s (Chan et al, 1998;Klukovits et al, 2010).…”

Alteration of the mu opioid receptor: Ca2+channel signaling pathway in a subset of rat sensory neurons following chronic femoral artery occlusion

“…In one set of experiments (see Fig. 2E), the brain and stellate ganglion (SG) tissue were isolated (Margas et al 2008) and used as a positive control for G␣ i1 and G␣ O protein detection, respectively.…”