Coupling specificity of NOP opioid receptors to pertussis-toxin-sensitive Galpha proteins in adult rat stellate ganglion neurons using small interference RNA.
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.
Department of Anesthesiology, Penn State University College of Medicine, Hershey, PA 17033-0850, USA.
Wojciech
W
Margas
Khaled
K
Sedeek
Victor
V
Ruiz-Velasco
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D044385:GTP-Binding Protein alpha Subunits / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D018847:Opioid Peptides / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D013233:Stellate Ganglion / Q000166:cytology*; D014162:Transfection / Q000379:methods; D020866:omega-Conotoxin GVIA / Q000494:pharmacology; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D000906:Antibodies; D002121:Calcium Channel Blockers; D044385:GTP-Binding Protein alpha Subunits; D009638:Norepinephrine; D018847:Opioid Peptides; D037342:Pertussis Toxin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011957:Receptors, Opioid; D020866:omega-Conotoxin GVIA; D000094942:Nociceptin Receptor; D000097629:Nociceptin; C000722285:Oprl1 protein, rat