Excitatory effects of orexin-A on nucleus tractus solitarius neurons are mediated by phospholipase C and protein kinase C.
Orexin (ORX)-A is a 33-amino acid peptide with demonstrated roles in the regulation of energy metabolism, autonomic control, and sleep. Orexin receptors (OXRs), OX1R and OX2R, and immunoreactive axons are present in the nucleus tractus solitarius (NTS). We demonstrated previously that bath application of ORX-A depolarizes NTS neurons through activation of a nonselective cationic conductance (NSCC) and inhibition of a sustained potassium current (IK). The present study examined the signaling pathways underlying the excitatory effects of ORX-A on NTS neurons using whole-cell patch-clamp recording techniques. Inclusion of guanosine 5'-O-(2-thiodiphosphate) in the internal pipette solution abolished the effects of ORX-A, confirming that the actions of ORX-A are mediated by G-protein-coupled receptors. The responses of ORX-A were also blocked by a phospholipase C (PLC) inhibitor, D609, and by a nonselective protein kinase (PK) inhibitor, H7, demonstrating the involvement of PLC and protein kinases. However, PKA appears not to play a role, because the depolarizing effects of ORX-A were still observed when the PKA inhibitor peptide (6-22) was included in the pipette solution, and bath application of 8-bromo-cAMP (a PKA agonist) was without effect on NTS neurons. In contrast, 12-O-tetradecanoylphorbol-13-acetate (a PKC agonist) depolarized NTS neurons, and bisindolylmaleimide (BIS), a PKC inhibitor, abolished the depolarizing effects of ORX-A. Finally, voltage-clamp experiments demonstrated that BIS also blocked the activation of NSCC and inhibition of IK by ORX-A in NTS neurons. These results therefore show that the excitatory effects of ORX-A on NTS neurons are mediated through activation of the PLC-PKC-NSCC and -IK signaling pathways, which probably result from OXR-coupled activation of Gq.
Yang
Bo
B
Department of Physiology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Samson
Willis K
WK
Ferguson
Alastair V
AV
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009474:Neurons / Q000187:drug effects* / Q000502:physiology*; D009479:Neuropeptides / Q000494:pharmacology*; D000068797:Orexins; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D017552:Solitary Nucleus / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002352:Carrier Proteins; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D000068797:Orexins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate