Regulation of OX1 orexin/hypocretin receptor-coupling to phospholipase C by Ca2+ influx.
OBJECTIVE
Orexin (OX) receptors induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and the "conventional" phospholipase C (PLC)-Ca2+ release-store-operated Ca2+ channel (SOC) pathways. In this study we assessed the ability of these different Ca2+ influx pathways to amplify OX1 receptor signalling to PLC in response to stimulation with the physiological ligand orexin-A.


METHODS
PLC activity was assessed in CHO cells stably expressing human OX1 receptors.


RESULTS
Inhibition of total Ca2+ influx by reduction of the extracellular [Ca2+] to 1 microM effectively inhibited the receptor-stimulated PLC activity at low orexin-A concentrations (by 93% at 1 nM), and this effect was gradually reduced by higher orexin-A concentrations. A similar but weaker inhibitory effect (84% at 1 nM) was obtained on depolarization to approximately 0 mV, which disrupts most of the driving force for Ca2+ entry. The inhibitor of the OX1 receptor-activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca2+] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg2+, and the SOC inhibitors, dextromethorphan, SKF-96365 (1-[beta-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole HCL) and 2-APB (2-aminoethoxydiphenyl borate), inhibited PLC activity at low concentrations of orexin-A, but were not as effective as TEA.


CONCLUSIONS
Both ROCs and SOCs markedly amplify the OX(1) receptor-induced PLC response, but ROCs are more central for this response. These data indicate the crucial role of ROCs in orexin receptor signalling.
Johansson
L
L
Department of Neuroscience, Division of Physiology, Uppsala University, Uppsala, Sweden.
Ekholm
M E
ME
Kukkonen
J P
JP
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D017136:Ion Transport; D064446:Orexin Receptors; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*
D064446:Orexin Receptors; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D010738:Type C Phospholipases; D002118:Calcium