| Orexins (also named hypocretins), including orexin-A (hypocretin-1) and orexin-B (hypocretin-2), are neuropeptides. Orexin-A and B are respectively composed of 33 and 28 residues and they are derived from a common precursor polypeptide, prepro-orexin. Orexins exert their actions through two closely related G-protein-coupled receptors orexin 1 and orexin 2 receptors (OX1R and OX2R). In the brain, orexin-producing neurons are specifically located within the lateral posterior hypothalamic region and widely project to the entire brain. It is well-established that orexin are implicated in regulation of various physiological processes, such as sleep/wake, circadian rhythm, neuronal firing, feeding behavior, etc. However, physiological functions of these peptides in the retina remain unknown. One and only study reported the expression of orexin-A and B in retinal neurons in human, but the authors did not definitely identify the orexin-positive cells by double-labeling experiments.In this work, we first examine the expression of orexin and their receptors in rat retina, and then investigate modulation of voltage-gated L-type Ca2+currents by orexin-A in rat retinal ganglion cells (GCs) and the underlying mechanisms.Reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunohistochemistry revealed the presence of mRNAs and proteins of orexin,OXiR and OX2R in rat retina. Immunostaining for orexin-A and B was observed in many cells in the inner nuclear layer and the ganglion cell layer. Double-labeling experiments demonstrated that in the outer retina horizontal cells, labeled by calbindin and bipolar cells, labeled by homeobox protein Chx10, were orexin-A-and B-positive. In the inner retina, two orexins were both found in GABAergic amacrine cells (ACs), including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase and choline acetyltransferase respectively. Glycinergic ACs, including All ACs, also expressed orexins. Additionally, orexins were expressed in almost all GCs retrogradely labeled by cholera toxin B (CTB) subunit. Morever, OX1R immunoreactivity was observed in most of GCs and all dopaminergic ACs, as well as in both outer and inner plexiform layers. In contrast, no obvious OX2R immunostaining was detectable in the rat retina. These results suggest that orexin may modulate the function of neurons, especially in the inner retina.Using whole-cell patch clamp recording, we first characterized L-type Ca2+ currents in GCs of rat retinal slice preparations. With Ba2+ as the substitution for Ca2+, the inward currents were induced from GCs by a depolarizing pulse from a holding potential of-40 mV to 0 mV in the precese of TTX and TEA. The currents were enhanced by application of L-type calcium channel activator BayK.8644, and abolished by L-type calcium channel blocker nifedipine. We further showed that application of orexin-A significantly increased the L-type Ca2+currents. The effect of orexin-A could be blocked by SB334867, a selective OXiR antagonist, but not by TCS OX229, a selective OX2R antagonist, indicating that orexin-A-induced potentiation was mediated by OXiR. Addtionally, application of orexin-A did not change the activation and steady-inactivation of the L-type calcium channels.Signaling pathways mediateing the orexin-A effect were further explored. Intracellular infusion of G-protein inhibitor GDP-β-S eliminated the orexin-A effect, suggesting the involvement of G-protein in the modulation. Internal dialysis of the phosphatidylinostiol (P1)-specific phospholipase C (PLC) inhibitor U73122, but not the phosphatidylcholine (PC)-PLC inhibitor D609, could block orexin-A-induced potentiation. Moreover, in intracellular Ca2+ -free solution (buffered with 10 mM BAPTA), application of orexin-A failed to increase the currents. Further more, intemal application of IP3 receptor antagonist heparin or xestospengins-C abolished the orexin-A effect. In contrast, in the presence of the ryanodine receptor modulator ryanodine, orexin-A persisted to increase the L-type Ca2+currents. Application of the protein kinase C (PKC) inhibitor bisindolymeleimide Ⅳ (Bis Ⅳ) and G66976 could also abolished the orexin-A effect. However, cAMP, protein kinases A (PKA) inhibitor KT5720, cGMP and protein kinase G (PKG) inhibitor KT5823 failed to block the orexin-A-induced potentiation in L-type Ca2+ currents.In conclusion, in the present work, the distribution of orexin-A, B and their receptors in rat retina are systematically studied. The augmentations of L-type Ca2+ currents by orexin-A via activation of OX1R may be mediated by a distinct G-protein/PI-PLC/IP3/Ca2+/PKC signaling pathway. |
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