OX1 orexin/hypocretin receptor activation of phospholipase D

Abstract

Background and purpose: Orexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling.

Experimental approach: Orexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX(1) orexin receptors using [(14) C]-oleic acid-prelabelling and the transphosphatidylation assay.

Key results: Orexin stimulation strongly increased PLD activity - even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration-response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCδ. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gα(i/o) nor Gβγ seemed to play an important role in the orexin response.

Conclusions and implications: Stimulation of OX(1) receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCδ but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCδ in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCδ.

Figure 10

The effect of Ca²⁺ on PLD activation. (A) The effect of reduction of the extracellular Ca²⁺ concentration to 3 µM or 140 nM from the regular 1 mM. The comparisons are to the corresponding control (same stimulus in the presence of 1 mM Ca²⁺). The experiments were performed in HBM instead of cell culture medium. (B) The ability of the Ca²⁺-elevating compounds, the Ca²⁺ ionophore, ionomycin and the SERCA (sarco-/endoplasmic reticulum Ca²⁺ ATPase) inhibitor, thapsigargin, to activate PLD. The comparisons are to basal. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 1

Orexins strongly stimulate PLD and PLC activation. (A) PtdBut generation as viewed on the radioactivity image of a TLC plate read from the imaging plate. The results are from a representative experiment. (B) Concentration–response curves of PLD activation for orexin-A and -B. The parameters for the two-site fit (eq. 2): orexin-A: pEC_50-1= 10.3 (24%), pEC_50-2= 7.7 ± 0.2 (76%); orexin-B: pEC_50-1= 9.0 (25%), pEC_50-2= 6.8 (75%). Data from a single representative experiment are presented in order to demonstrate that the biphasic shape of the curve is not an artefact resulting from averaging of data from different experiments. (C) Concentration–response curves of PLC activation for orexin-A and -B (n= 4). The parameters for the single-site fit with slope factor (eq. 1): orexin-A: pEC₅₀= 8.7, n_H= 0.69; orexin-B: pEC₅₀= 7.2, n_H= 1.0.

Figure 4

The role of PKC and PKD in PLD stimulation. (A) The effect of the nPKC inhibitor, GF109203X, and the cPKC and PKD inhibitor, Gö6076, on orexin- and TPA-induced PLD activities. (B) The effect of dominant-negative PKD constructs on orexin- and TPA-induced PLD activities. (C) PLD activity in response to the peptide PKC activators of PKCδ (KAD1-1), PKCε (KAE1-1) and cPKC (KAC1-1) as compared with orexin-A and TPA. (D) The effect of the peptide PKC inhibitors of PKCε (KIE1-1) and cPKC (KIC1-1) on orexin-, TPA- and KAE1-1-induced PLD activity. Please note that the effect of KIC1-1 on KAE1-1 was not tested, and thus that column is altogether missing. The comparisons are to the corresponding controls (same stimuli in the absence of the inhibitor) (A, B, D) or to the basal (C). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

The OX₁ orexin receptor antagonist SB-334867 effectively blocks the orexin-A-induced PLD activity. The comparisons are to the corresponding controls (same stimuli in the absence of the inhibitor). **P < 0.01; ***P < 0.001.

Figure 3

The effect of the PLD isoform-selective inhibitors, PLD1i and PLD2i, on orexin- and TPA-induced PLD activities. The comparisons are to the corresponding controls (same stimuli in the absence of the inhibitor). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

The PKCδ inhibitor, rottlerin, produces a concentration-dependent inhibition of the PLD responses to orexin-A and TPA. The comparisons are to the corresponding control (same stimulus in the absence of the inhibitor). **P < 0.01.

Figure 6

Investigations on the activation mechanisms for the PKC in PLD activation utilizing the PLC inhibitor U-73122 and the PLA₂ inhibitor MAFP. Neither treatment produced significant inhibition as compared with the corresponding controls.

Figure 7

Investigations on the involvement of the Rho family monomeric G-proteins in the PLD response to orexin and TPA. (A) The effect of the geranylgeranyl transferase I inhibitor GGTI-2133. The cells were incubated with GGTI-2133 for 24 h before the assay. (B) The effect of the dominant-negative (dn) Rho, Rac and cdc42 constructs. The cells were transfected with the constructs 24 h before the assay. (C) The effect of C3-exoenzyme (C3; inhibitor of Rho), Rhotekin-RBD (inhibitor of Rho), POSH-PBD (inhibitor of Rac) and Pak1-PBD (inhibitor of Rac and Cdc42). The cells were transfected with the constructs 24 h before the assay. No treatment/transfection produced significant inhibition as compared with the corresponding controls.

Figure 8

Investigations on the involvement of cAMP in the PLD response to orexin. (A) Orexin receptor stimulation of AC. The results are from a representative experiment. In the lack of apparent saturation in the concentration range used, the data are normalized to the response with 3 µM orexin-A. (B) Basal PLD activity in response to the AC activator forskolin. *P < 0.05.

Figure 9

Investigations on the involvement of Gα_i//o- and Gβγ-proteins in the PLD stimulation. (A) The effect of PTx pretreatment (100 ng·mL^–1, 24 h) on the orexin response. (B) The effect of exogenous Gβ₁γ₂- and Gβγ-scavengers Gα_t and T8-βARK on PLD activity. The cells were transfected with the constructs 24 h before the assay. The comparisons are to the corresponding controls (same stimuli in the ctrl cells). ns, not significant; *P < 0.05.