Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine

Abstract

Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX₁ and OX₂, Gq-coupled GPCRs. We examined the effect of a selective OX₁ agonist, OXA (17-33) on cytosolic calcium concentration, [Ca²⁺]_i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca²⁺]_i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX₁ receptors antagonist. In Ca²⁺-free saline, the OXA (17-33)-induced increase in [Ca²⁺]_i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP₃) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca²⁺]_i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca²⁺]_i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX₁ via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine-orexin A interaction in nucleus accumbens neurons.

Keywords: OX1 receptor; PLD; choline; orexin A; phospholipase D; reward.

Figure 1

OXA (17-33) increases cytosolic Ca²⁺ concentration, [Ca²⁺]_i, in nucleus accumbens neurons via OX₁ receptor activation. (A) Representative examples of fluorescence F340/380 ratio of Fura-2AM-loaded nucleus accumbens neurons in basal conditions (left) and after treatment with OXA (17-33) (10 nM), a selective OX₁ agonist, alone (top right) or in the presence of OX₁ antagonist, SB-334867 (1 µM) (bottom right). (B) OXA (17-33) (10 nM) produced a transient increase in [Ca²⁺]_i; the effect was abolished by SB-334867. (C) Comparison of the amplitude of [Ca²⁺]_i increase (mean + SD) produced by OXA (17–33) (0.1, 1, 10, and 100 nM); p < 0.05 as compared with the amplitude of [Ca²⁺]_i increase produced by each concentration (*) or by OXA (17-33) (10 nM) (**); n = 6 neurons/each concentration tested.

Figure 2

OXA (17-33) increases [Ca²⁺]_i via IP₃-dependent mechanism. (A) Illustration of average Ca²⁺ transients (± SD) induced in Ca²⁺-free saline by OXA (17-33) (10 nM) alone (left) and OXA (17-33) (10 nM) after pretreatment with 2-aminoethoxydiphenyl borate (2-APB, 100 µM) and xestospongin C (XeC, 10 µM, 15 min), IP₃ receptor antagonists (middle), or with bafilomycin A1 (Baf, 1 µM) (right). (B) Comparison of the amplitude of the increase in [Ca²⁺]_i (average + SD) in each condition. Pretreatment with 2-APB and xestospongin C abolished the Ca²⁺ response induced by OXA (17-33). * p < 0.05; n = 6 neurons/condition.

Figure 3

OXA (17-33) A increases [Ca²⁺]_i via choline-Sigma-1R-dependent mechanism. (A) Illustration of average Ca²⁺ transients (± SD) induced by OXA (17-33) (10 nM) alone (left) and in the presence of VU0155056 (1 μM), PLD inhibitor (middle), and BD1047 (50 µM) or NE-100 (5 µM), Sigma-1R antagonists (right). (B) Comparison of the amplitude of the increase in [Ca²⁺]_i (average + SD) in each condition. Inhibition of PLD or antagonism of Sig-1R reduces the Ca²⁺ response elicited by OXA (17-33) (10 nM); p < 0.05 as compared to amplitude of [Ca²⁺]_i increase produced by OXA (17-33) (*) or produced in the presence of the inhibitors (^#) (n = 6 neurons/condition).

Figure 4

Cocaine potentiates OXA (17-33)-induced increase in [Ca²⁺]_i via Sigma-1R activation. (A) Illustration of average Ca²⁺ responses (± SD) produced by application of cocaine (10 µM) alone (left, no response), cocaine (10 µM) and OXA (17-33) (10 nM) (middle), and cocaine and OXA (17-33) in the presence of Sigma-1R antagonists BD1047 (50 µM) or NE-100 (5 µM) (right). (B) Comparison of the amplitude of the increase in [Ca²⁺]_i (average + SD) in each condition. Cocaine potentiates the Ca²⁺ response induced by OXA (17-33), while antagonism of Sigma-1R abolished the potentiation produced by cocaine on the Ca²⁺ response elicited by OXA (17-33) (10 nM). * p < 0.05 (n = 6 neurons/condition).

Figure 5

Diagram illustrating the proposed model of potentiation of orexin by cocaine via Sigma-1R in nucleus accumbens neurons. (A) Orexin A acting on OX₁ receptor activates PLC and PLD. PLC increases IP₃ level and promotes the Ca²⁺ release from endoplasmic reticulum via IP₃ receptors (IP₃R). PLD produces choline (from hydrolysis of phosphatidylcholine) that acts on Sigma-1R to potentiate Ca²⁺ increase via IP₃ R (higher increase in [Ca²⁺]_i). (B) Inhibition of PLD or antagonism of Sigma-1R limits the orexin A-OX₁ receptor signaling to PLC-mediated IP₃-dependent increase in Ca²⁺ (smaller increase in [Ca²⁺]_i). (C) Cocaine, via Sigma-1R activation, potentiates the PLC- and PLD-mediated increase in [Ca²⁺]_i produced by orexin A acting on OX₁ (highest increase in [Ca²⁺]_i). The diagram was created using the Motifolio Illustration Toolkit Neuroscience (https://www.motifolio.com accessed on 7 March 2021).