In this present study, we describe a distinct effect of exogenous CCK-8 on the development of morphine dependence in vitro. Moreover, we provide the first piece of evidence that a CCK1 receptor antagonist can reverse the inhibitory effects of exogenous CCK-8 on morphine dependence. We also find that endogenous CCK exerts a potential facilitative effect via the CCK2 receptor. This suggests opposing roles of CCK1 and CCK2 receptors in the development of morphine dependence.
First, a suitable cell model was selected for this study. The SH-SY5Y cell line is derived from a human neuroblastoma cell line, SK-N-SH, by three rounds of subcloning. The SH-SY5Y cells are dopamine beta hydroxylase active, acetylcholinergic, glutamatergic and adenosinergic, and express abundant and functional μ- and δ-opioid receptors. SH-SY5Y cells have been extensively used for studies of opioid receptor regulation and intracellular signaling. Moreover, co-expression of the opioid and CCK systems in SH-SY5Y cells was confirmed, and CCK is endogenously expressed in SH-SY5Y cells. This system is useful for the study of the potential regulatory effects of exogenous CCK-8 on morphine dependence.
The interaction between CCK and opioids was first reported by Itoh et al. They showed that pre-treatment with CCK suppressed anti-nociception induced by β-endorphin . A subsequent in vivo microdialysis study found that the extracellular levels of CCK significantly increased after morphine administration, thus acting as a negative feedback modulator and a potent anti-opioid peptide [6, 7, 30]. Studies have confirmed that endogenous CCK potentiates, and the CCK antagonist attenuates the tolerance and dependence of opioids [31, 32]. The presence of opioid receptors in CCK-containing neurons suggest a potential direct influence of opioids on CCK release . However, earlier studies have failed to show an affinity of CCK for opioid receptors, indicating that CCK does not behave as a classical receptor antagonist via binding to opioid receptors . Han et al. found that the binding of CCK-8 to the CCK receptor reduces the binding affinity of μ-opioid receptor ligands, implying that receptor–receptor interaction between CCK and opioid systems may occur in an indirect manner . The molecular cloning of CCK receptor subtypes, one from the pancreas (type-1) and another from human brain (type-2), has confirmed the pharmacological classification of CCK receptors. The CCK2 receptor is predominantly localised in the CNS, and mainly mediates anxiety, panic attacks, pain and drug dependence [36–38]. The CCK1 receptor is present in discrete regions of the brain and has a low affinity for central CCK , and its function is poorly understood with only a few reports investigating the central role in food intake regulation . The use of highly selective receptor antagonists and antisense approaches has shown, at least in the rodent, that CCK2 receptors mediate the anti-opioid function of CCK [41–43]. The present study shows that co-pretreatment with LY-288,513 and morphine significantly inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells, but that co-pretreatment with L-364,718 displayed no effect. We verified that endogenous CCK played an anti-opioid role and potentiated the development of morphine dependence via the CCK2 receptor.
Together with our previous results, we found that exogenous CCK-8 pretreatment significantly inhibited morphine dependence in vitro and in vivo. These results show that the treatments of the CCK receptor agonist and antagonist demonstrated the same effect on morphine dependence. Moreover, CCK-8 treatment did not affect basal or forskolin-stimulated cAMP levels, suggesting that the effect of exogenous CCK-8 was not simply a direct action on cAMP. Several studies have reported that small doses of CCK inhibit the anti-nociceptive action of opioids, whereas large doses of CCK induce analgesia . We previously revealed that CCK-8 suppressed the binding affinity of the μ-opioid receptor in SH-SY5Y cells at concentrations of 1 nM, while it increased the expression of the endogenous opioid peptide from 0.1 to 1 μM . The dose–response curve of CCK-8 was inversely U-shaped, and CCK-8 displayed a dose-dependent, biphasic effect . We found that only high concentrations of CCK-8 were able to attenuate the cAMP overshoot in a dose-dependent manner. Thus, observations of exogenous CCK-8 may represent pharmacological effects, rather than physiological effects of endogenous CCK.
A previous study indicated that the CCK1 receptor was ineffective in the development of morphine dependence . Nevertheless, we found that a high dose of exogenous CCK-8 markedly attenuated the naloxone precipitated cAMP overshoot via the CCK1 receptor. We concluded that the CCK1 and CCK2 receptors play unique and distinct roles in physiology and pathophysiology. Moreover, data showing that CCK1 receptors mediate mnemonic effects, and that CCK2 receptors mediate amnestic effects, have been reported . Furthermore, CCK evokes [Ca2+
i signaling by the influx of extracellular calcium, likely through L-type calcium channels, and an antagonist for the CCK1 receptor blocked [Ca2+
i response to CCK-8 [49, 50]. CCK produces direct neuronal depolarisation via CCK1 receptors and inhibits GABAergic synaptic transmission , while CCK2 receptor activation augments long-term potentiation in hippocampal slices . CCK-8, the predominant central form of CCK, has a high affinity for the CCK2 receptor, but a low affinity for the CCK1 receptor. The CCK1 receptor is activated only in the presence of high CCK-8 levels, and exerts a different effect from the CCK2 receptor on morphine dependence. Due to the inhibitory function of LY-288,513 and CCK-8 on cAMP overshoot, the cumulative effect of LY-288,513 and exogenous CCK-8 on cAMP overshoot was not observed. The role of CCK2 receptor in the process of exogenous CCK-8 regulation on morphine dependence can not be ruled out.