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. 2024 Oct 15;85(5):704–712. doi: 10.15288/jsad.23-00377

Evidence for the Induction of Analgesic Cross-Tolerance Between Opioid and Apelin/APJ Systems in Male Rats

Elham Abbasloo a,,*, Saeed Esmaeili-Mahani b,,*, Firas Kobeissy c,,d, Theresa Currier Thomas e,,f
PMCID: PMC11533929  PMID: 38517751

Abstract

Objective:

Opioids are potent pain relievers for managing severe pain. However, their effectiveness is hindered by tolerance, which causes the need for higher doses and leads to adverse effects. In a previous study, we found that prolonged use of apelin, similar to opioids, results in a tolerance to its analgesic effects. It remains unclear whether there is a cross-tolerance between morphine and apelin, meaning if the analgesic effects of one can reduce the effectiveness of the other.

Method:

The tail-flick test was used to assess the nociceptive threshold. All experiments were carried out on 63 male Wistar rats, which received intrathecal apelin (3 μg/rat) or morphine (15 μg/rat) for 7 days. To determine cross-tolerance between the analgesic effect of morphine and apelin, the analgesic property of apelin or morphine was assessed in chronic morphine- or apelin-treated groups, respectively. To determine the role of apelin and opioid receptors signaling on the development of analgesic cross-tolerance, F13-A and naloxone, as apelin and opioid receptor antagonists, were injected simultaneously with morphine or apelin. At the end of the tests, the expression levels of apelin and μ-opioid receptors were evaluated by western blotting.

Results:

The data indicated that chronic apelin or morphine use produced tolerance to the antinociceptive effects of each other. F13-A and naloxone could inhibit the induction of such cross-tolerance. The molecular data showed that there was a significant downregulation of apelin receptors in chronic morphine-treated rats and vice versa.

Conclusions:

Chronic administration of apelin or morphine induces analgesic cross-tolerance that may, in part, be mediated through receptor interactions and downregulation. The demonstrated efficacy of F13-A in these experiments highlights its potential as a novel target for improving pain management through the inhibition of the apelin/APJ signaling pathway, meriting further investigation.

Opioids are valuable for managing acute moderate to severe pain caused by trauma, surgery, or cancer (Stein & Kopf, 2019). Opioids are also essential analgesics across various age groups (Thigpen et al., 2019). However, their use is limited because of the development of tolerance, which necessitates higher doses and leads to severe adverse effects like respiratory depression, nausea, vomiting, constipation, hyperalgesia, and dependence (Stein, 2020). Therefore, new strategies are required to overcome opioid analgesic tolerance and enhance the clinical utility and safety of these medications for pain treatment. Antinociceptive tolerance—associated with changes in opioid receptor signaling such as phosphorylation, desensitization, internalization, downregulation, or interaction with other receptors—contributes to heightened pain perception (Zhou et al., 2021).

In addition to developing tolerance, opioids can exhibit cross-tolerance with other analgesic medications. Crosstolerance refers to the phenomenon in which tolerance to one drug leads to tolerance to another drug. This commonly occurs between drugs with similar functions or effects, such as those targeting the same cell receptor or impacting the transmission of specific neurotransmitters. For instance, there is cross-tolerance in terms of analgesic effects between morphine and opioid peptides like beta-endorphin, ketocyclazocine, metenkephalin, leuenkephalin (Sivam & Ho, 1984), and morphine-3-glucuronide (a metabolite of morphine) (Blomqvist et al., 2020). Studies in mice have reported the development of analgesic cross-tolerance between morphine and nicotine (Zarrindast et al., 1999). Furthermore, cross-tolerance can also be observed between morphine and non-opioid drugs that possess analgesic properties. Chronic administration of morphine or orexin-A can induce crosstolerance to their analgesic effects (Azhdari-Zarmehri et al., 2018). Researchers have demonstrated the induction of cross-tolerance to cannabinoids in morphine-tolerant rhesus monkeys (Gerak et al., 2015).

Apelin, along with its receptors, the G-protein-coupled receptor APJ, is widely expressed in various peripheral tissues and the central nervous systems of both humans and animals. The apelin/APJ system plays a crucial role in numerous physiological and pathological processes. It has been implicated in depression, anxiety, memory, epilepsy, stroke, brain injury, and neuroprotection (Lv et al., 2020a). Lv and colleagues' study has highlighted the potent analgesic effect of the neuropeptide apelin (Lv et al., 2012).

Evidence supports the physiological significance of the interaction between APJ and opioid receptors in various physiological processes (Ilaghi et al., 2022). In our previous research, we reported that the neuropeptide apelin, similar to other analgesic drugs, can lead to analgesic tolerance as an undesired side effect. This tolerance is partly mediated through the activation of both apelin and opioid receptors (Abbasloo et al., 2016).

The objective of our current study was to investigate whether there is cross-tolerance between the opioidergic and apelinergic systems in terms of their analgesic effects. In Experiment I, we examined the analgesic effect of morphine in rats that had been chronically administered apelin. In Experiment II, we assessed whether analgesic doses of apelin also had analgesic effects in animals treated with chronic morphine. In Experiment III, we explored the role of apelin and morphine receptors using cross-antagonist administration.

Method

Animals

The study was conducted in compliance with the appropriate guidelines for investigating experimental pain in animals (Zimmermann, 1983), and it was approved by the Ethical Committee of Kerman University of Medical Sciences (IR.KMU.REC.1398.152). All experiments were carried out on male Wistar rats weighing between 200 g and 250 g, which were housed under standard controlled conditions (12-hour light/dark cycle, temperature of 22±1 °C) and provided free access to food and water.

Drugs

Apelin-13 and F-13-A were acquired from Phoenix Pharmaceuticals Inc. (Burlingame, CA). Morphine and naloxone were obtained from TMAD and Darou Pakhsh Pharmaceutical Company in Iran, respectively. The drugs were dissolved in artificial cerebrospinal fluid (ACSF: 148 mM NaCl, 3 mM KCl, 0.8 mM MgCl2, 1.4 mM CaCl2, 1.5 mM Na2HPO4, 0.2 mM NaH2PO4, 0.1 mg/ml of bovine serum albumin) (Jimenez Hamann et al., 2003). The administration of the drugs was conducted intrathecally using a microinjection syringe (Microliter #702, Hamilton Co., Reno, NV), with a total volume of 5 μl. Control animals received an equal volume (5 μl) of ACSF.

Antinociceptive test

We used the tail-flick test to evaluate acute sensitivity to pain, a method originally introduced by D'Amour and Smith in 1941 (D'Amour & Smith, 1941). The tail-flick latency for each rat was determined three times, and the mean was designated as the baseline latency before drug injection. The intensity of the beam was adjusted to produce a mean control reaction time between 4 and 6 seconds. The cutoff time was fixed at 10 seconds to avoid damage to the tail. By such modification, we were able to reveal potential subtle alterations that may occur in thermal nociception. Once the baseline latencies were established, the rats received intrathecal (IT) administration of the drugs, and the reaction latency was measured at the optimal time determined based on our previous study (45 minutes after injection) (Abbasloo et al., 2016). The tail-flick latencies were then converted to the percentage of antinociception using the following formula:

%Antinociception (%  MPE)=(reaction time of test-basal reaction time)/cutoff time-basal reaction time)

Intrathecal catheter implantation for drug delivery

Animals were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) intraperitoneally. An IT catheter (PE-10) was implanted in each rat according to a previously published method (Yaksh & Rudy, 1976). Animals that exhibited neurological deficits (e.g., paralysis) after the catheter implantation or during drug delivery were excluded from the experiments. All experiments were performed after a 7-day recovery period. At the end of the examination, the correct position of the cannula was confirmed by administering 15 μl of 2% lidocaine, which temporarily paralyzed the animals' hind limbs. In the molecular study, the lumbar region was immediately excised to prevent interference from lidocaine during blotting analysis, and the correct positioning of the IT cannula was macroscopically confirmed through visual inspection (Supplemental Material 1). (Supplemental material appears as an online-only addendum to this article on the journal's website.)

Experimental design

First, to evaluate the possible antinociceptive cross-tolerance, the effects of an analgesic dose of apelin-13 (3 μg/rat, IT) were evaluated on 7 days chronic ACSF (as a vehicle)- or morphine-treated rats (on the 8th day). Second, the analgesic level of an effective dose of morphine (15 μg/rat, IT) was assessed in chronically ACSF- or apelin-administrated rats (3 μg/rat, IT, for 7 days) on the 8th day. The antinociceptive effect was assessed on days 1, 3, 5, and 8 of the experiment. The nociceptive threshold was assessed both before and 45 minutes after the drug administration. Finally, animals were sacrificed 45 minutes after a final injection. Third, to investigate the contribution of APJ or opioid receptor signaling to the development of analgesic tolerance, antagonists targeting APJ (F13-A; 6 μg/rat) or opioids (naloxone; 6 μg/rat) were administered 20 minutes before the administration of drugs on the days when nociceptive testing was conducted (Abbasloo et al., 2016).

Tissue extraction and preparation

The rats were anesthetized using carbon dioxide and then decapitated. The spinal column was cut through the pelvic girdle. To access the sacral vertebral canal, a 16-gauge needle was inserted, and ice-cold saline was used for hydraulic extrusion of the spinal cord. The dorsal half of the lumbar cord was promptly dissected from the spinal cord and placed on ice. The tissue samples were weighed, immediately frozen in liquid nitrogen, and stored at -70 °C until they were ready for analysis.

Western blot analysis

To assess the modifications in the apelinergic and opioid systems, the density of APJ and μ-opioid receptors was measured using the western blot test. The spinal tissues from rats that received chronic IT injections of saline or drugs for 7 days were dissected. The tissues were homogenized in 700 μl ice-cold RIPA (radioimmunoprecipitation assay) lysis buffer (Sigma-Aldrich, Inc., St. Louis, MO; R0278), 1 mM protease inhibitors (Sigma-Aldrich, Inc., St. Louis, MO; P2714-IBTL), and 1 mM sodium orthovanadate using a tissue homogenizer (Hielscher UP200; Hielscher Ultrasonics, Teltow, Germany). The homogenate was centrifuged at 17,000×g for 15 minutes at 4 °C13,41,42. The supernatant was collected, and its protein concentrations were determined using the Bradford method (Bio-Rad Laboratories, München, Germany). On a 10% SDS-PAGE gel, equal amounts of protein (40 g) were separated electrophoretically and transferred to nitrocellulose membranes (Hybond ECL, GE HealthCare Bio-Sciences Corp., Piscataway, NJ). The membranes were blocked for 2 hours with 5% nonfat milk, then incubated overnight at 4°C with the following antibodies: apelin receptor (APLR: sc33823, 1:1000) or opioid mu1 receptor (MOR1(Q-18): sc27072, 1:1000). The membranes were incubated for 2 hours at room temperature with the secondary antibodies: goat anti-rabbit (sc2004, 1:10000) and mouse anti-goat (sc2354, 1:10000) followed by three times washing with tris-buffered saline with Tween (TBST; 15 minutes each). All antibodies were diluted in a blocking buffer containing 5% nonfat dried milk and TBST. Control of loading was performed using β-actin immunoblotting (antibody from Cell Signaling Technology Inc., Danvers, MA; 1:200). Antibody-antigen complex was visualized via the enhanced chemiluminescence (ECL) method and exposed to Lumi-Film Chemiluminescent Detection Film (Roche Germany). The density of the bands was calculated using Labworks software (Houshmandi et al., 2016; Najafipour et al., 2015).

Statistical analysis

The results are presented as mean ± SEM. To determine the differences in %MPE (antinociception) among groups throughout the study period, a two- or one-way analysis of variance was performed, followed by the Newman-Keuls test. A significance level of p < .05 was used to determine statistical significance. The APJ/β-actin or MOR1/β-actin band density ratios were calculated for each sample for the blotting values. The average ratios between the control and apelin groups were compared using the Student's t test. A significance level of p < .05 was considered statistically significant.

Results

Measurement of the development of tolerance to the analgesic effect of morphine and apelin-13

First, to determine the possible antinociceptive cross-tolerance between morphine and apelin, the occurrence of tolerance to the IT analgesic dose of morphine (15 μg/rat) or apelin-13 (3 μg/rat) was examined following 8 days of injection. The maximum analgesic effect of morphine was elicited on days 1 and 3, which was dramatically decreased on the 5th and 8th day of the experiment. There was no significant difference between morphine and vehicle + morphine groups (Figure 1A). In addition, chronic apelin-13 treatment resulted in a significant reduction of pain response values during the time course of the experiment. There was no significant difference between the apelin and vehicle + apelin groups (Figure 1B). These data indicate the development of tolerance to the analgesic effect of both apelin and morphine after chronic administration.

Figure 1.

Figure 1.

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The effects of intrathecal (IT) administration of F13-A (apelin antagonist) (A) and naloxone (opioid antagonist [Nalox]) (B) on the development of morphine (Mor; 15 μg/rat, IT) or apelin-13 (3 μg/rat, IT) analgesic tolerance, respectively. Each value in the graph represents the mean ± SEM.

++p < .01 and +++p < .001 = significantly different from the first day of the experiment; **p < .01 and ***p < .001 = as compared with group that received apelin (n = 7).

Second, to answer the question of whether apelin or opioid receptors have a role in the occurrence of each other's analgesic tolerance, antagonists of their receptors, F13-A and naloxone, were injected 20 minutes before the morphine or apelin-13, respectively. The data showed that a high level of the possible maximum effect (%MPE) appeared in the F13-A + Mor group during the days of the experiment in comparison with the chronic morphine-treated group, which had no significant analgesic effects (Figure 1A). Furthermore, naloxone (Nalox) could suppress apelin antinociceptive tolerance efficiently so that pain response values in the Nalox + Apelin group significantly increased as compared with the apelin-treated group during the experiment course (Figure 1B).

Surprisingly, use of cross-antagonists could significantly inhibit tolerance induction of drugs so that naloxone pretreatment prevented apelin tolerance (Figure 1B), and F13-A was able to block morphine analgesic tolerance (Figure 1A).

Evaluation of apelinergic and opioidergic system cross-tolerance

To examine the possible cross-talk between the apelinergic and the opioidergic system, a single analgesic dose of apelin-13 was intrathecally injected into the chronic saline or morphine-treated rats (7-day repeating injection). Our data showed that although apelin-13 analgesic effects were shown in the animals that had only received vehicle or F13-A + Morphine, it was not able to produce the analgesic effect in the chronic morphine-treated group. Because of the cross between the apelinergic and opioidergic systems, the tolerance to the analgesic effect of morphine could also prevent the appearance of the analgesic effect of apelin (Figure 2). Besides, no analgesic effect was observed after acute injection of an analgesic dose of morphine in rats that chronically received apelin (7-day repeating injection). However, the analgesic properties of morphine are properly expressed in chronic saline- or apelin-plus-naloxone–treated groups (Figure 3). It can be reasonably assumed that there is an analgesic cross-tolerance between opioid and apelin/APJ system (Figure 2).

Figure 2.

Figure 2.

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The analgesic effect of apelin-13 (3 μg/rat, IT) in chronic vehicle-, morphine-, and F13-A + Morphine-treated groups (7 days). Each value in the graph represents the mean ± SEM.

***p < .001 = as compared with the vehicle-treated group; ###p < .001 = significantly different versus morphine-treated group (n = 7).

Figure 3.

Figure 3.

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The analgesic effect of morphine (15 μg/rat, i.t) in chronic vehicle-, apelin-, and Naloxone+Apelin-treated groups (7 days). Each value in the graph represents the mean ± SEM. Nalox = naloxone.

***p < .001 = as compared with the vehicle-treated group; ##p < .01 significantly different versus the apelin-treated group (n = 7).

Effect of chronic administration of morphine on the level of APJ protein

Chronic morphine administration significantly downregulated APJ expression by 60% in the spinal cords of animals as compared with the chronic vehicle-treated group (p < .001) (Figure 4).

Figure 4.

Figure 4.

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Shows the impact of administering morphine chronically (15 mg/kg for 7 days) on the expression of the apelin receptor, APJ, in the dorsal portion of the rat's lumbar spinal cord, compared with the vehicle group. The predicted sizes of the APJ protein bands were 65 kDa. Beta-actin was used as an internal control. Each data point on the graph represents the mean ± SEM band density ratio for each group (n = 6). Statistical analysis revealed a significant difference (***p < .001) between the chronic morphine-treated group (chronic morphine) and the chronic vehicle-treated group (chronic vehicle).

Effect of chronic administration of apelin on the level of MOR protein

The level of the μ-opioid receptor (MOR) was significantly reduced by 76% in the chronic apelin-treated group as compared with the chronic vehicle (ACSF)-treated group (p < .001) (Figure 5).

Figure 5.

Figure 5.

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Illustrates the decrease in the expression of the μ-opioid receptor (MOR) in the dorsal portion of the lumbar spinal cord following chronic administration of apelin (3 μg/rats for 7 days), compared with the vehicle group. Beta-actin was used as an internal control. Each data point on the graph represents the mean ± SEM band density ratio for each group (n = 6). Statistical analysis revealed a significant difference (***p < .05) between the chronic apelin-13-treated group (chronic apelin-13) and the chronic vehicle-treated group (chronic vehicle).

Discussion

The present study demonstrated that the acute IT analgesic doses of apelin-13 administration could not overwhelm the development of morphine analgesic tolerance on the 8th day in chronic morphine–treated rats for 7 days; and so forth, there was an interruption with the chronic administration of apelin (7 days) and the acute analgesic dose of morphine on day 8. Furthermore, apelin and morphine anti-nociceptive analgesic tolerance were suppressed by antagonists F13-A and naloxone, respectively. Downregulation of receptors APJ and μ-opioid occurred by the effect of chronic morphine and apelin, respectively.

The presence of apelin, its receptor, APJ, and MOR expression within the descending pain modulatory pathway, which includes the ventrolateral periaqueductal gray, rostral ventromedial medulla, amygdala, hypothalamus, dorsal raphe nucleus, and the dorsal horn of the spinal cord had been shown (Hosoya et al., 2000; Lueptow et al., 2018; Reaux et al., 2001). This pathway plays a crucial role in modulating analgesia induced by endogenous and exogenous opioids. In addition, cellular and molecular changes following repeated opioids occur and lead to the development of tolerance (Lueptow et al., 2018).

The expression of MOR is known to play a role in both opioid-induced antinociception and the development of opioid tolerance (Bobeck et al., 2009; Fairbanks & Wilcox, 1997; Fang et al., 1989; Morgan et al., 2006; Tortorici et al., 2001). In rats, it has been observed that morphine tolerance is primarily mediated by central mechanisms involving the brain and spinal cord rather than peripheral opioid receptors (Blomqvist et al., 2022).

Several studies have investigated the APJ/opioid system in the regulation of pain transmission. For example, Lv et al. conducted a study in which they observed that the administration of (pyr) apelin-13 led to an increase in the expression of the prodynorphin gene, which is responsible for producing endogenous opioid peptides. They also observed an increase in the levels of mRNA and protein of dynorphin and kappa opioid receptor (KOR) in the prefrontal cortex of mice. The researchers further discovered that the antinociceptive effects of (pyr) apelin-13 were blocked by opioid receptor antagonists such as naloxone and a specific KOR antagonist called nor-binaltorphimine (nor-BNI; Lv et al., 2020b). They also illustrated that supraspinal co-administration of apelin and morphine enhanced their analgesic effect, with nalox-one effectively blocking the potentiated analgesic response (Lv et al., 2012). Researchers also found that the antinociceptive effect of apelin-13 was significantly diminished by β-funaltrexamine hydrochloride (β-FNA), the μ-opioid receptor antagonist, indicating the involvement of both the APJ receptor and μ-opioid receptor in this mechanism (Lv et al., 2012; Xu et al., 2009).

In line with those discoveries, Soleimani and colleagues found that injecting naloxone before administering apelin-13 led to a substantial reduction in apelin-induced analgesia in pulpal pain, as a common orofacial health issue, implying that an opioid receptor plays a role in apelin-13's inhibitory effects on periaqueductal grey matter (Soleimani et al., 2023). Befort et al.'s (2008) study reported decreased apelin transcript levels in the lateral hypothalamus of morphine-dependent mice, providing further evidence of pathway interactions.

Together, these experiments reveal a novel pharmacological cross-talk between apelin and morphine signaling, significantly affecting the development of antinociceptive tolerance and the modulation of descending pain pathways. The cause-and-effect relationship demonstrated in our study strongly suggests the potential co-expression of MOR and APJ receptors on neurons within pain-related pathways, encouraging further speculation and investigation into this complex interplay.

Regarding the interaction of apelinergic and opioidergic systems, our previous study showed that chronic IT administration of apelin-13 induces antinociceptive tolerance in rats, an effect inhibited by naloxone and F13-A. Moreover, chronic apelin-13 administration caused the downregulation of APJ, as a Class A (rhodopsin-like) G-protein coupled receptor (GPCR), which had occurred in the spinal cord of animals (Abbasloo et al., 2016). The interplay between GPCRs remains a subject of ongoing debate and discussion in the field of pharmacology. The mechanisms by which these receptors can mutually influence or regulate each other are still not fully understood and continue to raise questions (Fasciani et al., 2022). The performance of these GPCRs is impacted by the diversity of their agonists. Studies have shown that the apelin receptors, when combined with opioid receptors, cause signal transduction and cell proliferation in response to agonists of those receptors (Li et al., 2012). Considering such an effect, it is possible that in our study, both morphine and apelin are activated by each other's receptors, which is revealed by the absence of opioid signaling in apelin treatment or vice versa (p < .001) (Figures 4 and 5). Long-term use of some analgesic drugs can lead to a reduction of the receptors population, which is a known factor in developing tolerance (He et al., 2002; Sim-Selley et al., 2006). However, how chronic morphine can decrease the apelin receptor and vice versa may be related to similar mechanisms affecting the activation of GPCRs by these two substances. For example, one of the probable events is the apelinergic and opioidergic cross-talk in cAMP-dependent protein kinase A (PKA)-induced receptor phosphorylation (Bernstein & Welch, 1998; Ilaghi et al., 2022).

Morphine and other opioids produce pain relief by activating μ-opioid receptors, which trigger inhibitory G protein pathway and ion channel interactions. These pathways and interactions decrease cellular activity and cause hyperpolarization. Gi proteins primarily inhibit adenylyl cyclase activity, which decreases the production of cAMP from ATP and ultimately leads to the inhibition of PKA, a cAMP-dependent protein kinase. It is well known that chronic morphine administration results in a decrease in the PKA-induced phosphorylation of the mu-receptor, causing a decline in the effectiveness of the morphine receptor (Bernstein & Welch, 1998) where it is shown that antisense to PKA can completely block tolerance induced by morphine (Shen et al., 2000). In this regard, it is shown that the coupling of APJ to Gi/o blocks adenylyl cyclase, resulting in cAMP production and a consequent reduction in PKA-mediated phosphorylation of cytosolic and membrane proteins (Chapman et al., 2014; Ilaghi et al., 2022; Masri et al., 2006). Therefore, downregulation of APJ and MOR receptors observed in our study may be mediated through similar downstream signaling pathways involving these receptors.

Another common signaling mechanism between MOR and APJ that could lead them to internalization is the β-arrestin dependent pathway. Both MOR and APJ receptors have been reported to be internalized upon stimulation and activation by their agonists, mediated by β-arrestin (Bohn et al., 1999, 2000; Lee et al., 2010; Zhou et al., 2003), which unfortunately limits the clinical efficacy of apelin or morphine (Read et al., 2016; van Gastel et al., 2018).

The role of the serotonergic system in the cross-talk between apelinergic and opioidergic systems is another mechanism that warrants consideration. Evidence shows that zimelidine, a serotonin reuptake inhibitor, attenuates the development of morphine tolerance in rats (Ozdemir et al., 2012). Turtay and colleagues indicated that increased serotonergic activity might be responsible for the apelin-13 analgesic effect so that hindering the five-hydroxytryptamine-three (5-HT3) receptor by ondansetron reduced apelin-13 antinociception (Turtay et al., 2015).

In our study groups, it is also possible that the antagonists can influence the other's GPCRs (F13-A + Mor or Nalox + Apelin). According to Barki-Harrington et al. (2003), one type of receptor antagonist effectively hinders the trafficking and signaling of two distinct receptors at the same time. In line with this, Yeganeh et al. (2017) demonstrated that the effects of apelin on the cardiovascular system are influenced by naloxone or co-administration of naloxone and F13-A. This is another clear example indicating the involvement of opioidergic system antagonists in apelinergic system function.

Several reports demonstrate that antagonists are capable of acting not only on receptors but also on post-receptor signaling cascades. For example, naloxone and F13-A can inhibit the phosphorylation of MOR by G protein-coupled receptor kinases (GRKs), recruitment of β-arrestin, and internalization of their own receptors, thus affecting MOR and APJ function, respectively (Lowe et al., 2015; Zhang et al., 2023). Since the apelin receptor itself is a target for GRK-mediated phosphorylation following receptor activation, leading to desensitization and internalization, as observed in our previous study (Lee, 2006), it is probable that in our current study, naloxone has prevented the phosphorylation of the apelin receptor and the subsequent desensitization and internalization of the APJ receptor in the Nalox + Apelin group by inhibiting GRKs, β-arrestin, or other proteins. However, the level of changes in these proteins and kinases has not yet been measured.

Therefore, it is plausible to speculate that the respective inhibition of tolerance to the analgesic effects of morphine and apelin by F13-A and naloxone may involve one or several of the mechanisms previously discussed, in addition to potentially complex interactions within downstream signaling pathways. However, the distinct mechanisms underlying the observed outcomes warrant further investigation.

Conclusion

In this study, it was found that the effectiveness of apelin-13's pain relief was hindered by chronic morphine treatment. In addition, the antinociceptive tolerance to apelin-13 disrupted the effectiveness of morphine antinociception in rats. Furthermore, using an antagonist for both morphine and apelin-13 eliminated the antinociceptive tolerance induced by apelin-13 and morphine, respectively. This suggests that one possible mechanism contributing to the tolerance to the analgesic effects of morphine or apelin-13 may involve interactions through each other's receptors.

Footnotes

This study was supported by Physiology Research Center and Medical Faculty of Kerman University of Medical Sciences Grant Number 97000868 to Elham Abbasloo. Salary for Theresa Currier Thomas is supported, in part, by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number R01NS100793.

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