The opioid system is a key pain-relief mechanism that functions by inhibiting pain signals. It involves three receptor types: µ, $\delta$, and $\kappa$. When these receptors are activated, they reduce neuronal excitability, block the potential of action transmission, and prevent the release of excitatory neurotransmitters, thereby decreasing pain sensation [1, 2, 3]. In addition to mechanisms in the brain and spinal cord, opioids can also offer pain relief at the peripheral level [4, 5, 6]. Peripheral opioid receptors are located on sensory neurons and interact with both natural and synthetic opioid ligands [7, 8].

Although opioid analgesics are some of the most powerful medications for treating severe pain, their long-term use can lead to analgesic tolerance, addictive behaviors, and other adverse effects such as respiratory depression and constipation [9, 10, 11]. Therefore, it is essential to find alternative drugs that provide effective pain relief with fewer side effects. A significant number of pre- and clinical investigations highlight the role of the dopaminergic system in modulating nociception. Dysfunction of dopaminergic neurotransmission may be associated with heightened pain sensitivity. Positron emission tomography examinations used to evaluate patients with fibromyalgia syndrome revealed decreased dopamine (DA) synthesis and release in presynaptic neurons [12, 13]. Additionally, clinical research indicates that administering levodopa, the precursor of DA, relieves pain in individuals with Parkinson’s disease [14, 15]. In preclinical trials, injecting levodopa into the intrathecal space and specific regions of the central nervous system produced an antinociceptive effect [16, 17]. Peripherally, intraplantar administration of DA reversed Prostaglandin E2-induced hyperalgesia in mice [18].

Recent research indicates that higher endogenous dopamine levels improve the cannabinoid agonist 2-AG (2-Arachidonoylglycerol)-induced peripheral antinociception. It is also important to understand that the interaction between the dopaminergic and endocannabinoid systems plays a key role in the inhibitory regulation of nociception at the peripheral level [19]. Similar to the endocannabinoid system, the opioid system appears to have an additive or synergistic effect with the dopaminergic system in the relief of pain. Activation of µ-opioid receptors in the periaqueductal gray (PAG) depends on the activation of dopaminergic receptors [20]. These receptors are widely found in the ventrolateral PAG and contribute to antinociception caused by systemically administered morphine [21, 22].

This study explores whether DA-induced peripheral antinociception involves engaging the opioid system and identifies which opioid agonists and receptors may trigger it. It also investigates if the dopaminergic system plays a role in opioid-mediated peripheral antinociception. The results indicate a possible approach to improve the effectiveness of lower opioid doses in pain management, reducing side effects, while also clarifying the peripheral mechanisms of dopaminergic antinociception.

This study used a pharmacological approach to clarify how DA and opioids promote peripheral antinociception by examining how these two systems interact in suppressing pain at the peripheral level. The increase in nociceptive response was caused by PGE2, which sensitizes primary afferent neurons and leads to hyperalgesia to mechanical stimulation [27]. Previous studies have shown that DA blocks the effects of PGE2 in this model by activating D2 family receptors [18]. In addition to the classic roles assigned to the dopaminergic system [28], scientific literature increasingly emphasizes DA’s role in pain modulation [5, 29, 30]. Meanwhile, opioid drugs are well-established in medicine for treating severe pain. Although highly effective, these drugs are linked to analgesic tolerance, addiction, and physical dependence [11]. Therefore, developing new drugs with good analgesic effects and fewer side effects, or drugs that can enhance existing analgesics’ effectiveness while reducing adverse effects, is a promising strategy.

Although dopaminergic analgesia is well documented, its mechanisms are not fully understood. Therefore, our study aimed to examine the influence of the opioid system on DA-mediated antinociception at the peripheral level. The interaction between the opioid and dopaminergic systems in producing analgesia in the central nervous system has already been described, as these systems are closely connected anatomically [31]. The periaqueductal gray (PAG) plays a key role in nociception, often linked to opioid activity [32]. Dopaminergic neurons in this region may be involved in opioid analgesia [33]. Additionally, antinociception caused by activating D2 receptors in the PAG requires the activation of opioid receptors [20].

At the peripheral level, we observed that antinociception mediated by DA administration was reversed by antagonism of $\delta$ and $\kappa$ opioid receptors, but not by blocking the µ receptor, demonstrating that dopaminergic antinociception does not depend on activation of this receptor. In a similar study, peripheral antinociception induced by the antipsychotic aripiprazole, a partial agonist of dopamine D2 receptors, was reversed by naloxone and naltrindole, non-selective and selective $\delta$-opioid receptor antagonists, respectively, but not by nor-BNI, a $\kappa$-opioid antagonist [34]. Consistent with our findings, blocking µ-opioid receptors did not affect aripiprazole’s antinociceptive effects. It is important to note that most opioids used clinically are µ-opioid receptor agonists, and the main adverse effects of this drug class are related to activating this receptor [11]. Therefore, our results indicate that dopaminergic antinociception involves activating the opioid system, but without the adverse effects linked to µ-opioid receptor activation.

Another important finding from our study was that intermediate antinociception caused by DA was enhanced by bestatin, a drug that prevents the breakdown of endogenous opioids. Since different opioid peptides prefer specific receptors, we can infer from our results that, in our model, the opioids involved in dopaminergic antinociception are likely enkephalins and dynorphins, because these peptides can activate $\delta$ and $\kappa$-opioid receptors [11, 35], which are involved in DA antinociception. By exclusion, since µ-type receptors were not involved, $\beta$-endorphin does not seem to be related to this effect.

In the next part of our study, we explored how endogenous DA influences opioid antinociception. The opioid system operates through three receptor types: µ, $\delta$, and $\kappa$, which can produce analgesia not only via supraspinal and spinal mechanisms but also at the peripheral level [3, 7, 8]. To evaluate the opioid system, we used the agonists DAMGO, SNC 80, and bremazocine. Previous research has assessed the peripheral antinociceptive effects of these agonists in hyperalgesia models induced by PGE2 or carrageenan [23, 36, 37], demonstrating that their antinociception depends on activating the noradrenergic system, along with the L-arginine/NO/cGMP pathway. Our findings showed that submaximal doses of these agonists caused moderate antinociception, which was enhanced by increasing endogenous DA availability (by inhibiting its reuptake with GBR 12783). Additionally, dopamine D2 and D3 receptor antagonists, remoxipride and U 99194, reversed the antinociceptive effects of the highest doses of these opioids. Consistent with our results, other studies suggest that D2 receptors interact with opioid receptors and can amplify the analgesic effects of µ-opioid agonists [38]. Moreover, administration of quinpirole, a nonselective D2 family receptor agonist, increased the antinociceptive effect of DAMGO in models of inflammatory and neuropathic pain [39]. Furthermore, systemic delivery of R-VK4-40, a selective D3 receptor antagonist, reduced oxycodone’s tolerance and dependence issues (with oxycodone being the most commonly prescribed opioid of abuse) without diminishing its pain-relieving effects [40].

The results of this study, along with the literature review, highlight the critical role of the dopaminergic system in pain modulation, both centrally and peripherally. Additionally, considering the additive and/or synergistic interactions between D2 receptors and opioid receptors, D2 agonists may serve as adjuvants to enhance analgesic effects and decrease the adverse effects of opioids. Overall, these findings suggest that targeting the dopaminergic system could offer a fresh approach for more effective management of pain. This study has some limitations. First, only the mechanical withdrawal test was employed, and additional methods such as the thermal test were not included. Second, only male animals were used in the present experiments. Potential sex differences, as well as the inclusion of additional behavioral tests, will be considered in future studies.

This study showed how the body’s own pain relief systems work together to influence the blocking of pain signals at the peripheral site. Our data suggest that the opioid system is involved in the peripheral pain relief caused by DA, and that the dopaminergic system contributes to the analgesia produced by opioids. Understanding these mechanisms provides a promising path for creating new treatments that could be more effective than traditional options.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

BFGQ: Investigation, Methodology, Writing – original draft; WCPB: Investigation, Methodology; ALI: Investigation, Methodology; FCSF: Investigation, Methodology; TRLR: Investigation, Formal analysis, Resources; IDGD: Investigation, Resources, Formal analysis, Conceptualization, Writing review & editing, Supervision. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The Animal Experimentation Ethics Committee at the Federal University of Minas Gerais approved this study under protocol numbers 196/2018 and 129/2021, and followed ARRIVE 2.0 guidelines. Use Committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Not applicable.

This work was supported by grants from Comissão de Aperfeiçoamento de Pessoal do Nível Superior (CAPES), Conselho Nacional de Pesquisa (CNPq), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).

The authors declare no conflict of interest.