**4. Neuroanatomical Distribution of the** µ**-Opioid and Angiotensin Receptors in Areas Related to Pain**

#### *4.1. The µ-Opioid Receptor*

The neuroanatomical distribution of the MOR is now well-established by immunohistochemistry, autoradiography, in situ hybridization, and fluorescence techniques [109–113]. Accordingly, MORs can be found at supraspinal, spinal, and peripheral levels [114–116]. MORs are enriched in the descending pain modulatory pathway, involving the periaqueductal gray (PAG) matter, rostral ventromedial medulla (RVM), locus coeruleus (LC), and the dorsal horn of the spinal cord [115,117]. In addition, they can be found in brain regions that are strongly related to pain perception and integration, such as the cerebral cortex, thalamus, striatum, amygdala, hippocampus, nucleus accumbens, and the ventral tegmental area (VTA) [115,117]. Within the dorsal horn of the spinal cord, MORs are densely localized in the lamina I-II superficial layers on interneurons and projection neurons [115,118]. The dorsal root ganglia are also a significant locus for MORs attributed to pain [115,119]. MORs can also be found on C- and A-fibers and near primary afferent nociceptors [117].

#### *4.2. Angiotensin Receptors and Endogenous Angiotensin Ligands*

The components of neuronal angiotensin system are found in anatomical regions hosting different key points in pain pathways, including the dorsal horn of the spinal cord, dorsal root ganglia (DRG and identical structures, such as the spinal trigeminal tract and trigeminal ganglion), or peripheral nerves. Angiotensinogen mRNA can be found ubiquitously in the mammalian brain [120], spinal cord [99], and almost all cells in the DRG [42,43]. The angiotensinogen level in the CNS is not affected by STZ treatmentinduced diabetes; however, it is elevated following peripheral inflammation [22,121].

There are contradictory data in the literature about the localization of neuronal renin, the primary activating enzyme of the renin–angiotensin system [42,43,100]. AngI mRNA is present in the human DRG and trigeminal ganglion (TG) [42,43], whereas its protein form was described in rat DRG [121]. AngII was found in rat and human DRG [18,37,43,106,107], TG [42], neurons, satellite cells, and CD3+ T-cells [106]. The colocalization of AngII alongside components involved in pain sensation, such as substance P (SP) and vanilloid transient receptor potential channels, was reported as well on small and medium neurons [18,37,42,43,106]. In rodent, AngII can be found ubiquitously in the spinal cord; its level was highest in the superficial laminae of the dorsal horn, which could suggest a possible role of AngII in nociception [41,99]. Furthermore, AngII levels have been reported to be increased following mono- or polyneuropathic pain evoked by CCI [106] or

STZ, respectively [41,99]. Furthermore, this change in AngII levels was also seen in pain conditions induced by intraplantar formalin injection [41,99] or in bone cancer pain [105].

With respect to the receptors, several studies have reported on the distribution of AT1R on key points related to nociceptive transmission both in mice [39,40] and rats [31,36,43,122–129]. These areas include sciatic nerve [31,127,130], DRG [36,43,123, 125,127–131], and spinal cord [22,39,40,129,132]. Moreover, it can be found in different brain regions, such as the spinal trigeminal tract and raphe nuclei [122]. These data also provide strong evidence on a large amount of AT1aR, and smaller amounts of AT1bR mRNA [43,127,129,132], and the receptor protein [31,36,39,40,123,125,127–129] was also shown in the mentioned regions. In the DRG, the receptor protein was found on satellite cells and neurons of all sizes with a greater extent on smaller ones [36,110,128,130]. In the spinal cord, similarly to AngII, AT1R level was the highest in the superficial dorsal horn [39,128].

In contrast to AT1R, AT2R localization and the above-mentioned function in relation to nociception are controversial subjects. At present, little data are available on the ganglional or sensory neural expression of AT2R as many of the currently commercially available AT2R antibodies used for immunohistochemistry seem to show inappropriate specificity [131]. Therefore, it is important to evaluate the results of studies using antibodies with appropriate criticism—especially in case of earlier works.

Early autoradiographic studies found significant inhibition of AngII binding by AT1R but not by AT2R antagonists on the sciatic nerve, spinal cord, and (upper cervical) sensory ganglion [31,128]. AT2 mRNA was found in the DRG and sciatic nerve of rats [43,127]. The receptor protein was found by many research groups on neurons (IB4+ [132]), satellite cells [106,127], and CD3+ T-cells [106] and in the rat DRG as well [101,106,107,110,125,130]. Indeed, in a few studies, the AT2 antibody specificity was verified on AT2R KO mice, further reinforcing the results [37,123]. On the other hand, Shepherd and colleagues were not able to find AT2R mRNA or protein in the DRG of mice or humans [80]. In their study using Agtr2GFP reporter mice, the AT2 positivity in the sciatic nerve was detectable and increased after SNI but because of macrophage infiltration instead of neural expression. Taken together, Shepherd's group claims that AT2R is not expressed on sensory neurons involved in nociception [107]. In contrast, Benitez et al., found AT2 immunoreactivity in rat DRG mostly on non-peptidergic (IB4+) C- and Aδ-fibers showing high colocalization to AT1 yet using an antibody with specificity verified on AT2R KO mice. In their study, the level of AT2 increased in an inflammatory state following treatment with CFA [123]. It is important to mention that mice were used in the study conducted by Shepherd in contrast to rats used by Benitez. A very recent review published in 2021 by Danigo et al., provides detail on how to solve this contradiction and lists species differences as well as the possible gene duplication of AT2R (similar to AT1R) in mice which could cause a lack of signal in the reporter mice [24].

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase enzyme regulating the local levels of AngII and Ang 1-7 (metabolizes AngII to Ang 1-7). Its mRNA and protein were found in human DRG samples, colocalizing with nociceptor neuronal markers [133]. It is also expressed in mouse spinal cord, where it is localized on neurons and microglia but not on astrocytes [93]. Finally, MASR expression was shown in rat DRG [91,92], PAG [134] and in mouse spinal cord [93]. However, to the best of our knowledge, the localization of the Ang (1-7) peptide has not been fully described. The neuroanatomical localization of key elements of the RAS and µ-opioid receptors have been summarized in Table 1.

**Table 1.** Neuroanatomical distribution of ligands and receptors in the renin–angiotensin system with importance in pain transmission and the µ-opioid receptor (MOR).



**Table 1.** *Cont.*


**Table 1.** *Cont.*

Abbreviations: p: peptide/protein; r: mRNA; IHC: immunohistochemistry; autorad: autoradiography; PCR: polymerase chain reaction; ISH: in situ hybridization; RIA: radioimmunoassay; WB: Western blot; DM: diabetes mellitus; DH: dorsal horn; VH: ventral horn; SP: substance P; CGRP: calcitonin gene-related peptide; NF200: neurofilament protein 200; TRPV1: transient receptor potential cation channel subfamily V member 1; IB4: isolectin B4; NK1: neurokinin 1; NMDA: N-methyl D-aspartate. A hyphen indicates no assessment by the indicated studies.

#### **5. Possible Link between MOR Analgesics and Ligands Affecting Angiotensin Receptors in Relation to Pain**

Rather than dose escalation of MORs analgesics which is associated with an increase in the incidence of side effects, augmenting MORs-mediated analgesia would be an important strategy in the management of neuropathic pain. In regard to the interaction between opioid and angiotensin systems, to the best of our knowledge, the first study published in 1983 by Haulica et al., described that AngII produced naloxone reversible analgesia following icv. administration in rat tail-flick test; therefore, these results showed the implication of endogenous opioid system in the effect of AngII [68]. In a later study, the same research group also reported that naloxone or saralasin attenuates stress analgesia in rats [70]. Based on another study by Han et al., icv. administered AngII was able to reverse the antinociceptive action of sc. morphine [76]. Similarly, Yamada et al., showed

that AT2R activation decreases the analgesic effect of morphine [79]. On the other hand, a previous study by Mojaverian et al., reported that orally administered ACE inhibitor enalapril failed to influence morphine analgesia [143]. Recently, Taskiran and Avci reported that systemic captopril alone was able to increase tail-flick and hot plate latency, and it also increased the analgesic effect of systemic morphine. Furthermore, the co-treatment with captopril reduced morphine-induced analgesic tolerance development. Captopril also reduced the inflammatory and endoplasmatic stress response in the DRG caused by acute or chronic morphine treatment [32]. It is important to note however, that ACE inhibition could result in a diverse molecular effect, partly independent from RAS—such as the inhibition of the catabolism of endogenous opioids and peptide mediators, among others. Next, connection between Ang (1-7), MASRs and the opioid system is unclear as to the best of our knowledge there are little data available at present. In this respect, Costa et al., reported that endogenous opioids do not play a role in the analgesic action of Ang (1-7) as it was not sensitive to naloxone [95]. This does not necessarily mean that there are no possible interactions between the two systems. Indeed, there are several reports, indicating opioids are capable of changing physiological parameters, most notably changes in the blood pressure [144–148] or drinking-response to AngII [149–151]. However, regarding the relationship between RAS and the opioid system only a small proportion of these address the role of interactions in analgesia. We have summarized the outcomes of relevant studies in Table 2.


**Table 2.** Reported connections between the opioid and renin–angiotensin systems in relation to pain.

Abbreviations: ELISA: enzyme-linked immunosorbent assay.

With respect to neuropathic pain, Khan and coworkers showed that allodynia caused by CCI of the sciatic nerve was attenuated by a systemic single dose of EMA300, a small molecule AT2R antagonist [106]. In this study, the authors also proved that the nerve growth factor (NGF) level was significantly reduced in the ipsilateral lumbar DRGs of neuropathic rats. In addition, treatment with EMA300 could restore the decreased NGF level. Furthermore, several studies have shown that MOR reserve in the spinal cord and DRG is decreased in rodents with neuropathic pain. It is worth noting that administration of exogenous NGF does restore both MOR numbers and their analgesia at main relay points along the pain pathways, such as the spinal cord [58]. These results support a hypothesis on the possible existence of a link between MORs and angiotensin receptor affecting ligands which may provide a new strategy for the treatment of neuropathic pain. Namely, AT2R blockade was reported to restore pathologically decreased NGF levels in neuropathy, which, in turn, could positively influence the MOR number in the DRG and spinal cord, thus restoring the analgesic effect of MOR agonists (Figure 3). An opposing

viewpoint is the implication of NGF in pain induction which is not the scope of the present review but has been reported by other researchers [152–154]. Finally, whether activation or blockade of AT2R would be of value in managing neuropathic pain, we could propose that AT2R inhibition attenuates pain mediated by largely unidentified pathways. On the other hand, the neural growth and remodeling induced by AT2R activation may be beneficial for neuroregeneration, though undesired effects on the symptoms of neuropathy may occur.

To the best of our knowledge, so far, no publication has investigated the possible connections between the opioid system and the Ang (1-7)—MAS receptor branch of the RAS.

**Figure 3.** Possible links between neuropathy, the renin–angiotensin system, MORs and NGF. Red arrows indicate a reducing effect, while the blue ones indicate an increasing effect. In neuropathic conditions, the MOR reserve is decreased, resulting in impaired opioid analgesia. The receptor number can be restored by administration of NGF, the level of which is also reduced in the spinal cord in neuropathy. AT2 antagonists are capable of restoring the lowered NGF level, thus possibly restoring the analgesic effect of opioids. To the best of our knowledge, there is no evidence of the direct connection between MORs and the renin–angiotensin system. The figure was constructed based on literature discussed in Section 5.

#### **6. Concluding Remarks and Future Directions**

MOR analgesics alleviate neuropathic pain; however, high doses are needed, which, in turn, result in serious side effects both in preclinical and human studies. Current evidence indicates that AT1, AT2, and MASRs are involved in the control of neuropathic pain; however, their mechanism of action related to neuropathic pain has not yet been fully verified. Nevertheless, AT1, AT2, and MASRs are expressed in key areas related to pain where MORs agonists halt pain sensation. In neuropathic conditions, peripheral and central AT1 blockade and spinal MASRs activation appear to be beneficial. Data on the impact of AT2R in neuropathic pain are contradictory, though its activation or inhibition can result in neuroprotection or analgesia, respectively; however, future studies are needed to justify this issue. So far, there are no angiotensin receptor affecting agents that have been utilized clinically; however, there are clinical studies on AT2R inhibitors that have entered phase II trials but did not proceed further due to their toxicity. It is important to note that these clinical studies prove that such AT2R inhibitors showed equipotent efficacy with gabapentin. In neuropathic pain, the MOR receptors and NGF levels are decreased. Treatment with NGF results in restoring MOR and their analgesic activity in preclinical pain studies. On the other hand, there are studies reporting the increase in pain sensation upon NGF use, which is not the scope of the present review. Furthermore, some studies revealed that angiotensin AT2R inhibitors do increase NGF in neuropathic pain and thus normalize MOR levels. Therefore, we can speculate that drugs affecting angiotensin receptors could restore the effect of MOR analgesics, which results in avoiding dose escalation of opioids

upon the treatment of neuropathic pain. Finally, these strategies might offer a bridge upon titration of drugs with delay in onset used in the treatment of neuropathic pain.

**Author Contributions:** Conceptualization, K.K., D.Á.K., M.A.-K.; writing—original draft preparation K.K., D.Á.K., M.A.-K., F.Z. writing—review and editing, K.K., D.Á.K., M.A.-K., F.Z., Z.S.Z., A.R.G., T.T., A.M., É.S., M.B., P.R., S.F.; visualization, K.K., D.Á.K., M.A.-K.; supervision, M.A.-K.; project administration, K.K., M.A.-K.; funding acquisition, M.A.-K., T.T., É.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the "Competitiveness and excellence cooperations" project (2018-1.3.1-VKE-2018-00030) provided by the National Research, Development and Innovation Fund. Ferenc Zádor's work was supported by Bolyai János Research Fellowship (BO/00476/20/5) and Bolyai+ Fellowship for Education and Research (ÚNKP-20-5-SE-28).

**Conflicts of Interest:** The authors declare no conflict of interest.
