Involvement of P2X7 receptors in chronic pain disorders

Chronic pain is caused by cellular damage with an obligatory inflammatory component. In response to noxious stimuli, high levels of ATP leave according to their concentration gradient, the intracellular space through discontinuities generated in the plasma membrane or diffusion through pannexin-1 hemichannels, and activate P2X7Rs localized at peripheral and central immune cells. Because of the involvement of P2X7Rs in immune functions and especially the initiation of macrophage/microglial and astrocytic secretion of cytokines, chemokines, prostaglandins, proteases, reactive oxygen, and nitrogen species as well as the excitotoxic glutamate/ATP, this receptor type has a key role in chronic pain processes. Microglia are equipped with a battery of pattern recognition receptors that detect pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) from bacterial infections or danger associated molecular patterns (DAMPs) such as ATP. The co-stimulation of these receptors leads to the activation of the NLRP3 inflammasome and interleukin-1β (IL-1β) release. In the present review, we invite you to a journey through inflammatory and neuropathic pain, primary headache, and regulation of morphine analgesic tolerance, in the pathophysiology of which P2X7Rs are centrally involved. P2X7R bearing microglia and astrocyte-like cells playing eminent roles in chronic pain will be also discussed.

Opioid Analgesia and Opioid-Induced Adverse Effects: A Review

Opioids are widely used as therapeutic agents against moderate to severe acute and chronic pain. Still, these classes of analgesic drugs have many potential limitations as they induce analgesic tolerance, addiction and numerous behavioural adverse effects that often result in patient non-compliance. As opium and opioids have been traditionally used as painkillers, the exact mechanisms of their adverse reactions over repeated use are multifactorial and not fully understood. Older adults suffer from cancer and non-cancer chronic pain more than younger adults, due to the physiological changes related to ageing and their reduced metabolic capabilities and thus show an increased number of adverse reactions to opioid drugs. All clinically used opioids are μ-opioid receptor agonists, and the major adverse effects are directly or potentially connected to this receptor. Multifunctional opioid ligands or peripherally restricted opioids may elicit fewer adverse effects, as shown in preclinical studies, but these results need reproducibility from further extensive clinical trials. The current review aims to overview various mechanisms involved in the adverse effects induced by opioids, to provide a better understanding of the underlying pathophysiology and, ultimately, to help develop an effective therapeutic strategy to better manage pain.

Delta Opioid Receptor in Astrocytes Contributes to Neuropathic Cold Pain and Analgesic Tolerance in Female Mice

Background: The delta opioid receptor (DOR) contributes to pain control, and a major challenge is the identification of DOR populations that control pain, analgesia, and tolerance. Astrocytes are known as important cells in the pathophysiology of chronic pain, and many studies report an increased prevalence of pain in women. However, the implication of astrocytic DOR in neuropathic pain and analgesia, as well as the influence of sex in this receptor activity, remains unknown.Experimental Approach: We developed a novel conditional knockout (cKO) mouse line wherein DOR is deleted in astrocytes (named GFAP-DOR-KO), and investigated neuropathic mechanical allodynia as well as analgesia and analgesic tolerance in mutant male and female mice. Neuropathic cold allodynia was also characterized in mice of both sexes lacking DOR either in astrocytes or constitutively.Results: Neuropathic mechanical allodynia was similar in GFAP-DOR-KO and floxed DOR control mice, and the DOR agonist SNC80 produced analgesia in mutant mice of both sexes. Interestingly, analgesic tolerance developed in cKO males and was abolished in cKO females. Cold neuropathic allodynia was reduced in mice with decreased DOR in astrocytes. By contrast, cold allodynia was exacerbated in full DOR KO females.Conclusions: These findings show that astrocytic DOR has a prominent role in promoting cold allodynia and analgesic tolerance in females, while overall DOR activity was protective. Altogether this suggests that endogenous- and exogenous-mediated DOR activity in astrocytes worsens neuropathic allodynia while DOR activity in other cells attenuates this form of pain. In conclusion, our results show a sex-specific implication of astrocytic DOR in neuropathic pain and analgesic tolerance. These findings open new avenues for developing tailored DOR-mediated analgesic strategies.

The Guts of the Opioid Crisis

Bidirectional interactions of the gut epithelium with commensal bacteria are critical for maintaining homeostasis within the gut. Chronic opioid exposure perturbs gut homeostasis through a multitude of neuro-immune-epithelial mechanisms, resulting in the development of analgesic tolerance, a major underpinning of the current opioid crisis. Differences in molecular mechanisms of opioid tolerance between the enteric and central pain pathways pose a significant challenge for managing chronic pain without untoward gastrointestinal effects.

Agonist dependency of the second phase access of β-arrestin 2 to the heteromeric µ-V1b receptor

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to β-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that β-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and β-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, β-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of β-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of β-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

The Expression and Function of Nitric Oxide Synthase Enzyme in Atorvastatin Effects on Morphine-Induced Dependence in Mice

Background: Atorvastatin exerts neuroprotective effects on the treatment of central nervous system disorders. Morphine analgesic tolerance and dependence remain as major concerns in medicine. Nitric oxide (NO) pathway mediates the development of opioid analgesic tolerance and dependence, as well as atorvastatin neuroprotection. Objectives: The present study aimed to assess the possible involvement of the NO/cGMP pathway in the process of the effects of atorvastatin on morphine physical dependence. Methods: Dependence was induced by repetitive injection of morphine sulfate. Naloxone was injected at the dose of 4 mg/kg on the last day of the experiment to assess withdrawal signs. Animals received atorvastatin (1, 5, 10, and 20 mg/kg, orally). Nitric oxide synthase (NOS) inhibitors and ODQ were injected before protective dose atorvastatin. The gene expression of NOS isoforms was evaluated by real time-PCR. Thereafter, the hippocampal levels of cGMP and nitrite were measured. Results: Treatment with atorvastatin 10 mg/kg significantly attenuated naloxone-induced withdrawal behaviours. The administration of L-NAME, aminoguanidine, and ODQ before atorvastatin enhanced its effects. The treatment with atorvastatin significantly decreased the nitrite and cGMP levels as well as NOS gene expression in the hippocampus of dependent animals. Conclusions: It can be concluded that atorvastatin, possibly, through inducible NOS, could alleviate morphine dependence and withdrawal signs.

Paroxetine effects on morphine analgesic tolerance in rats

Objectives To alleviate different pain intensities, morphine administration has been extensively used. However, prolonged administration of morphine leads to a progressive decline of its analgesic effect which limits their overall utility. Morphine tolerance is considered as a challenging issue for the treatment of both acute and chronic pain. We conducted this study in rats to investigate the effect of paroxetine on morphine tolerance when used preemptively or after morphine tolerance had developed. Methods Male Wistar rats (weight 250–300 g, n=10) were used to evaluate the effects of paroxetine on tolerance to morphine. In order to induce tolerance, daily intraperitoneal injection of morphine (7 mg/kg) was done. After tolerance induction, a group of animals received intraperitoneal injection of 10 mg/kg paroxetine 30 min prior to each morphine dose. In another trial, to investigate the potential of paroxetine to prevent tolerance to morphine, animals were pretreated with 10 mg/kg paroxetine 30 min before morphine administration. In the control groups, 10 mL/kg of saline was injected. The behavioral test (tail-flick test) was done for all groups. Results Our data showed that paroxetine significantly reversed tolerance to morphine when used after tolerance induction (p<0.001). However, administration of paroxetine before occurrence of tolerance had no effect. Conclusions We conclude that paroxetine could decrease tolerance to morphine when used after the occurrence of morphine tolerance, while it was not able to prevent morphine tolerance when administered preemptively. Ethical committee number IRIB.SBMU.MSP.REC.1394.098.

Profiling the Effects of Repetitive Morphine Administration on Motor Behavior in Rats

Efficient repetitive clinical use of morphine is limited by its numerous side effects, whereas analgesic tolerance necessitates subsequent increases in morphine dose to achieve adequate levels of analgesia. While many studies focused on analgesic tolerance, the effect of morphine dosing on non-analgesic effects has been overlooked. This study aimed to characterize morphine-induced behavior and the development and progression of morphine-induced behavioral tolerance. Adult male Sprague–Dawley rats were repetitively treated with subcutaneous morphine for 14 days in two dose groups (A: 5 mg/kg/day (b.i.d.) → 10 mg/kg/day; B: 10 mg/kg/day (b.i.d.) → 20 mg/kg/day). Motor behavior was assessed daily (distance traveled, speed, moving time, rearing, rotation) in an open-field arena, before and 30 min post-injections. Antinociception was measured using tail-flick and hot-plate assays. All measured parameters were highly suppressed in both dosing groups on the first treatment day, followed by a gradual manifestation of behavioral tolerance as the treatment progressed. Animals in the high-dose group showed increased locomotor activity after 10 days of morphine treatment. This excitatory phase converted to an inhibition of behavior when a higher morphine dose was introduced. We suggest that the excitatory locomotor effects of repetitive high-dose morphine exposure represent a signature of its behavioral and antinociceptive tolerance.

Pharmacological and genetic manipulations at the µ-opioid receptor reveal arrestin-3 engagement limits analgesic tolerance and does not exacerbate respiratory depression in mice

Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.