Opioid-Based Haptens: Development of Immunotherapy.

Over the past decades, extensive preclinical research has been conducted to develop vaccinations to protect against substance use disorder caused by opioids, nicotine, cocaine, and designer drugs. Morphine or fentanyl derivatives are small molecules, and these compounds are not immunogenic, but when conjugated as haptens to a carrier protein will elicit the production of antibodies capable of reacting specifically with the unconjugated hapten or its parent compound. The position of the attachment in opioid haptens to the carrier protein will influence the specificity of the antiserum produced in immunized animals with the hapten-carrier conjugate. Immunoassays for the determination of opioid drugs are based on the ability of drugs to inhibit the reaction between drug-specific antibodies and the corresponding drug-carrier conjugate or the corresponding labelled hapten. Pharmacological studies of the hapten-carrier conjugates resulted in the development of vaccines for treating opioid use disorders (OUDs). Immunotherapy for opioid addiction includes the induction of anti-drug vaccines which are composed of a hapten, a carrier protein, and adjuvants. In this review we survey the design of opioid haptens, the development of the opioid radioimmunoassay, and the results of immunotherapy for OUDs.

Peripheralization Strategies Applied to Morphinans and Implications for Improved Treatment of Pain.

Opioids are considered the most effective analgesics for the treatment of moderate to severe acute and chronic pain. However, the inadequate benefit/risk ratio of currently available opioids, together with the current 'opioid crisis', warrant consideration on new opioid analgesic discovery strategies. Targeting peripheral opioid receptors as effective means of treating pain and avoiding the centrally mediated side effects represents a research area of substantial and continuous attention. Among clinically used analgesics, opioids from the class of morphinans (i.e., morphine and structurally related analogues) are of utmost clinical importance as analgesic drugs activating the mu-opioid receptor. In this review, we focus on peripheralization strategies applied to N-methylmorphinans to limit their ability to cross the blood-brain barrier, thus minimizing central exposure and the associated undesired side effects. Chemical modifications to the morphinan scaffold to increase hydrophilicity of known and new opioids, and nanocarrier-based approaches to selectively deliver opioids, such as morphine, to the peripheral tissue are discussed. The preclinical and clinical research activities have allowed for the characterization of a variety of compounds that show low central nervous system penetration, and therefore an improved side effect profile, yet maintaining the desired opioid-related antinociceptive activity. Such peripheral opioid analgesics may represent alternatives to presently available drugs for an efficient and safer pain therapy.

Insights into the Current and Possible Future Use of Opioid Antagonists in Relation to Opioid-Induced Constipation and Dysbiosis.

Opioid receptor agonists, particularly those that activate µ-opioid receptors (MORs), are essential analgesic agents for acute or chronic mild to severe pain treatment. However, their use has raised concerns including, among others, intestinal dysbiosis. In addition, growing data on constipation-evoked intestinal dysbiosis have been reported. Opioid-induced constipation (OIC) creates an obstacle to continuing treatment with opioid analgesics. When non-opioid therapies fail to overcome the OIC, opioid antagonists with peripheral, fast first-pass metabolism, and gastrointestinal localized effects remain the drug of choice for OIC, which are discussed here. At first glance, their use seems to only be restricted to constipation, however, recent data on OIC-related dysbiosis and its contribution to the appearance of several opioid side effects has garnered a great of attention from researchers. Peripheral MORs have also been considered as a future target for opioid analgesics with limited central side effects. The properties of MOR antagonists counteracting OIC, and with limited influence on central and possibly peripheral MOR-mediated antinociception, will be highlighted. A new concept is also proposed for developing gut-selective MOR antagonists to treat or restore OIC while keeping peripheral antinociception unaffected. The impact of opioid antagonists on OIC in relation to changes in the gut microbiome is included.

Shedding Light on the Pharmacological Interactions between µ-Opioid Analgesics and Angiotensin Receptor Modulators: A New Option for Treating Chronic Pain.

The current protocols for neuropathic pain management include µ-opioid receptor (MOR) analgesics alongside other drugs; however, there is debate on the effectiveness of opioids. Nevertheless, dose escalation is required to maintain their analgesia, which, in turn, contributes to a further increase in opioid side effects. Finding novel approaches to effectively control chronic pain, particularly neuropathic pain, is a great challenge clinically. Literature data related to pain transmission reveal that angiotensin and its receptors (the AT1R, AT2R, and MAS receptors) could affect the nociception both in the periphery and CNS. The MOR and angiotensin receptors or drugs interacting with these receptors have been independently investigated in relation to analgesia. However, the interaction between the MOR and angiotensin receptors has not been excessively studied in chronic pain, particularly neuropathy. This review aims to shed light on existing literature information in relation to the analgesic action of AT1R and AT2R or MASR ligands in neuropathic pain conditions. Finally, based on literature data, we can hypothesize that combining MOR agonists with AT1R or AT2R antagonists might improve analgesia.

Comparisons of In Vivo and In Vitro Opioid Effects of Newly Synthesized 14-Methoxycodeine-6-O-sulfate and Codeine-6-O-sulfate.

The present work represents the in vitro (potency, affinity, efficacy) and in vivo (antinociception, constipation) opioid pharmacology of the novel compound 14-methoxycodeine-6-O-sulfate (14-OMeC6SU), compared to the reference compounds codeine-6-O-sulfate (C6SU), codeine and morphine. Based on in vitro tests (mouse and rat vas deferens, receptor binding and [35S]GTPγS activation assays), 14-OMeC6SU has µ-opioid receptor-mediated activity, displaying higher affinity, potency and efficacy than the parent compounds. In rats, 14-OMeC6SU showed stronger antinociceptive effect in the tail-flick assay than codeine and was equipotent to morphine, whereas C6SU was less efficacious after subcutaneous (s.c.) administration. Following intracerebroventricular injection, 14-OMeC6SU was more potent than morphine. In the Complete Freund's Adjuvant-induced inflammatory hyperalgesia, 14-OMeC6SU and C6SU in s.c. doses up to 6.1 and 13.2 µmol/kg, respectively, showed peripheral antihyperalgesic effect, because co-administered naloxone methiodide, a peripherally acting opioid receptor antagonist antagonized the measured antihyperalgesia. In addition, s.c. C6SU showed less pronounced inhibitory effect on the gastrointestinal transit than 14-OMeC6SU, codeine and morphine. This study provides first evidence that 14-OMeC6SU is more effective than codeine or C6SU in vitro and in vivo. Furthermore, despite C6SU peripheral antihyperalgesic effects with less gastrointestinal side effects the superiority of 14-OMeC6SU was obvious throughout the present study.

On the Role of Peripheral Sensory and Gut Mu Opioid Receptors: Peripheral Analgesia and Tolerance.

There is growing evidence on the role of peripheral µ-opioid receptors (MORs) in analgesia and analgesic tolerance. Opioid analgesics are the mainstay in the management of moderate to severe pain, and their efficacy in the alleviation of pain is well recognized. Unfortunately, chronic treatment with opioid analgesics induces central analgesic tolerance, thus limiting their clinical usefulness. Numerous molecular mechanisms, including receptor desensitization, G-protein decoupling, ß-arrestin recruitment, and alterations in the expression of peripheral MORs and microbiota have been postulated to contribute to the development of opioid analgesic tolerance. However, these studies are largely focused on central opioid analgesia and tolerance. Accumulated literature supports that peripheral MORs mediate analgesia, but controversial results on the development of peripheral opioid receptors-mediated analgesic tolerance are reported. In this review, we offer evidence on the consequence of the activation of peripheral MORs in analgesia and analgesic tolerance, as well as approaches that enhance analgesic efficacy and decrease the development of tolerance to opioids at the peripheral sites. We have also addressed the advantages and drawbacks of the activation of peripheral MORs on the sensory neurons and gut (leading to dysbiosis) on the development of central and peripheral analgesic tolerance.

The Peripheral Versus Central Antinociception of a Novel Opioid Agonist: Acute Inflammatory Pain in Rats.

Opioid analgesics devoid of central side effects are unmet medical need in the treatment of acute pain (e.g. post-operative pain). Recently, we have reported on 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), a novel opioid agonist of high efficacy producing peripheral antinociception in subchronic inflammatory pain in certain doses. The present study focused on the antinociceptive effect of 14-O-MeM6SU compared to morphine in formalin test of an early/acute (Phase I) and late/tonic (Phase II) pain phases. Subcutaneous 14-O-MeM6SU (253-1012 nmol/kg) and morphine (3884-31075 nmol/kg) dose dependently reduced the pain behaviors of both phases. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid antagonist, abolished the antinociceptive effect of 506 nmol/kg 14-O-MeM6SU. On the other hand, the effects of 14-O-MeM6SU (1012 nmol/kg) and morphine (15538 nmol/kg) were only partially affected by NAL-M, indicating the contribution of CNS to antinociception. Locally injected test compounds into formalin treated paws caused antinociception in both phases. Locally effective doses of test compounds were also injected into contralateral paws. Morphine showed effects in both phases, 14-O-MeM6SU in certain doses failed to produce antinociception in either phase. A NAL-M reversible systemic dose of 14-O-MeM6SU and the lowest systemic effective dose of morphine were evaluated for their sedative effects following isoflurane-induced sleeping (righting reflex). In contrast to morphine, 14-O-MeM6SU in certain antinociceptive doses showed no impact on sleeping time. These data highlight that high efficacy opioids of limited CNS penetration in certain doses mitigate somatic and inflammatory pain by targeting MOR at the periphery.

New Morphine Analogs Produce Peripheral Antinociception within a Certain Dose Range of Their Systemic Administration.

Growing data support peripheral opioid antinociceptive effects, particularly in inflammatory pain models. Here, we examined the antinociceptive effects of subcutaneously administered, recently synthesized 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU) compared with morphine-6-O-sulfate (M6SU) in a rat model of inflammatory pain induced by an injection of complete Freund's adjuvant and in a mouse model of visceral pain evoked by acetic acid. Subcutaneous doses of 14-O-MeM6SU and M6SU up to 126 and 547 nmol/kg, respectively, produced significant and subcutaneous or intraplantar naloxone methiodide (NAL-M)-reversible antinociception in inflamed paws compared with noninflamed paws. Neither of these doses significantly affected thiobutabarbital-induced sleeping time or rat pulmonary parameters. However, the antinociceptive effects of higher doses were only partially reversed by NAL-M, indicating contribution of the central nervous system. In the mouse writhing test, 14-O-MeM6SU was more potent than M6SU after subcutaneous or intracerebroventricular injections. Both displayed high subcutaneous/intracerebroventricular ED50 ratios. The antinociceptive effects of subcutaneous 14-O-MeM6SU and M6SU up to 136 and 3043 nmol/kg, respectively, were fully antagonized by subcutaneous NAL-M. In addition, the test compounds inhibited mouse gastrointestinal transit in antinociceptive doses. Taken together, these findings suggest that systemic administration of the novel compound 14-O-MeM6SU similar to M6SU in specific dose ranges shows peripheral antinociception in rat and mouse inflammatory pain models without central adverse effects. These findings apply to male animals and must be confirmed in female animals. Therefore, titration of systemic doses of opioid compounds with limited access to the brain might offer peripheral antinociception of clinical importance.

Involvement of the peripheral sensory and sympathetic nervous system in the vascular endothelial expression of ICAM-1 and the recruitment of opioid-containing immune cells to inhibit inflammatory pain.

Endogenous opioids are known to be released within certain brain areas following stressful stimuli. Recently, it was shown that also leukocytes are a potential source of endogenously released opioid peptides following stress. They activate sensory neuron opioid receptors and result in the inhibition of local inflammatory pain. An important prerequisite for the recruitment of such leukocytes is the expression of intracellular adhesion molecule-1 (ICAM-1) in blood vessels of inflamed tissue. Here, we investigated the contribution of peripheral sensory and/or sympathetic nerves to the enhanced expression of ICAM-1 simultaneously with the increased recruitment of opioid peptide-containing leukocytes to promote the inhibition of inflammatory pain. Selective degeneration of either peripheral sensory or sympathetic nerve fibers by their respective neurotoxins, capsaicin or 6-hydroxydopamime, significantly reduced the subcutaneous immigration of ß-endorphin- (END-) and met-enkephalin- (ENK-)-containing polymorphonuclear leukocytes (PMN) (in the early phase) and mononuclear cells (in the late phase) during painful Freund's complete adjuvant (FCA) rat hind paw inflammation. In contrast, this treatment did not alter the percentage of opioid peptide-containing leukocytes in the circulation. Calcitonin gene-related peptide- (CGRP-) and tyrosine hydroxylase- (TH-) immunoreactive (IR) nerve fibers were in close contact to ICAM-1 IR blood vessels within inflamed subcutaneous tissue. The selective degeneration of sensory or sympathetic nerve fibers attenuated the enhanced expression of vascular endothelial ICAM-1 after intraplantar (i.pl.) FCA and abolished endogenous opioid peptide-mediated peripheral analgesia. Our results suggest that, during localized inflammatory pain, peripheral sensory and sympathetic nerve fibers augment the expression of vascular endothelial ICAM-1 simultaneously with the increased recruitment of opioid peptide-containing leukocytes which consequently promotes the endogenous opioid peptide-mediated inhibition of inflammatory pain. They support existing evidence about a close link between the nervous and the immune system.

Peri, pre and postnatal morphine exposure: exposure-induced effects and sex differences in the behavioural consequences in rat offspring.

This study investigated the behavioural consequences of peri, pre and postnatal morphine (MO) exposure in rats. From gestational day 1 dams were treated with either saline or MO subcutaneously once a day (5 mg/kg on the first 2 days, 10 mg/kg subsequently). Spontaneous locomotor activity in a new environment (habituation) and antinociceptive effects of MO were measured separately in male and female pups after weaning and also in late adolescence or adulthood. The rewarding effect of MO was assessed by conditioned place preference in adult animals. Both exposure-induced and sex differences were observed. A significant delay in habituation to a new environment and decreased sensitivity to the antinociceptive effect of MO were found in male offspring of MO-treated dams. In contrast, the place preference induced by MO was enhanced in the MO-exposed adult animals and this effect was more marked in females. Prenatal exposure to MO resulted in more marked changes than the postnatal exposure through maternal milk. The results indicate that a medium MO dose administered once-daily results in long-term consequences in offspring and may make them more vulnerable to MO abuse in adulthood.

Geroprotection in the future. In memoriam of Joseph Knoll: The selegiline story continues.

In memoriam of Joseph Knoll: the selegiline story continues.

Efficacy-Based Perspective to Overcome Reduced Opioid Analgesia of Advanced Painful Diabetic Neuropathy in Rats.

Reduction of the opioid analgesia in diabetic neuropathic pain (DNP) results from µ-opioid receptor (MOR) reserve reduction. Herein, we examined the antinociceptive and antiallodynic actions of a novel opioid agonist 14-O-methymorphine-6-O-sulfate (14-O-MeM6SU), fentanyl and morphine in rats with streptozocin-evoked DNP of 9-12 weeks following their systemic administration. The antinociceptive dose-response curve of morphine but not of 14-O-MeM6SU or fentanyl showed a significant right-shift in diabetic compared to non-diabetic rats. Only 14-O-MeM6SU produced antiallodynic effects in doses matching antinociceptive doses obtained in non-diabetic rats. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid receptor antagonist failed to alter the antiallodynic effect of test compounds, indicating the contribution of central opioid receptors. Reduction in spinal MOR binding sites and loss in MOR immunoreactivity of nerve terminals in the spinal cord and dorsal root ganglia in diabetic rats were observed. G-protein coupling assay revealed low efficacy character for morphine and high efficacy character for 14-O-MeM6SU or fentanyl at spinal or supraspinal levels (E max values). Furthermore, at the spinal level only 14-O-MeM6SU showed equal efficacy in G-protein activation in tissues of diabetic- and non-diabetic animals. Altogether, the reduction of spinal opioid receptors concomitant with reduced analgesic effect of morphine may be circumvented by using high efficacy opioids, which provide superior analgesia over morphine. In conclusion, the reduction in the analgesic action of opioids in DNP might be a consequence of MOR reduction, particularly in the spinal cord. Therefore, developing opioids of high efficacy might provide analgesia exceeding that of currently available opioids.

Capsaicin-sensitive sensory neurons regulate myocardial function and gene expression pattern of rat hearts: a DNA microarray study.

We have previously shown that capsaicin-sensitive sensory nerves contribute to the regulation of normal cardiac function and to the development of cardiac adaptation to ischemic stress; however, the underlying molecular mechanisms remain unknown. Therefore, here we assessed cardiac functional alterations and relative gene expression changes by DNA microarray analysis of 6400 genes in rat hearts 7 days after the end of systemic capsaicin treatment protocol leading to selective sensory chemodenervation. Capsaicin pretreatment resulted in a cardiac dysfunction characterized by elevation of left ventricular end-diastolic pressure and led to altered expression of 80 genes of known function or homology to known sequences. Forty-seven genes exhibited significant up-regulation and 33 genes were down-regulated (changes ranged from -3.9 to +4.8-fold). The expression changes of 10 selected genes were verified, and an additional 11 genes were examined by real-time quantitative PCR. This is the first demonstration that gene expression changes in the heart due to capsaicin pretreatment included vanilloid receptor-1 (capsaicin receptor), transient receptor potential protein, GABA receptor rho-3 subunit, 5-hydroxytryptamine 3 receptor B, neurokinin receptor 2, endothelial nitric oxide synthase, matrix metalloproteinase-13, cytochrome P450, farnesyl-transferase, ApoB, and leptin. None of the genes have been previously shown to be involved in the mechanism of the cardiac functional effects of sensory chemodenervation by capsaicin. We conclude that capsaicin-sensitive sensory nerves play a significant role in the regulation of a variety of neuronal and non-neuronal genes in the heart and possibly in other tissues as well.

DAMGO and 6beta-glycine substituted 14-O-methyloxymorphone but not morphine show peripheral, preemptive antinociception after systemic administration in a mouse visceral pain model and high intrinsic efficacy in the isolated rat vas deferens.

Peripheral micro-opioid receptors (MOR) have emerged as important components of inhibitory nociceptive pathways. Here, the antinociceptive effects of MOR agonists, the 6beta-glycine derivative of 14-O-methyloxymorphone (HS-731), DAMGO and morphine were evaluated in a mouse model of visceral pain. The abdominal acetic acid-induced writhing test was used to examine the peripheral, preemptive antinociceptive opioid action on visceral nociception. HS-731 administered subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) dose-dependently and completely inhibited writhing, being 24-598-fold more potent, depending on the administration route, than two selective MOR agonists, the enkephalin analogue [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO) and morphine. A longer duration of action (2-3 h) was induced by HS-731 given before acetic acid, while shorter effect was produced by morphine (30-60 min) and DAMGO (30-45 min). The antinociceptive effects of systemic opioids were reversed by the s.c. opioid antagonist, naloxone. Blocking of central MOR by the selective MOR antagonist D-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP, i.c.v.) resulted in a significant reduction of antinociception of s.c. morphine, whereas it completely failed to antagonize the effects of systemic HS-731 or DAMGO. In in vitro studies, HS-731 and DAMGO, but not morphine showed high intrinsic efficacy, naltrexone-sensitive agonist effect at MOR of the rat vas deferens. These data demonstrate that selective activation of peripheral MOR by systemic s.c. HS-731 or DAMGO produces potent peripheral, preemptive visceral antinociception, while morphine's effects are mediated primarily through central mechanisms. Our findings support the role of peripheral MOR in the pathology of pain states involving sensitization of peripheral nociceptors.

14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [35S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (Emax) and potency (EC50) than morphine in MVD, RVD or [35S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for µ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value.

New opioid receptor antagonist: Naltrexone-14-O-sulfate synthesis and pharmacology.

Opioid antagonists, naloxone and naltrexone have long been used in clinical practice and research. In addition to their low selectivity, they easily pass through the blood-brain barrier. Quaternization of the amine group in these molecules, (e.g. methylnaltrexone) results in negligible CNS penetration. In addition, zwitterionic compounds have been reported to have limited CNS access. The current study, for the first time gives report on the synthesis and the in vitro [competition binding, G-protein activation, isolated mouse vas deferens (MVD) and mouse colon assay] pharmacology of the zwitterionic compound, naltrexone-14-O-sulfate. Naltrexone, naloxone, and its 14-O-sulfate analogue were used as reference compounds. In competition binding assays, naltrexone-14-O-sulfate showed lower affinity for µ, δ or κ opioid receptor than the parent molecule, naltrexone. However, the µ/κ opioid receptor selectivity ratio significantly improved, indicating better selectivity. Similar tendency was observed for naloxone-14-O-sulfate when compared to naloxone. Naltrexone-14-O-sulfate failed to activate [35S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile5,6deltorphin II and U69593), similarly to the reference compounds. Schild plot constructed in MVD revealed that naltrexone-14-O-sulfate acts as a competitive antagonist. In mouse colon, naltrexone-14-O-sulfate antagonized the inhibitory effect of morphine with lower affinity compared to naltrexone and higher affinity when compared to naloxone or naloxone-14-O-sulfate. In vivo (mouse tail-flick test), subcutaneously injected naltrexone-14-O-sulfate antagonized morphine's antinociception in a dose-dependent manner, indicating it's CNS penetration, which was unexpected from such zwitter ionic structure. Future studies are needed to evaluate it's pharmacokinetic profile.

Regulation of human recombinant P2X3 receptors by ecto-protein kinase C.

The whole-cell patch-clamp technique was used to record current responses to nucleotides and nucleosides in human embryonic kidney HEK293 cells transfected with the human purinergic P2X3 receptor. When guanosine 5'-O-(3-thiodiphosphate) was included into the pipette solution, UTP at concentrations that did not alter the holding current facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. ATP and GTP, but not UDP or uridine, had an effect similar to that of UTP. Compounds known to activate protein kinase C (PKC) acted like the nucleoside triphosphates investigated, whereas various PKC inhibitors invariably reduced the effects of both PKC activators and UTP. The substitution by Ala of Ser/Thr residues situated within PKC consensus sites of the P2X3 receptor ectodomain either abolished (PKC2 and PKC3; T134A, S178A) or did not alter (PKC4 and PKC6; T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. Both the blockade of ecto-protein kinase C activity and the substitution of Thr-134 or Ser-178 by Ala depressed the maximum of the concentration-response curve for alpha,beta-meATP without altering the EC50 values. Molecular simulation of the P2X3 receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. alpha,beta-meATP-induced currents through native homomeric P2X3 receptors of rat dorsal root ganglia were also facilitated by UTP. In conclusion, it is suggested that low concentrations of endogenous nucleotides in the extracellular space may prime the sensitivity of P2X3 receptors toward the effect of subsequently applied (released) higher agonistic concentrations. The priming effect of nucleotides might be attributable to a phosphorylation of PKC sites at the ectodomain of P2X3 receptors.

Decrease of current responses at human recombinant P2X3 receptors after substitution by Asp of Ser/Thr residues in protein kinase C phosphorylation sites of their ecto-domains.

The whole-cell patch-clamp technique was used to record current responses to nucleotides in HEK 293 cells transiently transfected with the human (h) P2X(3) receptor. When GDP-beta-S was included into the pipette solution, UTP at concentrations which did not alter the holding current, facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. The substitution of Ser/Thr residues situated within protein kinase C (PKC) consensus phosphorylation sites of the P2X(3) receptor ecto-domain by the neutral amino acid Ala either abolished (T134A, S178A) or did not alter (T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. The substitution of the same Ser/Thr residues in all four PKC sites by the negatively charged Asp prevented the potentiation by UTP. The Asp mutations abolished the first, fast offset time-constant, but did not alter, or in the case of S269D even increased, the second, slow offset time-constant; at the same time such mutations invariably increased the onset time-constant and massively depressed the peak current amplitude. None of the Ala mutations (with the exception of S269A) influenced the time-course of desensitisation or the peak current amplitude. It is concluded that constitutive activation of PKC sites at the ecto-domain of the hP2X(3) receptor both abolishes the UTP-induced potentiation of the alpha,beta-meATP current and accelerates its rate of desensitisation.

Membrane-bound glucocorticoid receptors on distinct nociceptive neurons as potential targets for pain control through rapid non-genomic effects.

Glucocorticoids were long believed to primarily function through cytosolic glucocorticoid receptor (GR) activation and subsequent classical genomic pathways. Recently, however, evidence has emerged that suggests the presence of rapid non-genomic GR-dependent signaling pathways within the brain, though their existence in spinal and peripheral nociceptive neurons remains elusive. In this paper, we aim to systemically identify GR within the spinal cord and periphery, to verify their putative membrane location and to characterize possible G protein coupling and pain modulating properties. Double immunofluorescence confocal microscopy revealed that GR predominantly localized in peripheral peptidergic and non-peptidergic nociceptive C- and Aδ-neurons and existed only marginally in myelinated mechanoreceptive and proprioreceptive neurons. Within the spinal cord, GR predominantly localized in incoming presynaptic nociceptive neurons, in pre- and postsynaptic structures of the dorsal horn, as well as in microglia. GR saturation binding revealed that these receptors are linked to the cell membrane of sensory neurons and, upon activation, they trigger membrane targeted [35S]GTPγS binding, indicating G protein coupling to a putative receptor. Importantly, subcutaneous dexamethasone immediately and dose-dependently attenuated acute nociceptive behavior elicited in an animal model of formalin-induced pain hypersensitivity compared to naive rats. Overall, this study provides firm evidence for a novel neuronal mechanism of GR agonists that is rapid, non-genomic, dependent on membrane binding and G protein coupling, and acutely modulates nociceptive behavior, thus unraveling a yet unconsidered mechanism of pain relief.

Inhibition of N-type voltage-activated calcium channels in rat dorsal root ganglion neurons by P2Y receptors is a possible mechanism of ADP-induced analgesia.

Patch-clamp recordings from small-diameter rat dorsal root ganglion (DRG) neurons maintained in culture demonstrated preferential inhibition by ATP of high-voltage-activated, but not low-voltage-activated, Ca2+ currents (I(Ca)). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the omega-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetraammonium, two P2Y1 receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X3 and P2Y1 receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5'-O-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y1 receptors appear to block I(Ca) by a pathway involving the betagamma subunit of a G(q/11) protein. Less efficient buffering of the intracellular Ca2+ concentration ([Ca2+]i) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca2+]i by a Galpha-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca2+]i by inhibiting I(Ca) via Gbetagamma. Adenosine 5'-O-(2-thiodiphosphate) inhibited dorsal root-evoked polysynaptic population EPSPs in the hemisected rat spinal cord and prolonged the nociceptive threshold on intrathecal application in the tail-flick assay. These effects were not antagonized by PPADS. Hence, P2Y receptor activation by ADP, which is generated by enzymatic degradation of ATP, may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effect of ATP.