Mohammed Shaqura

Rapid upregulation of μ opioid receptor mrna in dorsal root ganglia in response to peripheral inflammation depends on neuronal conduction

S.c. painful inflammation leads to an increase in axonal transport of opioid receptors from dorsal root ganglia (DRG) toward the periphery, thus causing a higher receptor density and enhanced opioid analgesia at the injured site. To examine whether this increase is related to transcription, the mRNA of Delta- (DOR) and mu-opioid receptor (MOR) in lumbar DRG was quantified by real time Light Cycler polymerase chain reaction (LC-PCR), and correlated to ligand binding in DRG and sciatic nerve. In normal DRG, DOR mRNA was seven times less abundantly expressed than MOR mRNA. After induction of unilateral paw inflammation, mRNA content for DOR remained unchanged, but a bi-phasic upregulation for MOR mRNA with an early peak at 1-2 h and a late increase at 96 h was found in ipsilateral DRG. As no changes were observed in DRG of the non-inflamed side, this effect was apparently not systemically mediated. A significant increase in binding of the MOR ligand DAMGO was detected after 24 h in DRG, and after early and late ligation in the sciatic nerve, indicating an enhanced axonal transport of MOR in response to inflammation. The early increase in MOR mRNA could be completely prevented by local anesthetic blockade of neuronal conduction in sciatic nerve. These data suggest that mRNA of the two opioid receptors DOR and MOR is differentially regulated in DRG during peripheral painful inflammation. The apparently increased axonal transport of MOR in response to this inflammation is preceded by upregulated mRNA-transcription, which is dependent on neuronal electrical activity.

Chronic morphine use does not induce peripheral tolerance in a rat model of inflammatory pain

Although opioids are highly effective analgesics, they are also known to induce cellular adaptations resulting in tolerance. Experimental studies are often performed in the absence of painful tissue injury, which precludes extrapolation to the clinical situation. Here we show that rats with chronic morphine treatment do not develop signs of tolerance at peripheral mu-opioid receptors (micro-receptors) in the presence of painful CFA-induced paw inflammation. In sensory neurons of these animals, internalization of mu-receptors was significantly increased and G protein coupling of mu-receptors as well as inhibition of cAMP accumulation were preserved. Opioid receptor trafficking and signaling were reduced, and tolerance was restored when endogenous opioid peptides in inflamed tissue were removed by antibodies or by depleting opioid-producing granulocytes, monocytes, and lymphocytes with cyclophosphamide (CTX). Our data indicate that the continuous availability of endogenous opioids in inflamed tissue increases recycling and preserves signaling of mu-receptors in sensory neurons, thereby counteracting the development of peripheral opioid tolerance. These findings infer that the use of peripherally acting opioids for the prolonged treatment of inflammatory pain associated with diseases such as chronic arthritis, inflammatory neuropathy, or cancer, is not necessarily accompanied by opioid tolerance.

Endogenous peripheral antinociception in early inflammation is not limited by the number of opioid-containing leukocytes but by opioid receptor expression

&NA; Endogenous inhibition of inflammatory pain is mediated by leukocytes that secrete opioid peptides upon exposure to stress (cold water swim stress, CWS) or after local injection of corticotropin releasing factor (CRF). Since in early inflammation few opioid‐containing leukocytes are detected and since peripheral opioid‐mediated antinociception is low we examined whether antinociception could be augmented by increased recruitment of opioid‐containing polymorphonuclear cells (PMN). Rats were intraplantarly (i.pl.) injected with Freunds complete adjuvant (FCA) and with the PMN‐recruiting chemokine macrophage inflammatory protein‐2 (MIP‐2, 1–10 &mgr;g; control: saline) for 2 h. Intraplantar leukocytes were quantified by flow cytometry. Paw pressure threshold (PPT) was determined before and after exposure to CWS, i.pl. injection of CRF and opioid peptides. Opioid receptors (OR) were measured by binding studies in dorsal root ganglia (DRG) and by immunohistochemistry in the paw. Our studies showed that (i) MIP‐2 injection dose‐dependently augmented recruitment of PMN and opioid‐containing leukocytes (5‐fold increase in cells/paw, P<0.05), (ii) PPT was not different between groups at baseline and after CWS or CRF (maximum MPE: 20±2.3–29±7.2%, P<0.05), (iii) injection of opioid peptides dose‐dependently increased the PPT (P<0.05, maximum MPE: and 18±2.6–21±3.6%), (iv) MOR (&mgr; OR, MOP) binding sites in the ipsilateral DRG were unchanged (24±2–22±1.2 fmol/mg protein, P>0.05, ANOVA) and (v) the number of MOR and DOR (&dgr; OR, DOP) stained nerve fibers in peripheral tissue were unaltered (both P>0.05, t‐test). In summary, antinociception during early inflammation is apparently not limited by the number of opioid‐containing leukocytes but by OR availability.

Peripheral Antinociceptive Effects of Exogenous and Immune Cell-Derived Endomorphins in Prolonged Inflammatory Pain

Endomorphins (EMs) are endogenous selective μ-opioid receptor agonists. Their role in inflammatory pain has not been fully elucidated. Here we examine peripheral antinociception elicited by exogenously applied EM-1 and EM-2 and the contribution of EM-containing leukocytes to stress- and corticotropin-releasing factor (CRF)-induced antinociception. To this end, we applied behavioral (paw pressure) testing, radioligand binding, immunohistochemistry, and flow cytometry in rats with unilateral hindpaw inflammation induced with Freund’s adjuvant. EMs injected directly into both hindpaws produced antinociception exclusively in inflamed paws. This was blocked by locally applied μ-receptor-selective (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2) but not κ-receptor-selective (nor-binaltorphimine) antagonists. δ-Receptor antagonists (naltrindole and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu) did not influence EM-1-induced but dose-dependently decreased EM-2-induced antinociception. Antibodies against β-endorphin, methionine-enkephalin, or leucine-enkephalin did not significantly change EM-2-induced antinociception. Both EMs displaced binding of [3H]-[d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to μ-receptors in dorsal root ganglia (DRG). Using [3H]-naltrindole or [125I]-[d-Pen2,5]-enkephalin, no detectable δ-binding was found in DRG of inflamed hindlimbs. Numerous β-endorphin-containing and fewer EM-1- and EM-2-containing leukocytes were detected in subcutaneous tissue of inflamed paws. Leukocyte-depleting serum decreased the number of immigrating opioid-containing immune cells and attenuated swim stress- and CRF-induced antinociception in inflamed paws. Both forms of antinociception were strongly attenuated by anti-β-endorphin and to a lesser degree by anti-EM-1 and anti-EM-2 antibodies injected into inflamed paws. Together, exogenously applied and immune cell-derived EMs alleviate prolonged inflammatory pain through selective activation of peripheral opioid receptors. Exogenous EM-2 in addition to μ-receptors also activates peripheral δ-receptors, which does not involve actions via other opioid peptides.

Relative contributions of peripheral versus supraspinal or spinal opioid receptors to the antinociception of systemic opioids

The contribution of supraspinal, spinal or peripheral mu‐opioid receptors (MORs) to the overall antinociception of systemic centrally penetrating versus peripherally restricted opioids has not been thoroughly investigated. Therefore, we examined paw pressure thresholds in Wistar rats with complete Freunds adjuvant hindpaw inflammation following different doses of intraplantar (i.pl.) as well as intravenous (i.v.) fentanyl (6.25–50 μg/kg), morphine (1–7.5 mg/kg) or loperamide (1–7.5 mg/kg). Antagonism of the i.v. mu‐opioid agonists by intracerebroventricular (i.c.v.), intrathecal (i.t.) or i.pl. naloxone‐methiodide (NLXM) revealed the relative contributions of supraspinal, spinal and peripheral MOR to the overall antinociceptive effects. In parallel, the MOR density at these three levels of pain transmission was assessed by radioligand binding. Antinociceptive effects of i.v. fentanyl and morphine, but not of the peripherally restricted loperamide were two‐ to threefold greater and longer lasting compared with their i.pl. administration. I.c.v. but not i.pl. NLXM significantly antagonized fentanyls and morphines antinociception by 70–80%, whereas i.t. NLXM reduced it by 20–30%. In contrast, antinociception of i.v. loperamide was abolished by i.pl. but not by i.c.v. or i.t. NLXM. In parallel, a respective 32‐ and sixfold higher MOR density in supraspinal and spinal versus peripheral sensory neurons was detected. In conclusion, in comparison with supraspinal and spinal opioid receptors, peripheral opioid receptors do not significantly contribute to the antinociception of systemic fentanyl and morphine during inflammatory pain. Antinociception of their i.v. administration was superior over both i.v and i.pl. loperamide, acting exclusively via peripheral MOR. These findings may guide the future development of novel peripherally restricted opioids.

Rab7 Silencing Prevents μ-Opioid Receptor Lysosomal Targeting and Rescues Opioid Responsiveness to Strengthen Diabetic Neuropathic Pain Therapy

Painful diabetic neuropathy is poorly controlled by analgesics and requires high doses of opioids, triggering side effects and reducing patient quality of life. This study investigated whether enhanced Rab7-mediated lysosomal targeting of peripheral sensory neuron μ-opioid receptors (MORs) is responsible for diminished opioid responsiveness in rats with streptozotocin-induced diabetes. In diabetic animals, significantly impaired peripheral opioid analgesia was associated with a loss in sensory neuron MOR and a reduction in functional MOR G-protein-coupling. In control animals, MORs were retained mainly on the neuronal cell membrane. In contrast, in diabetic rats, they were colocalized with upregulated Rab7 in LampI-positive perinuclear lysosome compartments. Silencing endogenous Rab7 with intrathecal Rab7-siRNA or, indirectly, by reversing nerve growth factor deprivation in peripheral sensory neurons not only prevented MOR targeting to lysosomes, restoring their plasma membrane density, but also rescued opioid responsiveness toward better pain relief. These findings elucidate in vivo the mechanisms by which enhanced Rab7 lysosomal targeting of MORs leads to a loss in opioid antinociception in diabetic neuropathic pain. This is in contrast to peripheral sensory neuron MOR upregulation and antinociception in inflammatory pain, and provides intriguing evidence that regulation of opioid responsiveness varies as a function of pain pathogenesis.

The presence of mu-, delta-, and kappa-opioid receptors in human heart tissue.

Abstract Functional evidence suggests that the stimulation of peripheral and central opioid receptors (ORs) is able to modulate heart function. Moreover, selective stimulation of either cardiac or central ORs evokes preconditioning and, therefore, protects the heart against ischemic injury. However, anatomic evidence for OR subtypes in the human heart is scarce. Human heart tissue obtained during autopsy after sudden death was examined immunohistochemically for mu- (MOR), kappa- (KOR), and delta- (DOR) OR subtypes. MOR and DOR immunoreactivity was found mainly in myocardial cells, as well as on sparse individual nerve fibers. KOR immunoreactivity was identified predominantly in myocardial cells and on intrinsic cardiac adrenergic (ICA) cell-like structures. Double immunofluorescence confocal microscopy revealed that DOR colocalized with the neuronal marker PGP9.5, as well as with the sensory neuron marker calcitonin gene-related peptide (CGRP). CGRP-immunoreactive (IR) fibers were detected either in nerve bundles or as sparse individual fibers containing varicose-like structures. Our findings offer the first hint of an anatomic basis for the existence of OR subtypes in the human heart by demonstrating their presence in CGRP-IR sensory nerve fibers, small cells with an eccentric nucleus resembling ICA cells, and myocardial cells. Taken together, this suggests the role of opioids in both the neural transmission and regulation of myocardial cell function.

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 β-endorphin- (END-) and met-enkephalin- (ENK-)-containing polymorphonuclear leukocytes (PMN) (in the early phase) and mononuclear cells (in the late phase) during painful Freunds 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.

Identification of μ- and κ-opioid receptors as potential targets to regulate parasympathetic, sympathetic, and sensory neurons within rat intracardiac ganglia

Recent interest has been focused on the opioid regulation of heart performance; however, specific allocation of opioid receptors to the parasympathetic, sympathetic, and sensory innervations of the heart is scarce. Therefore, the present study aimed to characterize such specific target sites for opioids in intracardiac ganglia, which act as a complex network for the integration of the hearts neuronal in‐ and output. Tissue samples from rat heart atria were subjected to RT‐PCR, Western blot, radioligand‐binding, and double immunofluorescence confocal analysis of μ (M)‐ and κ (K)‐opioid receptors (ORs) with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene‐related peptide (CGRP), and substance P (SP). Our results demonstrated MOR‐ and KOR‐specific mRNA, receptor protein, and selective membrane ligand binding. By using immunofluorescence confocal microscopy, MOR and KOR immunoreactivity were colocalized with VAChT in large‐diameter parasympathetic principal neurons, with TH‐immunoreactive small intensely fluorescent (SIF) cells, and on nearby TH‐IR varicose terminals. In addition, MOR and KOR immunoreactivity were identified on CGRP‐ and SP‐IR sensory neurons throughout intracardiac ganglia and atrial myocardium. Our findings show that MOR and KOR are expressed as mRNA and translated into specific receptor proteins on cardiac parasympathetic, sympathetic, and sensory neurons as potential binding sites for opioids. Thus, they may well play a role within the complex network for the integration of the hearts neuronal in‐ and output. J. Comp. Neurol. 518:3836–3847, 2010.

Developmental expression of δ-opioid receptors during maturation of the parasympathetic, sympathetic, and sensory innervations of the neonatal heart: Early targets for opioid regulation of autonomic control

Evidence is accumulating regarding the local opioid regulation of heart function. However, the exact anatomical location of δ‐opioid receptors (DORs) and expression during maturation of the autonomic and sensory innervations of the neonatal heart is unknown. Therefore, we aimed to characterize target sites for opioids in neonatal rat heart intracardiac ganglia at postnatal day (P)1, P7 and adulthood (P56–P84). Rat heart atria were subjected to reverse‐transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis of DORs with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene‐related peptide (CGRP), and substance P (SP). Our results demonstrated DOR mRNA, protein, and binding sites that gradually increased from P1 toward adulthood. Immunofluorescence confocal microscopy showed DOR co‐localized with VAChT in large‐diameter principal neurons, TH‐immunoreactive (IR) small intensely fluorescent (SIF) catecholaminergic cells, and CGRP‐ or SP‐IR afferent nerve terminals arborizing within intracardiac ganglia and atrial myocardium. Co‐expression of DOR with VAChT‐IR neurons was observed from the first day of birth (P1). In contrast, DORs on TH‐IR SIF cells or CGRP‐IR fibers were not observed in intracardiac ganglia of P1, but rather in P7 rats. The density of nerve fibers in atrial myocardium co‐expressing DORs with different neuronal markers increased from neonatal age toward adulthood. In summary, the enhanced DOR expression parallel to the maturation of cardiac parasympathetic, sympathetic, and sensory innervation of the heart suggests that the cardiac opioid system is an important regulator of neonatal and adult heart function through the autonomic nervous system. J. Comp. Neurol. 519:957–971, 2011.