Ian Kitchen

Disruption of the κ‐opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective κ‐agonist U‐50,488H and attenuates morphine withdrawal

μ‐, δ‐ and κ‐opioid receptors are widely expressed in the central nervous system where they mediate the strong analgesic and mood‐altering actions of opioids, and modulate numerous endogenous functions. To investigate the contribution of the κ‐opioid receptor (KOR) to opioid function in vivo, we have generated KOR‐deficient mice by gene targeting. We show that absence of KOR does not modify expression of the other components of the opioid system, and behavioural tests indicate that spontaneous activity is not altered in mutant mice. The analysis of responses to various nociceptive stimuli suggests that the KOR gene product is implicated in the perception of visceral chemical pain. We further demonstrate that KOR is critical to mediate the hypolocomotor, analgesic and aversive actions of the prototypic κ‐agonist U‐50,488H. Finally, our results indicate that this receptor does not contribute to morphine analgesia and reward, but participates in the expression of morphine abstinence. Together, our data demonstrate that the KOR‐encoded receptor plays a modulatory role in specific aspects of opioid function.

Development of opioid systems: peptides, receptors and pharmacology.

The understanding of opioid system function in the adult has progressed markedly in the last decade. This in turn has led to an increased knowledge of how opioid function develops in the embryo and in the postnatal period. With respect to the ontogenetic profile of the opioid peptide products of the 3 opioid precursors, all are detectable during gestation and their development in rodents is not completed until well after birth, the third postnatal week often exhibiting the most marked increases. There is not an exact parallel development of the precursors and there may be regional fluctuations for some of the peptides in the postnatal period. All of the indications are that the ontogenesis of the 3 precursors occurs independent ly, and that their opioid peptide products have distinct ontogenetic profiles (Section 2.2.–2.4.). Indeed in a comparative immunocytochemical study for dynorphin-A and enkephalin36 the patterns of deveopment in the hippocampal formation were dissimilar. In addition the differences observed in ontogenetic patterns for the precursor products may well reflect differential activities of the processing cleavage enzymes in the developing animal. Indeed a comparative ontogenetic study of ACTH and β-endorphin64,65 showed a dissimilar profile during early development in rat brain and pituitary. Whilst these may be due to different processing patterns, the possibility of differential release, secretion or degradation of these peptides cannot be excluded and may be independent of the synthesis of the precursor. There is also a differential ontogeny of the opioid receptor subtypes and the recent evidence with highly selective binding ligands has provided a clear pattern, μ- And κ-sites are the first to appear though δ-receptors are absent until the second postnatal week in the rat. In this species the full development of μ- and δ-sites occurs in the third and fourth weeks respectively. There is still some argument about the ontogenetic profile for κ-sites though, like the δ-receptors, full development is probably later than for the μ-sites. Ontogenetic profiles of opioid receptors have also been demonstrated in the chick, sheep and mouse. Too few studies have looked at peptide and recep tor development in parallel and it is still unclear if specific peptide development is directly linked with development of a single receptor site. The postnatal development of both opioid peptides and receptors is mirrored by pharmacological actions of opioids which differ in the neonate. Their pharmacological effects are not solely dependent on the number of receptors but also on the ontogenetic pattern of the metabolising enzymes and on bloodbrain barrier development. There are some anomalous responses to opioids in the neonate and these include locomotion, respiratory depressant activity and effects on corticosterone release. There are also indications that the opioid system is particularly sensitive to insult during the developing period. In particular toxic effects at low doses have been shown for lead exposure, diazepam and haloperidol treatment. It may be that the complexity of opioid function increases the risk of disruption or it may reflect a ubiquitous involvement of the endogenous opioid peptides in the control of several neurochemica in the brain. The future needs more studies in animals other than the rat and a clearer picture of the genetic profile of the precursors and the processing of the same during development. This information should not be long in coming.

Interaction with vesicle luminal protachykinin regulates surface expression of delta-opioid receptors and opioid analgesia

Opioid and tachykinin systems are involved in modulation of pain transmission in the spinal cord. Regulation of surface opioid receptors on nociceptive afferents is critical for opioid analgesia. Plasma-membrane insertion of delta-opioid receptors (DORs) is induced by stimulus-triggered exocytosis of DOR-containing large dense-core vesicles (LDCVs), but how DORs become sorted into the regulated secretory pathway is unknown. Here we report that direct interaction between protachykinin and DOR is responsible for sorting of DORs into LDCVs, allowing stimulus-induced surface insertion of DORs and DOR-mediated spinal analgesia. This interaction is mediated by the substance P domain of protachykinin and the third luminal domain of DOR. Furthermore, deletion of the preprotachykinin A gene reduced stimulus-induced surface insertion of DORs and abolished DOR-mediated spinal analgesia and morphine tolerance. Thus, protachykinin is essential for modulation of the sensitivity of nociceptive afferents to opioids, and the opioid and tachykinin systems are directly linked by protachykinin/DOR interaction.

Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry. Implications for drug addiction, sleep and pain

Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A)receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.

Characterization of P1-purinoceptors on rat duodenum and urinary bladder.

1 The P1‐purinoceptors mediating relaxation of the rat duodenum and inhibition of contraction of the rat urinary bladder were characterized by use of adenosine and its analogues 5′‐N‐ethylcarboxamidoadenosine (NECA), N6‐cyclopentyladenosine (CPA) and 2‐p‐((carboxyethyl)phenethylamino)‐5′‐carboxamidoadenosine (CGS 21680), as well as the A1‐selective antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX). The stable analogue of adenosine 5′‐triphosphate (ATP), adenylyl 5′‐(β,γ‐methylene)diphosphonate (AMPPCP), was also used as previous work had indicated that it has a direct action on some P1 receptors in addition to its P2‐purinoceptor activity. 2 In the rat duodenum, the order of potency of the adenosine agonists was NECA ≥ CPA > AMPPCP = adenosine > CGS 21680, and DPCPX antagonized CPA and AMPPCP at a concentration of 1 nm whereas equivalent antagonism of NECA and adenosine required a concentration of 1 μm. This suggests the presence of a mixture of A1 and A2 receptors in this tissue, with CPA and AMPPCP acting on the A1 and NECA and adenosine acting on the A2 receptors. 3 In the rat bladder, the order of potency of the adenosine agonists for inhibition of carbachol‐induced contractions was NECA ≫ adenosine > CPA = CGS 21680, and a concentration of DPCPX of 1 μm was required to antagonize responses to NECA and adenosine. This suggests the presence of A2 receptors in this tissue. ATP and AMPPCP each caused contractions which were not enhanced by DPCPX (1 μm) which suggests that in this tissue AMPPCP was acting only via P2 receptors and had no P1 agonist activity. That AMPPCP was active on the A1 receptors in the duodenum but inactive on the A2 receptors in the bladder implies that it has selectivity for the A1 subtype. 4 That CGS 21680, which has been reported to bind selectively to the high affinity A2a subclass of A2 receptors, had a very low potency on the A2 receptors in the duodenum and in the bladder suggests that these receptors are of the low affinity A2b subclass.

Quantitative autoradiographic mapping of opioid receptors in the brain of δ-opioid receptor gene knockout mice

Using quantitative receptor autoradiography we have determined if deletion of the delta-opioid receptor gene (Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse delta-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. Delta-opioid receptors were labeled with [(3)H]deltorphin I (7 nM), mu- with [(3)H]DAMGO (4 nM), and kappa- with [(3)H]CI-977 (2.5 nM) or [(3)H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [(3)H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [(3)H]deltorphin I binding correlated significantly with those demonstrating high levels of the mu-receptor and were abolished in the presence of the mu-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [(3)H]DAMGO and [(3)H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [(3)H]bremazocine binding were higher in brains from -/- than +/+ mice. Our findings suggest that residual [(3)H]deltorphin I binding in the brain of delta-receptor gene knockout mice is the result of cross-reactivity with mu-sites and that there are no delta-receptor subtypes derived from a different gene. Changes in mu- and kappa-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the delta-receptor. The differences in [(3)H]CI-977 and [(3)H]bremazocine binding indicate these ligands show differential recognition of the kappa-receptor.

Antagonism of swim-stress-induced antinociception by the δ-opioid receptor antagonist naltrindole in adult and young rats

1 The availability of the non‐peptide δ‐opioid receptor antagonist naltrindole has provided the possibility for in vivo studies on the function of δ‐opioid receptors. We have studied the effects of naltrindole on swim‐stress‐induced antinociception in adult and neonatal rats. 2 Adult, 25 and 20 day old rats were stressed by warm water (20°C) swimming for 3 min periods and antinociception was assessed by the tail immersion test (50°C). 3 Naltrindole (0.5 and 1 mg kg−1) antagonized swim‐stress‐induced antinociception in adult and 25 day old rats but in 20 day old rats naltrindole (1 mg kg−l) was without effect. 4 Antinociception induced by the highly μ‐opioid receptor selective agonist alfentanil was completely antagonized by naloxone (1 mg kg−l) but virtually unaffected by naltrindole (1 mg kg−1). 5 Neither naloxone nor naltrindole (1 mg kg−1) antagonized swim‐stress‐induced rises in plasma corticosterone in adult rats at the time of peak antinociception. 6 In conclusion, naltrindole shows in vivo antagonism of opioid‐mediated responses. Swim‐stress‐induced antinociception is mediated through the δ‐opioid receptor in 25 day old and adult rats and through the μ‐opioid site in 20 day old animals.

Quantitative autoradiography of μ-,δ- and κ1 opioid receptors in κ-opioid receptor knockout mice

Abstract Mice deficient in the κ-opioid receptor (KOR) gene have recently been developed by the technique of homologous recombination and shown to lack behavioural responses to the selective κ1-receptor agonist U-50,488H. We have carried out quantitative autoradiography of μ-, δ- and κ1 receptors in the brains of wild-type (+/+), heterozygous (+/−) and homozygous (−/−) KOR knockout mice to determine if there is any compensatory expression of μ- and δ-receptor subtypes in mutant animals. Adjacent coronal sections were cut from the brains of +/+, +/− and −/− mice for the determination of binding of [ 3 H ]CI-977, [ 3 H ]DAMGO ( d -Ala2-MePhe4-Gly-ol5 enkephalin) or [ 3 H ]DELT-I ( d -Ala2 deltorphin I) to κ1-, μ- and δ-receptors, respectively. In +/− mice there was a decrease in [ 3 H ]CI-977 binding of approximately 50% whilst no κ1-receptors could be detected in any brain region of homozygous animals confirming the successful disruption of the KOR gene. There were no major changes in the number or distribution of μ- or δ-receptors in any brain region of mutant mice. There were, however some non-cortical regions where a small up-regulation of δ-receptors was observed in contrast to an opposing down-regulation of δ-receptors evident in μ-knockout brains. This effect was most notable in the nucleus accumbens and the vertical limb of the diagonal band, and suggests there may be functional interactions between μ- and δ-receptors and κ1- and δ-receptors in mouse brain.

Analysis of [3H] bremazocine binding in single and combinatorial opioid receptor knockout mice

Despite ample pharmacological evidence for the existence of multiple mu-, delta- and kappa-opioid receptor subtypes, only three genes encoding mu-(MOR), delta-(DOR) and kappa-(KOR) opioid receptor have been cloned. The KOR gene encodes kappa(1)-sites, which specifically bind arylacetamide compounds, and the possible existence of kappa-opioid receptor subtypes derived from another kappa-opioid-receptor gene, yet to be characterized, remains a very contentious issue. kappa(2)-Opioid receptors are described as binding sites typically labelled by the non-selective benzomorphan ligand [3H]bremazocine in the presence of mu-, delta- and kappa(1)-opioid receptor blocking ligands. To investigate the genetic origin of kappa(2)-opioid receptors, we have carried out homogenate binding experiments with [3H]bremazocine in brains of single MOR-, DOR-, KOR- and double MOR/DOR-deficient mice. Scatchard analysis showed that 68+/-12% of the binding sites arise from the MOR gene, 27+/-1% from the DOR gene and 14.5+/-0.2% from the KOR gene, indicating that the three known genes account for total [3H]bremazocine binding. Experiments in the presence of mu-, delta- and kappa(1)-opioid receptor suppressor ligands further showed that non-kappa(1)-opioid receptor labelling can be accounted for by binding to both the mu- and delta-opioid receptors. Finally, [3H]bremazocine binding experiments performed on brain membranes from the triple MOR/DOR/KOR-deficient mice revealed a complete absence of binding sites, confirming definitively that no additional gene is required to explain the total population of [3H]bremazocine binding sites. Altogether the data show that the putative kappa(2)-opioid receptors are in fact a mixed population of KOR, DOR and predominantly MOR gene products.

Behavioural effects of selective μ-, κ-, and δ-opioid agonists in neonatal rats

The behavioural effects of selective μ-, κ- and δ-opioid agonists in 5-, 10- and 20-day-old rats were investigated by observational analysis. The predominant response to μ-agonists was behavioural depression. High doses (10 mg/kg IP) of morphine and DAGO (d-Ala2, NMe-Phe4, Glyol5-enkephalin) produced overt sedation in all the age groups and also induced catalepsy which was particularly apparent in the 5- and 10-day-old animals. These compounds did not produce any signs of behavioural activation in the neonatal rats. In contrast, rat pups treated with the κ-agonists U50,488H and PD 117,302 (1,10 mg/kg IP) exhibited marked hyperactivity with increases in wall-climbing and locomotion. Sedative effects of the highest dose of the κ-agonists began to emerge, however, as the animals grew older, resulting in significant decreases in behaviours such as gnawing and grooming at 20 days of age. The κ-agonist (+)-tifluadom (0.1–10 mg/kg), but not its corresponding (-)-isomer, produced an increase in activity in 5-day-old rats, thus extending the observations made with U50,488H and PD 117,302 and establishing the stereoselective nature of the response. The involvement of κ-receptors in opioid-induced hyperactivity was further substantiated by using a variety of opioid antagonists. In this context, the increase in activity induced by U50,488H (10 mg/kg) in 5-day-old neonates was attenuated by naltrexone (1 mg/kg IP) but not by larger doses (10 mg/kg) of either M8008 (which has low affinity for κ-receptors) or the selective δ-receptor antagonist ICI 174,864. Finally, DPDPE (d-Pen2, d-Pen5-enkephalin) which acts selectively at δ-opioid receptors, did not exert any behavioural effects in either the 5-, 10- or 20-day-old rat pups at doses of up to 10 mg/kg. These results demonstrate behavioural effects of μ- and κ-but not δ-agonists in neonatal rats. There is a clear differentiation between μ- and κ-receptor effects and both μ- and κ-mediated behaviours show dissimilarities from the adult profile.