Postnatal development of δ-opioid receptor subtypes in mice

1. The density and affinity of binding sites for the δ-selective opioid ligands [³H]-[D-Ala², Asp⁴]deltorphin (DELT-I), [³H]-[D-Ala²Glu⁴]-deltorphin (DELT-II), [³H]-[D-Pen²,D-Pen⁵]enkephalin (DPDPE), and [³H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice.
2. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (B_max) labelled by [³H]-NTI was about one half that labelled by [³H]-DELT-I, [³H]-DELT-II and [³H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE B_max was 25% less than the deltorphin-II B_max.
3. In saturation experiments, specific binding of [³H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34% high affinity site, K_d=1.08 nM and 66% low affinity sites, K_d=39.9 nM).
4. DPDPE produced a biphasic inhibition of specific [³H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice.
5. In adult mouse brain labelled with [³H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites. DELT-I and DPDPE produced monophasic inhibition of specific [³H]-DELT-II binding to brain homogenates of adult mice.
6. These data suggest that a sub-population of δ-sites (probably the δ₂-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward.
7. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three δ-agonists was: DELT-I> DELT-II> DPDPE in 10 day-old mice, and: DELT-I= DELT-II> DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the δ₂-subtype.

     

Effects of κ-opioid receptor ligands on intracranial self-stimulation in rats

Rationale Elevations in cAMP response element binding protein (CREB) function within the mesolimbic system of rats reduce cocaine reward in place conditioning studies and increase immobility in the forced swim test. Each of these behavioral adaptations can be interpreted as a depressive-like effect (i.e., anhedonia, despair) that may reflect reduced activity of brain reward systems. Furthermore, each effect appears due to increases in CREB-mediated expression of dynorphin, since each is attenuated by intracranial injections of the -opioid receptor antagonist norBNI.Objectives Intracranial self-stimulation (ICSS) studies were conducted in rats to determine whether administration of a -agonist would have depressive-like effects on brain stimulation reward, and whether pretreatment with a -antagonist would attenuate any such effects. Conditions that have depressive effects in people (e.g., drug withdrawal) increase the threshold amounts of stimulation required to sustain ICSS in rats.Methods Sprague-Dawley rats with lateral hypothalamic stimulating electrodes were tested in a curve-shift variant of the ICSS procedure after systemic administration of the -agonist U-69593 alone, the novel -antagonist 5-acetamidinoethylnaltrindole (ANTI) alone, or co-administration of both drugs.Results U-69593 dose dependently increased ICSS thresholds, suggesting that activation of -receptors reduced the rewarding impact of the brain stimulation. ANTI had no effects on its own, but it attenuated increases in ICSS thresholds caused by the agonist.Conclusions These data provide further evidence that stimulation of brain -receptors may trigger certain depressive-like signs, and that antagonists may have efficacy as antidepressants without having reward-related actions of their own.     

Role of µ-opioid receptor reserve and µ-agonist efficacy as determinants of the effects of µ-agonists on intracranial self-stimulation in rats

The net effect of μ-opioid receptor agonists on intracranial self-stimulation (ICSS) in rats reflects an integration of rate-increasing and rate-decreasing effects. Previous opioid exposure is associated with tolerance to rate-decreasing effects and the augmented expression of abuse-related rate-increasing effects. This finding was replicated here with morphine. Subsequent studies then tested the hypothesis that opioid agonist-induced rate-decreasing effects require the activation of a larger relative fraction of μ receptors, and hence are more vulnerable to tolerance-associated reductions in receptor density than rate-increasing effects. Two sets of experiments were conducted to test this hypothesis. First, the effects of morphine on ICSS were examined after pretreatment with the irreversible μ antagonist β-funaltrexamine to reduce the density of available μ receptors. Second, effects were examined for a range of μ opioids that varied in relative efficacy at μ receptors. The hypothesis predicted that (a) morphine, after β-funaltrexamine treatment, or (b) low-efficacy μ agonists would mimic the effects of morphine tolerance to produce the reduced expression of rate-decreasing effects and enhanced expression of rate-increasing effects. Neither of these predictions were supported. These results indicate that μ agonist-induced facilitation and depression of ICSS may be mediated by distinct populations of μ receptors that respond differently to regimens of opioid exposure.     

Decreased µ-opioid receptor binding in the globus pallidus of rats treated with chronic haloperidol

RATIONALE: Chronic neuroleptic treatment produces a movement disorder in rats characterized by vacuous chewing movements (VCMs). Neuroleptics also produce a variety of changes in opioid neurotransmission in several regions of the basal ganglia. Rats with the VCM syndrome show elevated mRNA for enkephalin in striatopallidal neurons, suggesting a possible role for enkephalin in the pathophysiology of VCMs. OBJECTIVE: This study investigated the role of mu-opioid receptor density in the basal ganglia on the expression of VCMs. METHODS: Rats were treated with haloperidol for 24 weeks and withdrawn for 9 weeks. Mu (m) receptors were labeled with [3H]-DAMGO. RESULTS: Haloperidol treatment produced a significant reduction in mu-receptor binding in the globus pallidus (P<0.05). There was, however, no relationship between mu-opioid receptor density and VCMs in this or any other region of the basal ganglia. CONCLUSION: These results replicate prior findings of a neuroleptic-induced reduction in [3H]-DAMGO binding in the globus pallidus. The lack of association between VCMs and [3H]-DAMGO binding in the globus pallidus or any other region suggests that prior reports of enkephalinergic mRNA changes in the striatum are not accompanied by compensatory changes in postsynaptic neurons.     

Involvement of δ-opioid receptors in pentylenetetrazol kindling development and kindling-related processes in rats

The role of delta-opioid receptors on the development of kindling induced by the convulsant pentylenetetrazol (37.5 mg kg(-1) i.p.) was investigated in rats. Besides the seizure development, the kindling induced enhancement of glutamate binding and the kindling-induced learning deficit were examined. A clear depression of kindling development by blocking of delta-opioid receptors by intracerebroventricular administration of naltrindole (10 nmol/5 microl) was found. In an acute convulsion test performed 8 days after kindling completion, animals pretreated with naltrindole during kindling induction showed lower seizure stages compared to saline-pretreated kindled animals. The kindling-induced increase in hippocampal glutamate binding was completely prevented by naltrindole, whereas the kindling-induced learning deficit was not influenced. The learning performance of control animals pretreated with naltrindole was very low. It was hypothesized that the various consequences of kindling induction could be influenced separately. Summarizing the results, an involvement of the delta-opioid system in mechanisms underlying chemical kindling was clearly demonstrated. Interactions of endogenous opioid systems with glutamatergic transmission were suggested.     

The effect of pretreatment with a δ2-opioid receptor antisense oligodeoxynucleotide on the recovery from acute antinociceptive tolerance to δ2-opioid receptor agonist in the mouse spinal cord

1. An intrathecal (i.t.) injection of a selective δ₂-opioid receptor agonist, [D-Ala²]deltorphin II, produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D-Ala²]deltorphin II. This acute tolerance lasted 3 to 9 h and completely subsided by 12 h. The experiments were designed to examine the effect of pretreatment with an antisense oligodeoxynucleotide to δ₂-opioid receptor mRNA (δ-AS oligo) on the recovery from tolerance to [D-Ala²]deltorphin II-induced antinociception in male ICR mice.
2. Pretreatment with δ-AS oligo (1.63 to 163 pmol, i.t.), but not mismatched oligo (MM oligo) (163 pmol), prevented the recovery from acute tolerance to [D-Ala²]deltorphin II-induced antinociception in a dose-dependent manner. However, treatment with δ-AS oligo (163 pmol) did not prevent the recovery from tolerance to either the μ-opioid receptor agonist [D-Ala²,NMePhe⁴,Gly(ol)⁵]enkephalin (DAMGO) or the κ-opioid receptor agonist U50,488H, indicating subtype specificity in the mechanism by which δ-AS oligo inhibits recovery from δ₂-opioid tolerance.
3. Treatment with [D-Ala²]deltorphin II (i.t.) significantly reduced the binding of [tyrosyl-3,5-³H(N)]-Tyr-D-Ser-Gly-Phe-Leu-Thr ([³H]-DSLET), a δ₂-opioid receptor agonist ligand, in the spinal cord 3 h after treatment, but binding returned to control levels by 24 h after treatment. However, [³H]-DSLET binding in the spinal cord remained significantly reduced at 24 h if δ-AS oligo (163 pmol) was coadministered with [D-Ala²]deltorphin II (6.4 nmol).
4. Based on these findings, it is concluded that a single stimulation of spinal cord δ₂-opioid receptors by intrathecally-administered [D-Ala²]deltorphin II induces a long-lasting desensitization of δ₂-opioid receptors to [D-Ala²]deltorphin II. Recovery from δ₂-opioid receptor-mediated antinociceptive tolerance apparently depends on replenishment by newly synthesized δ₂-opioid receptor protein rather than immediate reversal of δ₂-opioid receptors.

     

Lack of involvement of δ-opioid receptor in mediating physical dependence at the hypothalamus-pituitary-adrenocortical (HPA) axis in the rat

1. In previous studies, we have demonstrated that δ-opioid receptors are involved both in the acute control of hypothalamus-pituitary-adrenocortical (HPA) axis activity and in the development of neuroendocrine opioid tolerance. In the present work we studied whether central δ-opioid receptors play a role in the development of neuroendocrine physical dependence to opioids in the rat.2. Intracerebroventricular (i.c.v.) administration of the δ-selective agonist DPDPE ([D-Pen²,D-Pen²]enkephalin) produced stimulation of HPA activity, as shown by an increase in corticosterone release. This effect was antagonized by i.c.v. co-administration of ICI 174,864, a selective δ-receptor antagonist, which provide direct evidence that the activation of the HPA axis produced by DPDPE is mediated by central δ-opioid receptor.3. Chronic pretreatment with i.c.v. DPDPE resulted in tolerance to its neuroendocrine effect. Intracerebroventricular injection of ICI 174,864 to DPDPE-tolerant rats produced neither alteration in corticosterone release nor behaviour signs of dependence.4. It was concluded that δ-opioid receptors do not play a role in the development of opioid neuroendocrine physical dependence at the HPA axis.     

μ-, δ- and κ-opioid receptor agonists do not alter the discriminative stimulus effects of cocaine or d-amphetamine in rats

Opioid receptor agonists can modulate the activity of dopamine neurons and could therefore, modify the behavioral effects of drugs that act through the dopamine systems, such as d-amphetamine and cocaine. We tested the ability of agonists selective for the μ- (morphine, methadone, buprenorphine, nalbuphine and heroin), δ- (DPDPE and SCH32615), and κ- (U69593 and bremazocine) opioid receptors to alter the discriminative stimulus effects of d-amphetamine and cocaine in rats. Separate groups of male Sprague-Dawley rats were trained to discriminate between 1.0 mg/kg d-amphetamine or 10 mg/kg cocaine from saline. Rats were pretreated with vehicle or an agonist, then dose-response curves for d-amphetamine or cocaine were generated. None of the opioid agonists changed significantly the ED₅₀ values of cocaine and d-amphetamine. As a positive control, we tested for antagonism of these effects by the D1 and D2 dopamine receptor antagonists, SCH23390 and eticlopride, respectively. Both antagonists at least partially attenuated the stimulus effects of both training drugs. Our results suggest that any modulation of dopaminergic neurotransmission by the agonists tested in the present study is not sufficient to affect the stimulus effects of d-amphetamine and cocaine in rats.     

Pharmacogenomics of the human µ-opioid receptor

The μ-opioid receptor is a primary target for clinically important opioid analgesics, including morphine, fentanyl and methadone. Many genetic variations have been identified in the human μ-opioid receptor MOP gene (OPRM1), and their implications have been reported in the effects of opioid drugs and susceptibility to drug dependence. Interestingly, agonistic and antagonistic opioid effects are inversely associated with the A118G polymorphism genotype. The A118G polymorphism may also be associated with substance dependence and susceptibility to other disorders, including epilepsy and schizophrenia. The IVS1+A21573G, IVS1-T17286C, and TAA+A5359G polymorphisms in the OPRM1 gene may be associated with alcohol, opioid and tobacco dependence, respectively. However, some studies have failed to confirm the correlations between the polymorphisms and opioid effects and substance dependence. Further studies are needed to elucidate the molecular mechanisms underlying the effects of OPRM1 polymorphisms.     

Agonists at the δ-opioid receptor modify the binding of µ-receptor agonists to the µ-δ receptor hetero-oligomer

BACKGROUND AND PURPOSE

µ- and δ-opioid receptors form heteromeric complexes with unique ligand binding and G protein-coupling profiles linked to G protein α z-subunit (Gα_z) activation. However, the mechanism of action of agonists and their regulation of the µ–δ receptor heteromer are not well understood.

EXPERIMENTAL APPROACH

Competition radioligand binding, cell surface receptor internalization in intact cells, confocal microscopy and receptor immunofluorescence techniques were employed to study the regulation of the µ–δ receptor heteromer in heterologous cells with and without agonist exposure.

KEY RESULTS

Gα_z enhanced affinity of some agonists at µ–δ receptor heteromers, independent of agonist chemical structure. δ-Opioid agonists displaced µ-agonist binding with high affinity from µ–δ heteromers, but not µ receptor homomers, suggestive of δ-agonists occupying a novel µ-receptor ligand binding pocket within the heteromers. Also, δ-agonists induced internalization of µ-opioid receptors in cells co-expressing µ- and δ-receptors, but not those expressing µ-receptors alone, indicative of µ–δ heteromer internalization. This dose-dependent, Pertussis toxin-resistant and clathrin- and dynamin-dependent effect required agonist occupancy of both µ- and δ-opioid receptors. In contrast to µ-receptor homomers, agonist-induced internalization of µ–δ heteromers persisted following chronic morphine exposure.

CONCLUSIONS AND IMPLICATIONS

The µ–δ receptor heteromer may contain a novel δ-agonist-detected, high-affinity, µ-receptor ligand binding pocket and is regulated differently from the µ-receptor homomer following chronic morphine exposure. Occupancy of both µ- and δ-receptor binding pockets is required for δ-agonist-induced endocytosis of µ–δ receptor heteromers. δ-Opioid agonists target µ–δ receptor heteromers, and thus have a broader pharmacological specificity than previously identified.     

The selective κ-opioid receptor antagonist JDTic attenuates the alcohol deprivation effect in rats

The mechanisms behind relapse to ethanol intake in recovering alcoholics are still unclear. The negative reinforcing effects contributing to ethanol addiction, including relapse, are considered to be partly driven by the κ-opioidergic system. As the κ-opioidergic system interacts with the mesolimbic reward pathway, the aim of the study was to clarify the role of nucleus accumbens shell κ-opioidergic mechanisms in relapse to ethanol intake by using the alcohol deprivation effect (ADE) paradigm. The ADE is defined as a transient increase in voluntary ethanol intake after a forced period of abstinence. Male Long-Evans rats were trained to voluntarily consume 10% (v/v) ethanol solution. Ethanol access and deprivation cycles were initiated after stable ethanol intake baselines had been reached and bilateral guide cannulas had been implanted above the nucleus accumbens shell. One cycle consisted of 10 days of 90 min access to ethanol followed by 6 days of ethanol deprivation. The ADE was measured in the beginning of a new cycle. Rats received JDTic, a selective κ-antagonist, either subcutaneously (10 mg/kg) or intra-accumbally (15 µg/site) or, as a reference substance, systemic naltrexone (0.3 mg/kg) before ethanol re-access, and the effects on the ADE were evaluated. Systemic and intra-accumbal JDTic significantly attenuated the ADE on the first day of ethanol re-access, as did systemic naltrexone. Additionally, naltrexone decreased ethanol intake levels. These results suggest that nucleus accumbens shell κ-opioidergic mechanisms may have a role in mediating relapse to ethanol intake. Additionally, κ-antagonism could be a valuable adjunct in ethanol relapse prevention.     

Effect of serotonergic lesion on “anxious” behaviour measured in the elevated plus-maze test in the rat

5,7-Dihydroxytryptamine (250 µg) was administered intracerebroventricularly to rats to lesion central serotonergic neurones. Fourteen days later the rats were tested in the elevated plus-maze anxiety model in comparison to sham lesioned animals. Twenty-four hours later, the rats were killed and serotonin levels and [³H]paroxetine binding measured in cortical and hippocampal membranes. The lesion destroyed 81% of the serotonergic innervation in the cortex and 99% in the hippocampus as determined by endogenous serotonin levels. Lesioned rats had an increased ratio of open/total arm entries in the elevated plus-maze, reflecting a decreased level of anxiety. These results are compatible with the implication of serotonin in the control of anxiety and suggest that an anxiolytic effect may be induced by lowering the level of serotonergic activation.     

Corticotropin-releasing factor antagonist attenuates the “anxiogenic-like” effect in the defensive burying paradigm but not in the elevated plus-maze following chronic cocaine in rats

Rationale: Chronic cocaine abuse is associated with the development of anxiogenic states in humans. Corticotropin-releasing factor (CRF) is an endogenous neurotropic factor well known to modulate stress responses. It has been postulated that CRF is involved in the neurobiological mechanisms underlying the anxiety and/or stress responses associated with removal of cocaine after chronic administration. Objective: The present study investigated the role of endogenous CRF in mediating the “anxiety-like” effect 48 h after the cessation of saline or chronic cocaine treatment in rats, using the defensive burying paradigm and the elevated plus-maze. Methods: Rats received daily injections of cocaine (20 mg/kg IP, for 14 consecutive days) or vehicle. Forty-eight hours after the last injection, animals were tested in the plus-maze and then in the defensive burying paradigm. In a second experiment, intracerebroventricular (ICV) cannulae were implanted at the lateral ventricle. Animals were allowed a 1-week period for recovery before starting the chronic drug treatment. The defensive burying testing took place 48 h after cessation of the treatment. The CRF antagonist [DPhe¹², Nle^21,38, C^αMe Leu³⁷] r/h CRF_(12–41), (also known as D-phe CRF_(12–41)) (0.04, 0.2 and 1.0 μg/5 μl) was injected 5 min before the 15-min testing. Results: An “anxiogenic-like” effect following chronic cocaine treatment was demonstrated with the defensive burying paradigm, but not with the elevated plus-maze. This “anxiety-like” response was attenuated by ICV pretreatment with the CRF antagonist D-Phe CRF_(12–41), with the highest dose of the CRF antagonist reversing the observed “anxiogenic-like” response. Conclusions: These data suggest that brain CRF may be substantially involved in the development of “anxiety-like” responses related to cocaine withdrawal and could be important for future drug dependence treatments. Received: 11 September 1997/Final version: 4 January 1999     

Respiratory and cardiovascular effects of the μ-opioid receptor agonist [Lys7]dermorphin in awake rats

1. Changes in respiratory variables, arterial blood pressure and heart rate were studied in awake rats after injection of the opioid peptide [Lys⁷]dermorphin and its main metabolites, [1-5]dermorphin and [1-4]dermorphin.
2. Fifteen minutes after injection, doses of [Lys⁷]dermorphin producing antinociception (i.c.v., 36–120 nmol; s.c., 0.12–4.7 μmol kg⁻¹) significantly increased respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. This respiratory stimulation was reversed to depression by the 5-HT receptor antagonist ritanserin (2 mg kg⁻¹, s.c.), was blocked by naloxone (0.1 mg kg⁻¹, s.c.), significantly reduced by the μ₁ opioid receptor antagonist naloxonazine (10 mg kg⁻¹, s.c., 24 h before) but unaffected by peripherally acting opioid antagonist naloxone methyl bromide (3 mg kg⁻¹, s.c.). Forty five minutes after injection, doses of the peptide producing catalepsy (s.c., 8.3–14.2 μmol kg⁻¹, i.c.v., 360 nmol) significantly reduced respiratory frequency and volume of rats breathing air and blocked the hypercapnic ventilator response of rats breathing from 4% to 10% CO₂. I.c.v. administration of [1-5]dermorphin and [1-4]dermorphin (from 36 to 360 nmol) never stimulated respiration but significantly reduced basal and CO₂-stimulated ventilation. Opioid respiratory depression was only antagonized by naloxone.
3. In awake rats, [Lys⁷]dermorphin (0.1–1 mg kg⁻¹, s.c.) decreased blood pressure. This hypotensive response was abolished by naloxone, reduced by naloxone methyl bromide and unaffected by naloxonazine.
4. In conclusion, the present study indicates that analgesic doses of [Lys⁷]dermorphin stimulate respiration by activating central μ₁ opioid receptors and this respiratory stimulation involves a forebrain 5-hydroxytryptaminergic excitatory pathway.

     

The involvement of μ- and κ- but not δ-opioid receptors in the body weight gain of suckling rats

The effects of the benzomorphan antagonist Mr 2266 and the selective -antagonist ICI 154,129 on the body weight gain of 6-day-old suckling rat pups was observed. Mr 2266 significantly reduced body weight gain in these animals, though ICI 154,129 had no affect on this variable. These findings suggest that - and - but probably not -opioic receptors are involved in the regulation of ingestive behaviours in infant rats. The results are discussed in relation to the development of opioid-receptor subtypes in the neonatal rat brain.     

The role of δ-opioid receptors in learning and memory underlying the development of addiction

Opioids are important endogenous ligands that exist in both invertebrates and vertebrates and signal by activation of opioid receptors to produce analgesia and reward or pleasure. The μ-opioid receptor is the best known of the opioid receptors and mediates the acute analgesic effects of opiates, while the δ-opioid receptor (DOR) has been less well studied and has been linked to effects that follow from chronic use of opiates such as stress, inflammation and anxiety. Recently, DORs have been shown to play an essential role in emotions and increasing evidence points to a role in learning actions and outcomes. The process of learning and memory in addiction has been proposed to involve strengthening of specific brain circuits when a drug is paired with a context or environment. The DOR is highly expressed in the hippocampus, amygdala, striatum and other basal ganglia structures known to participate in learning and memory. In this review, we will focus on the role of the DOR and its potential role in learning and memory underlying the development of addiction.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Effects of κ-opioid receptor stimulation in the heart and the involvement of protein kinase C

1. The role of protein kinase C (PKC) in mediating the action of κ-receptor stimulation on intracellular Ca²⁺ and cyclic AMP production was determined by studying the effects of trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ-receptor agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC agonist, on the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte.
2. U50,488H at 2.5–40 μM decreased both the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation dose-dependently, effects which PMA mimicked. The effects of the κ-agonist, that were blocked by a selective κ-antagonist, nor-binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of κ-receptor stimulation.
3. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca²⁺]_i transient induced by caffeine, that depletes the SR of Ca²⁺, was used as an indicator of Ca²⁺ content in the SR. The caffeine-induced [Ca²⁺]_i transient was significantly reduced by U50,488H at 20 μM. This effect of U50,488H on caffeine-induced [Ca²⁺]_i transient was significantly attenuated by 1 μM chelerythrine, indicating that the action of PKC involves mobilization of Ca²⁺ from the SR. When the increase in IP₃ production in response to κ-receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to κ-receptor stimulation does not involve IP₃. The observations suggest that PKC may act directly at the SR.
4. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of κ-receptor stimulation on Ca²⁺ in the SR and cyclic AMP production, both of which play an essential role in Ca²⁺ homeostasis in the heart.

     

Differential regulation of δ-opioid receptor trafficking after internalization by TIPP and DPDPE

AIM: To explore the mechanisms underlying the difference between TIPP and DPDPE in desensitization of the δ-opioidreceptors. METHODS: GH3 cells stably expressing HA-tagged δ-opioid receptors were treated with TIPP, DPDPE (1 μmol/L)or morphine (10 μmol/L) for different periods of time in the presence or absence of 50 μmol/L monensin or 10 nmol/L OA.Internalization of δ-opioid receptor was assessed using confocal