Antidepressive effects of the κ-opioid receptor agonist salvinorin A in a rat model of anhedonia

Salvinorin A (SalvA), the hallucinogenic derivative of the plant Salvia divinorum, is a selective κ-opioid receptor agonist that may also have antidepressant properties. Chronic mild stress (CMS) was applied to male and female Long-Evans rats to model anhedonia common in depression. The progressive loss in preference for a sucrose solution over plain water, a measure of anhedonia, and locomotor activity were monitored for 7 weeks. Because antidepressant medications often modify reproductive functions, endocrine glands and hormone-sensitive tissues were assessed at necropsy after the conclusion of the behavioral protocol. Three weeks of CMS exposure led to a decrease in sucrose preference. CMS was continued for 3 additional weeks and animals were randomly assigned to treatment with 1 mg SalvA/kg body weight or to a vehicle control group. The results indicate that SalvA reversed anhedonia whereas control animals continued to show a suppressed preference for the sucrose solution. In addition, no change in sucrose preference was observed in nonstressed rats that were exposed to the same dosage of SalvA. The results indicate that SalvA is an effective antidepressant agent when administered chronically to rats showing symptoms of depression similar to those observed in humans.     

Blockade of U50,488H analgesia by antisense oligodeoxynucleotides to a κ-opioid receptor

The recently cloned κ-opioid receptor has binding characteristics consistent with those of a κ₁-opioid receptor. Repeated intrathecal administration of an antisense oligodeoxynucleotide against the κ₁-opioid receptor selectively lowers U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetemide) analgesia (P < 0.02) without affecting μ or δ analgesia. A mismatched antisense oligodeoxynucleotide in which 4 bases had been switched is inactive against U50,488H analgesia. These studies confirm at the molecular level traditional pharmacological studies implying a distinct receptor mechanisms for κ₁ analgesia and demonstrate the utility of antisense approaches in studies of opioid pharmacology.     

Aversive properties of theκ opioid agonist U50,488 in the week-old rat pup

Opioids have been shown to produce analgesia and to be reinforcing during the first week of life in the rat. Opioids also have analgesic actions in both the infant and adult, but can be aversive in the mature animal. We examined the aversive effects of the opioid agonist U50,488 during the first postnatal week in the rat pup in three ways. In the first experiment, U50,488, injected peripherally (1.0–30.0 mg/kg), was paired with an odor and pups were tested 8 h later for positional preference for avoidance of that odor. This task is similar to conditioned preference/aversion tests used with adult animals. Both 3- and 7-day-old pups learned to avoid the odor adulterated side at the two higher doses. When exposed to odors previously associated with U50,488, pups at both ages decreased locomotor activity. In a second experiment, acute treatment with U50,488 increased ultrasonic distress vocalizations (USV) equally at 3 and 7 days of age, increased locomotor activity, and decreased rectal temperature. Neither of the latter two effects was correlated with the increase in USV production. The third experiment showed that conditioned odor cues increased USV 8 h later in 3- and 7-day-old pups at 1.0–10.0 mg/kg without changes in activity or rectal temperature. The results from these studies suggest that U50,488 can produce aversions in the neonatal rat pup as it does in the adult.Supported by U.S.P.H.S. grant DA-06600     

Possible involvement of the opioid receptor gene expression in the mechanisms of the analgesic action of melatonin

In the present study, we attempt to analyse the potential involvement of the opioid receptor gene expression in the mechanisms of the analgesic action of melatonin. A trauma-pain model was established in Wistar rats by combining right - hind limb amputation with 50℃ tail - flick test. Antinociception was determined by tail-flick latency to hot waster at 50℃. Melatonin produced the antinociceptive effect in dose-dependent manner after i. p or i. c.v. administration Injected i. c. v. to rats, naloxone（10μg） obviously antagonized the antinociceptive effect induced by i.p. melatonin.     

Modulation of κ-opioid receptor mediated tolerance in the guinea-pig ileum by chronic co-administration of dihydropyridines

The influence of the L-type Ca²⁺ channel modulators nimodipine (a Ca²⁺ blocker) and BAY K 8644 (a Ca²⁺ activator) on the expression of tolerance to the inhibitory effects of - and µ-opioid agonists in the guinea-pig ileum from guinea-pigs rendered tolerant to the -opioid receptor agonist U-50,488H was investigated. Tolerance to U-50,488H was induced by its administration (15 mg/kg twice a day) for 4 days. Control groups received saline at the same time schedule. Chronic infusion of guinea-pigs with nimodipine (2 /l/h for 7 days) or BAY K 8644 (0.5 /l/h for 7 days), did not cause any modification of the height of contractions induced by electrical stimulation of the myenteric plexus-longitudinal muscle (MPLM) preparation from naive guinea-pigs. The Ca²⁺ antagonist nimodipine increases the potency of U-50,488H (selective agonist) to reduce the amplitude of neurogenic contractions of the MPLM strip in naïve animals, whereas the Ca²⁺ activator BAY K 8644 induced the opposite effect. However, the effect of DAMGO (selective µ agonist) was not modified in guinea-pigs infused with nimodipine or BAY K 8644. Tolerance to the inhibitory effects of both U-50,488H and DAMGO was observed following administration of U-50,488H for 4 days and was revealed as a rightward shift of the concentration-response curves for the two agonists. Chronic infusion of guinea-pigs with nimodipine concurrently with chronic U-50,488H, markedly attenuated the expression of selective tolerance to U-50,488H as well as the cross-tolerance between U-50,488H and DAMGO. By the contrary, the magnitude of tolerance to U-50,488H and to DAMGO was enhanced by concomitant infusion of BAY K 8644. The results suggest that, in the GPI, -opioid receptor may be functionally linked to the dihydropyridine-sensitive Ca²⁺ channel: The blockade of the channel increased whereas its activation reduced the potency of U-50,488H. In chronic experiments, nimodipine prevented the expression of tolerance to U-50,488H and the cross-tolerance between U-50,488H and DAMGO, whereas BAY K 8644 produced the opposite effect. These results suggest that, in the GPI, selective tolerance to -agonist as well as cross-tolerance between - and µ-opioid agonists would involve activation of L-type Ca²⁺ channels, which could indicate that intracellular Ca²⁺ may be the final common pathway through which myenteric neurons adapt to the chronic opioid exposure.     

Effects of a newly synthesized κ-opioid receptor agonist, TRK-820, on the discriminative stimulus and rewarding effects of cocaine in rats

RATIONALE: We previously demonstrated that the prototypical kappa-opioid receptor agonist U-50,488H did not affect the discriminative stimulus effects of cocaine, and the dose of U-50,488H which significantly induced aversive effects attenuated the rewarding effects of cocaine. OBJECTIVES: In the present study, the effects of a newly synthesized kappa-opioid receptor agonist, TRK-820, on the discriminative stimulus and rewarding effects of cocaine were examined in rats. METHODS: In the drug discrimination procedure, the effects of TRK-820 on the discriminative stimulus effects of cocaine were examined in rats that had been trained to discriminate between 10 mg/kg cocaine and saline. TRK-820-induced place preference or place aversion and the effects of TRK-820 on the cocaine (4 mg/kg)-induced place preference were examined using a conditioned place preference procedure in rats. RESULTS: TRK-820 did not engender cocaine-like responding in rats trained to discriminate between 10 mg/kg cocaine and saline. In combination tests, low doses of TRK-820, which did not affect the response rate, significantly attenuated the discriminative stimulus effects of cocaine, and these effects of TRK-820 were reversed by a kappa-opioid receptor antagonist, nor-BNI. In the conditioned place preference procedure, low doses of TRK-820, which did not affect the response rate in the drug discrimination, did not produce either place preference or place aversion, whereas, higher dose (80 microg/kg) of TRK-820 slightly but significantly induced a place aversion. Under these conditions, the cocaine-induced place preference was completely attenuated by low doses of TRK-820. These results may prompt further investigation of the effectiveness of the new kappa-opioid receptor agonist TRK-820 as a novel pharmacotherapeutic compound for the treatment of cocaine addiction.     

Molecular determinants of selective agonist and antagonist binding to the histamine H₄ receptor

The deorphanization of the histamine H? receptor (H?R) has led to a significant number of scientific publications and patent applications. Whereas some histamine H?, H? and H? receptor ligands were found to have significant affinity for H?R, several agonists and antagonists with high affinity for H?R and selectivity over the other histamine receptors were successfully designed and synthesized. Moreover, site-directed mutation studies on H?R have been performed and reveal detailed information on receptor-ligand interactions. This review will focus on the most important H?R ligand scaffolds and their structure-activity relationships and selectivity over other histamine receptors and specific H?R functional activity. Experimental data are used to construct and validate high resolution three-dimensional receptor-ligand models and, vice versa, in silico models are used to design and rationalize experimental studies to probe receptor-ligand interactions.     

Effectiveness of nalbuphine,a κ-opioid receptor agonist and μ-opioid receptor antagonist,in the inhibition of INa,IK(M), and IK(erg) unlinked to interaction with opioid receptors

Nalbuphine (NAL) is recognized as a mixer with the κ-opioid receptor agonist and the μ-opioid receptor antagonist. However, whether this drug causes any modifications in neuronal ionic currents is unclear. The effects of NAL on ionic currents in mHippoE-14 hippocampal neurons were investigated. In the whole-cell current recordings, NAL suppressed the peak amplitude of voltage-gated Na⁺ current (I_Na) with an IC₅₀ value of 1.9 μM. It shifted the steady-state inactivation curve of peak I_Na to the hyperpolarized potential, suggesting that there is the voltage dependence of NAL-mediated inhibition of peak I_Na. In continued presence of NAL, subsequent application of either dynorphin A_1-13 (1 μM) or naloxone (30 μM) failed to modify its suppression of peak I_Na. Tefluthrin (Tef; 10 μM), a pyrethroid known to activate I_Na, increased peak I_Na with slowed current inactivation; however, further application of NAL suppressed Tef-mediated suppression of peak I_Na followed by an additional slowing of current inactivation. In addition, NAL suppressed the amplitude of M-type K⁺ current [I_K(M)] with an IC₅₀ value of 5.7 μM, while it slightly suppressed erg-mediated and delayed-rectifier K⁺ currents. In the inside-out current recordings, NAL failed to modify the activity of large-conductance Ca²⁺-activated K⁺ channels. In differentiated NG108-15 neuronal cells, NAL also suppressed the peak I_Na, and subsequent addition of Tef reversed NAL-induced suppression of I_Na. Our study highlights the evidence that in addition to modulate opioid receptors, NAL has the propensity to interfere with ionic currents including I_Na and I_K(M), thereby influencing the functional activities of central neurons.     

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.

     

The effects of spiradoline (U-62066E), a κ-opioid receptor agonist,on neuroendocrine function in man

1. Opioid drugs act on specific receptors which are principally classified into μ, δ and κ subtypes. Spiradoline (U-62066E) is a κ-selective agent which has been shown to possess potent anti-nociceptive effects but does not show cross tolerance with morphine.
2. We have assessed the neuroendocrine effects of spiradoline in healthy volunteers with two doses (1.6 and 4.0 μg kg⁻¹, i.m.) of the compound. Six male non-smokers aged 19–27 years were studied by use of a randomized, double-blind three-limb placebo-controlled cross-over design. Blood was taken from an in-dwelling venous cannula basally and at 15 min intervals for 2 h for determination of serum cortisol, prolactin, growth hormone (GH) and catecholamines.
3. Psychological function was assessed by the Stanford Sleepiness Scale (SSS) and the Addiction Research Centre Inventory (ARCI) administered before the medication and at 35 min, 1 h 25 min and 2 h afterwards. Cardiovascular variables were recorded at 10 min intervals. Results were analysed by analysis of variance.
4. Spiradoline showed a significant (P<0.05) dose-dependent increase in free water clearance, as predicted for a κ-opioid agonist. It also caused a dose-dependent stimulation of prolactin, (increment over baseline for higher dose 214%), GH (433%) and cortisol (215%) release (P<0.05). There were no significant drug-related changes in plasma catecholamines, blood pressure, pulse or psychological variables.
5. We have therefore confirmed that κ-opioids increase free-water clearance and may participate in the stimulation of prolactin and GH release. In contrast to μ and δ-opioid agonists, this novel κ-agonist stimulates cortisol release in man.

     

Estradiol and testosterone modulate the anesthetic action of the GABA-A agonist THIP,but not of the neurosteroid 3α,5β-pregnanolone in the rat

Rationale As sex steroids modify the number and distribution of brain -aminobutyric acid (GABA)_A receptor subunits, we investigated the potential modulation of anesthesia, induced by agents acting on the GABA_A receptor, by estrogen and androgen.Objectives To assess possible effects of sex and hormonal condition (i.e., intact vs castrate; estradiol vs testosterone treatment) on the anesthetic effect of a GABA_A agonist, THIP (4,5,6,7-tetrahydroisoxazolo[5,4,-c]pyridin-3-ol hydrochloride), and an allosteric modulator of the GABA_A receptor: 3-hydroxy-5-pregnan-20-one (epipregnanolone).Methods The potencies of THIP and epipregnanolone for inducing loss of the righting response were compared between: (a) female and male rats; (b) intact and castrated animals of each sex; (c) untreated castrates and castrates given estradiol or testosterone.Results Sex and endocrine condition influenced sensitivity to i.v. THIP for the induction of anesthesia. ED₅₀ values were: gonadectomized females, 80 mg/kg > intact males, 50 mg/kg > proestrous females, 35 mg/kg > gonadectomized males, 28 mg/kg. Estradiol benzoate (EB; 3 g/day for 5 days) significantly increased THIP sensitivity in gonadectomized females: THIP + EB: ED₅₀=26 mg/kg vs THIP + sesame oil: ED₅₀=94 mg/kg, while testosterone propionate (TP; 10 mg injected 24 h before THIP) decreased THIP sensitivity in orchidectomized males when compared with vehicle-injected animals (ED₅₀=72 mg/kg vs 22 mg/kg, respectively).Conclusions Results suggest that estrogen increases the density or availability of GABA_A receptor subtypes on which THIP acts, while testosterone exerts the opposite effect. Neither sex nor gonadal condition influenced the anesthetic action of epipregnanolone as a similar potency was found in intact and in gonadectomized males and females.     

The role of α2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent,clozapine, in the rat

1. The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat.
2. The close structural analogues of clozapine, loxapine (0.1 mg kg⁻¹ s.c.) and iso-clozapine (1 and 3 mg kg⁻¹ s.c.) induced catalepsy in rats. In contrast, clozapine and the regio-isomer of loxapine, iso-loxapine (up to 10 mg kg⁻¹ s.c.) did not produce catalepsy, but at a dose of 1 mg kg⁻¹ significantly inhibited catalepsy induced by loxapine (0.3 mg kg⁻¹ s.c.).
3. Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D₂/5-HT_1A, D₂/5-HT_1B/1D and D₂/α₂-receptor affinity (K_D) ratios: i.e. 30–100-fold higher ratios were calculated for loxapine and iso-clozapine, whereas the ratios were less than 1 for clozapine and iso-loxapine. The ratios of affinities for D₂ to 5-HT_2A, 5-HT_2C or D₁ did not reflect the grouping of cataleptic and non-cataleptic compounds.
4. Co-treatment with the α₂-adrenoceptor antagonists, yohimbine (1–10 mg kg⁻¹ s.c.), RX 821002 (1–10 mg kg⁻¹ s.c.) and MK-912 (0.3 and 1 mg kg⁻¹ s.c.) dose-dependently inhibited the cataleptic response to loxapine (0.3 mg kg⁻¹). Yohimbine (1–10 mg kg⁻¹ s.c.) also dose-dependently inhibited the cateleptic response to haloperidol (0.3 mg kg⁻¹ s.c.). The α₂-adrenoceptor antagonists had no effect per se.
5. Neither yohimbine (10 mg kg⁻¹) nor RX821002 (3 mg kg⁻¹) altered the cataleptic response to the D₁ receptor antagonist, SCH 23390 (1 mg kg⁻¹ s.c.), while, like clozapine, both compounds abolished the response to the 5-HT_2A receptor antagonist, MDL 100,151 (3 mg kg⁻¹ s.c.).
6. The present data strongly implicate α₂-adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

     

Inhibition of protein kinase C,but not of protein kinase A,blocks the development of acute antinociceptive tolerance to an intrathecally administered μ-opioid receptor agonist in the mouse

A specific protein kinase C inhibitor, calphostin C, which injected alone had no effect on the antinociception induced by intrathecal (i.t.) administration of a selective μ-opioid receptor agonist, [d-Ala²,NMePhe⁴,Gly(ol)⁵]enkephalin (DAMGO), dose-dependently attenuated the development of acute tolerance to the i.t. DAMGO-induced antinociception in male ICR mice. On the other hand, a selective protein kinase A inhibitor, KT5720, did not have any effect on the development of acute tolerance to DAMGO antinociception. These findings suggest that protein kinase C, but not protein kinase A, plays an important role in the development of acute tolerance to the μ-opioid receptor agonist-induced antinociception.     

Failure ofγ-hydroxybutyrate to alter the function of the GABAA receptor complex in the rat cerebral cortex

The present study was designed to evaluate the possible interaction of-hydroxybutyrate (GHB) with the GABA_A receptor complex in the rat cerebral cortex. To this purpose we studied the effect of in vitro addition and in vivo administration of GHB on the biochemical parameters currently used to evaluate the function of the GABAergic system. In vitro addition of increasing concentrations of GHB failed to modify [³H]flunitrazepam (³H]FNZ) binding and the modulatory action of GABA on this binding. Moreover, unlike diazepam, GHB did not modify in vitro both muscimol-stimulated³⁶Cl^– uptake and t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to rat cerebral cortex. In vivo administration of sedative and hypnotic doses of GHB (300–750 mg/kg IP) failed to induce in 60 min any significant change in the [³⁵S]TBPS binding to unwashed cortical membranes. Moreover, GHB also failed to antagonize the increase in [³⁵S]TBPS binding (+55%) induced by isoniazid (350 mg/kg SC). In contrast, at the highest doses used, this drug completely antagonized the seizure activity induced by isoniazid. In conclusion, our data show that GHB fails to alter the function of the GABA_A/benzodiazepine/ionophore receptor complex in the rat cerebral cortex.     

Anti-arrhythmic effects of levcromakalim in the ischaemic rat heart: a dual mechanism of action?

The action of pharmacological openers of K(ATP) channels depends on the availability and levels of various intracellular nucleotides. Since these are subject to change during myocardial ischaemia, K(ATP) channel openers may affect ischaemic and non-ischaemic tissue differentially. Using a recently developed dual coronary perfusion method, we investigated the effects on arrhythmias of the prototypical K(ATP) channel opener levcromakalim when applied selectively to ischaemic and/or non-ischaemic tissue. A novel perfusion cannula was used to independently perfuse the left and right coronary beds of hearts isolated from rats. Selective infusion of levcromakalim (3, 10 or 30 microM) into the left coronary bed in the absence of ischaemia did not induce ventricular arrhythmias. Regional zero-flow ischaemia was induced by cessation of flow to the left coronary bed and hearts received levcromakalim selectively into either the left, right, or both coronary beds. When applied selectively to the ischaemic left coronary bed, levcromakalim (3, 10 or 30 microM; n=10/group) delayed the onset of ventricular tachycardia in a dose-dependent manner (by 21*, 43* and 112%* at 3, 10 and 30 microM; *P<0.05 vs. control). When applied only to the non-ischaemic right coronary bed, levcromakalim reduced the incidence of ventricular tachycardia during later phases of ischaemia (from 100% in controls to 30%*). When present in both coronary beds, levcromakalim had a striking anti-arrhythmic effect--the overall incidence of ventricular tachycardia being reduced from 100% in controls to 20%*. We conclude that levcromakalim may have an anti-arrhythmic effect when applied either to ischaemic or non-ischaemic tissue but that the mechanisms may differ depending on the metabolic state of the heart.     

Haemodynamic and tolerance studies in man of a new,orally active,selectiveβ 1-adrenoceptor agonist H 80/62

Summary The selective ₁-adrenoceptor agonist H 80/62 was administered intravenously and orally to healthy subjects and its effects on systolic time intervals, arterial blood pressure and heart rate were studied. Side-effects were noted too, and continuous ECG-recordings were made in order to study its arrhythmogenic effect. After i.v. administration of H 80/62 20 µg/kg body weight there was shortening of total electromechanical systole, the pre-ejection period and of the left ventricular ejection time, systolic blood pressure tended to increase, and diastolic blood pressure and heart rate were essentially unchanged. When administered orally as a sustained-release preparation in doses between 20 and 40 mg the haemodynamic effects were qualitatively the same as after i.v. administration, but in some studies there was a slight increase in heart rate. During exercise the systolic blood pressure and heart rate were identical after H 80/62 and placebo. The effect of the drug was maximal immediately after cessation of the i.v. infusion and basal values were regained within 60 min. After oral administration of a sustained-release formulation the effect was maximal after one hour and persisted for at least five to seven hours. The drug was well tolerated on repeated administration. The incidence of ventricular extrasystoles was possibly increased in one subject out of eight (11 ventricular extrasystoles during 18 h). The results of this Phase I study of H 80/62 warrant further evaluation of the drug in man.     

Is inositol 145-trisphosphate a second messenger coupled to kappa-opioid receptor in cardiac myocytes of the rat?

AIM: To elucidate whether inositol 1,4, 5-trisphosphate (IP_3) is a second messenger linking activation of kappareceptor to the release of Ca~(2 ) from sarcoplasmic reticulum in rat cardiac myocytes. METHODS: Fura-2 fluorimetric     

Involvement of neuronal TGF-β activated kinase 1 in the development of tolerance to morphine-inducedantinociception in rat spinal cord

Background and Purpose

Tolerance induced by morphine and other opiates remains a major unresolved problem in the clinical management of pain. There is now good evidence for the importance of MAPKs in morphine-induced antinociceptive tolerance. A member of the MAPK kinase kinase family, TGF-β activated kinase 1 (TAK1) is the common upstream kinase of MAPKs. Here, we have assessed the involvement of TAK1 in the development of tolerance to morphine-induced analgesia.

Experimental Approach

The effects of an antagonist of TAK1 on morphine tolerance were investigated in vivo using the Randall–Selitto test, and the mechanism was investigated using Western blot and immunohistochemistry. The expression of TAK1 after chronic morphine exposure was also evaluated in vitro by immunohistochemistry.

Key Results

Chronic intrathecal morphine exposure up-regulated protein levels and phosphorylation of spinal TAK1. TAK1 immunoreactivity was co-localized with the neuronal marker NeuN. Intrathecal administration of 5Z-7-oxozeaenol (OZ), a selective TAK1 inhibitor, attenuated the loss of morphine analgesic potency and morphine-induced TAK1 up-regulation. Furthermore, OZ decreased the up-regulated expression of spinal p38 and JNK after repeated morphine exposure. In vitro studies demonstrated that sustained morphine treatment induced TAK1 up-regulation, which was reversed by co-administration of OZ. A bolus injection of OZ showed some reversal of established morphine antinociceptive tolerance.

Conclusions and Implications

TAK1 played a pivotal role in the development of morphine-induced antinociceptive tolerance. Modulation of TAK1 activation by the selective inhibitor OZ in the lumbar spinal cord may prove to be an attractive adjuvant therapy to attenuate such tolerance.     

Protein kinase A signalling is involved in the relaxant responses to the selective β-oestrogen receptor agonist diarylpropionitrile in rat aortic smooth muscle in vitro

Objectives The oestrogen receptor β (ERβ) selective agonist diarylpropionitrile (DPN) relaxes endothelium‐denuded rat aorta, but the signalling mechanism is unknown. The aim of this study was to assess whether protein kinase A (PKA) signalling is involved in DPN action. Methods cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry. Key findings DPN increased cAMP content and hyperpolarised cell membranes over the same range of concentrations as it relaxed phenylephrine‐precontracted aortic rings (10–300 µM). DPN‐induced vasorelaxation was largely reduced by the PKA inhibitors Rp‐8‐Br‐cAMPS (8‐bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Rp‐isomer) and H‐89 (N‐(2‐bromocynnamyl(amino)ethyl)‐5‐isoquinoline sulfonamide HCl) (?73%) and by the adenylate cyclase inhibitor MDL12330A (cis‐N‐(2‐phenylcyclopentyl)‐azacyclotridec‐1‐en‐2‐amine)) (?65.5%). Conversely, the PKG inhibitor Rp‐8‐Br‐cGMP was inactive against DPN vasorelaxation. In aortic smooth muscle segments, DPN increased PKA‐dependent HSP20 phosphorylation, an effect reversed by H‐89. Relaxant responses to DPN were modestly antagonised (?23 to ?48% reduction; n = 12 per compound) by the potassium channel inhibitors iberiotoxin, PNU‐37883A, 4‐aminopyridine, or BaCl₂. All four potassium channel inhibitors together reduced DPN relaxation by 86 ± 9% (n = 12) and fully blocked DPN hyperpolarisation. Conclusions ERβ‐dependent relaxation of rat aortic smooth muscle evokes an adenylate cyclase/cAMP/PKA signalling pathway, likely activating the cystic fibrosis transmembrane conductance regulator chloride channel and at least four potassium channels.     

Identification of human cytochrome P450 enzymes involved in the metabolism of a novel к-opioid receptor agonist, nalfurafine hydrochloride

Nalfurafine hydrochloride (TRK-820) exhibits strong к-opioid agonistic activity and is a new antipruritic agent for uremic pruritus. This study was performed to identify the human hepatic cytochrome P450 isoforms involved in the metabolic conversion of nalfurafine to the decyclopropylmethylated form, de-CPM, using human liver microsomes and E. coli membrane fractions expressing human P450 isoforms. Samples were analysed by liquid chromatography with a radioactivity detector and liquid chromatography-tandem mass spectrometry. The metabolism of nalfurafine by human liver microsomes exhibited a biphasic kinetic profile. Experiments examining the metabolism by E. coli membrane fractions expressing human P450 isoforms indicated that CYP1A1, 2C8, 2C19 and 3A4 had the ability to produce de-CPM. In experiments with human liver microsomes that examined the inhibition of nalfurafine metabolism by anti-human P450 antibodies, anti-CYP3A4 antibody predominantly, and anti-CYP2C8 and 2C19 antibodies moderately, inhibited de-CPM formation. From these results, CYP3A4 appeared to be the major isoform involved in the metabolic decyclopropylmethylation of nalfurafine, while CYP2C8 and 2C19 most likely play a minor role in the formation of de-CPM.