Effects of the local administration of selective μ-, δ-and κ-opioid receptor agonists on osteosarcoma-induced hyperalgesia

The stimulation of peripheral opioid receptors yields analgesic responses in a model of bone cancer-induced pain in mice. In order to know the type(s) of peripheral opiate receptors involved, the paw thermal withdrawal latencies were measured in C3H/HeJ mice bearing a tibial osteosarcoma, after administering selective agonists of μ-,δ-and κ-opiate receptors. The peritumoral administration of DAGO (0.6–6 μg) inhibited the osteosarcoma-induced hyperalgesia at doses ineffective in healthy animals, the highest one even increasing the withdrawal latencies over the control values. Naloxone-methiodide (2 mg/kg) and cyprodime (1 mg/kg), but not naltrindole (0.1 mg/kg) nor nor-binaltorphimine (10 mg/kg), antagonized DAGO-induced analgesic effects, these therefore probably being mediated through peripheral μ-opioid receptors. The peritumoral injection of DPDPE (100 μg) induced analgesia which was inhibited by naloxone-methiodide and naltrindole but not by nor-binaltorphimine. Cyprodime partially antagonized the analgesia induced by 100 μg of DPDPE, but did not modify the effect induced by 30 μg of this agonist—a dose that restores the hyperalgesic latencies up to the control values. The antihyperalgesic effect induced by the peritumoral administration of U-50,488H (1 μg) was antagonized by naloxone-methiodide and nor-binaltorphimine, but not by cyprodime nor naltrindole, thus suggesting the involvement of peripheral κ-opioid receptors. In conclusion, the stimulation of peripheral μ-, δ- and κ-opioid receptors is a pharmacological strategy useful for relieving this experimental type of bone cancer-induced pain, the greatest analgesic effect being achieved by stimulating peripheral μ-opioid receptors.     

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.     

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.     

Demonstration of the heterogeneity of the κ-opioid receptors in guinea-pig cerebellum using selective and nonselective drugs

In guinea-pig cerebellum, saturation studies reveal that the nonselective opioid [³H]ethylketazocine has a binding capacity (R) of 6.79 pmol/g tissue which is similar to the sum of the individual R values of the μ-, δ- and κ₁-selective opioids. Conversely, the binding parameters of the nonselective opioid [³H]bremazocine are best-fitted to a two-site model (K_d1 = 0.12 nM, R₁ = 11.3 pmol/g tissue; K_d2 = 6.03 nM, R₂ = 9.09 pmol/g tissue) with an R_TOTAL value of 20.3 pmol/g tissue which is statistically different from the R value of [³H]ethylketazocine or the sum of R_μ + R_δ + R_κ1. This suggests that [³H]bremazocine labels additional opioid binding sites. After suppression of the μ-, δ- and κ₁-receptors, [³H]bremazocine binding is then best-fitted to a one-site model with a K_d value of 1.48 nM and an R value of 11.2 pmol/g tissue. Competition studies done against the binding of [³H]U69593 indicate that the opioid receptors labelled with this ligand are related to the κ₁-receptor subtype. However, competition studies performed against the binding of [³H]bremazocine (under suppressed conditions) display a pharmacological profile related to another subtype of κ-receptors previously described in guinea-pig brain as the κ₂-receptors.     

Identification of a κ-opioid agonist as a potent and selective lead for drug development against human African trypanosomiasis

A resazurin-based cell viability assay was developed for phenotypic screening of the LOPAC 1280 ‘library of pharmacologically active compounds’ against bloodstream forms of Trypanosoma brucei in vitro identifying 33 compounds with EC₅₀ values <1 μM. Counter-screening vs. normal diploid human fibroblasts (MRC5 cells) was used to rank these hits for selectivity, with the most potent (<70 nM) and selective (>700-fold) compounds being suramin and pentamidine. These are well-known antitrypanosomal drugs which demonstrate the robustness of the resazurin cell viability assay. The most selective novel inhibitor was (+)-trans-(1R,2R)-U50,488 having an EC₅₀ value of 60 nM against T. brucei and 270-fold selectivity over human fibroblasts. Interestingly, (−)-U50,488, a known CNS-active κ-opioid receptor agonist and other structurally related compounds were >70-fold less active or inactive, as were several μ- and κ-opioid antagonists. Although (+)-U50,488 was well tolerated by the oral route and displayed good pharmaceutical properties, including high brain penetration, the compound was not curative in the mouse model of infection. Nonetheless, the divergence of antinociceptive and antitrypanosomal activity represents a promising start point for further exploratory chemistry. Bioinformatic studies did not reveal any obvious candidate opioid receptors and the target of this cytostatic compound is unknown. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism.     

Minimal contribution of P-gp on the low brain distribution of naldemedine,a peripherally acting μ-opioid receptor antagonist

Naldemedine tosylate, a peripherally acting μ-opioid receptor antagonist, is indicated for treatment of opioid induced constipation in both Japan and US. Naldemedine has limited ability to affect the central analgesic effect of opioid analgesics. In this study, we investigated the contribution of P-glycoprotein (P-gp) on the brain distribution of naldemedine. Naldemedine tosylate showed acceptable oral absorption in rats. Following a single oral administration of [¹⁴C]-naldemedine tosylate to rats and ferrets, little radioactivity was detected in the region protected by the blood-brain barrier (BBB). In the assessment using Caco-2 cells, it was determined that naldemedine is a substrate for P-gp. The contribution of P-gp to the brain distribution of naldemedine was assessed using multidrug resistance 1a/b (mdr1a/b) knockout mice. While the brain-to-plasma concentration ratio (brain Kp) of naldemedine in the mdr1a/b knockout mice was 4-fold of that in the wild-type mice, the brain Kp in the mdr1a/b knockout mice was quite low (brain Kp < 0.1). These results suggest that the low brain distribution of naldemedine was due to the limited ability to cross the BBB rather than efflux by P-gp and therefore brain distribution of naldemedine would not be affected by concomitant administration of P-gp inhibitors or functional disorder of P-gp.     

Lack of a rewarding effect and a locomotor-enhancing effect of the selective μ-opioid receptor agonist amidino-TAPA

Rationale and objectives

Psychological dependence is one of the worst side effects of morphine. It limits the clinical availability of morphine and non-patient morphine users suffer from addiction. An analgesic, which is more potent than morphine but without the liability of psychological dependence, has long been sought in the clinic. We have recently developed a new μ-opioid receptor agonist, N^α-amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA), as a potent analgesic with an antinociceptive profile that is distinct from morphine, including the release of endogenous κ-opioid peptides. The activation of κ-opioid receptors has been suggested to suppress the development of psychological dependence by μ-opioid receptor agonists. In the present study, the psychological dependence liability and the related locomotor-enhancing effect of amidino-TAPA were evaluated.

Results

Amidino-TAPA injected subcutaneously produced an extremely potent and longer lasting antinociception than morphine in ddY mice, prodynorphin-knockout mice, and wild-type C57BL/6J mice. Unlike subcutaneously injected morphine, which had potent locomotor-enhancing and rewarding effects at antinociceptive doses in ddY mice, amidino-TAPA injected subcutaneously did not induce significant locomotor-enhancing and rewarding effects at antinociceptive or even higher doses in ddY mice. In wild-type C57BL/6J mice, amidino-TAPA showed the same pharmacological profile (potent antinociception, lack of locomotor-enhancing and rewarding effects) as in ddY mice. However, amidino-TAPA produced potent locomotor-enhancing and rewarding effects at antinociceptive doses in prodynorphin-knockout mice.

Conclusions

The present results suggest that amidino-TAPA is a potent analgesic without the liability of psychological dependence because it releases endogenous κ-opioid peptides.     

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.

     

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

The behavioural effects of selective -, - and -opioid agonists in 5-, 10- and 20-day-old rats were investigated by observational analysis. The predominant response to -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 -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 -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 -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 -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 -receptors) or the selective -receptor antagonist ICI 174,864. Finally, DPDPE (d-Pen², d-Pen⁵-enkephalin) which acts selectively at -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 - and -but not -agonists in neonatal rats. There is a clear differentiation between - and -receptor effects and both - and -mediated behaviours show dissimilarities from the adult profile.     

Effect of the milk extract of Semecarpus anacardium nut on adjuvant arthritis—A dose-dependent study in wistar albino rats

• 1.1. Adjuvant-induced arthritis in rats is used as a pathologic model for chronic inflammatory disease to evaluate the efficacy of therapeutic agents.
• 2.2. In the present work, attempts have been made to study the potency of a milk extract of Semecarpus anacardium (Serankottai Nei), a Siddha preparation from Semecarpus anacardium nut, which has been shown to have antiarthritic effects.
• 3.3. Experimental arthritis induces a significant modification in lysosomal enzyme release and total carbohydrate components of glycoprotein.
• 4.4. Milk extract was administered at the dose level of 50, 100, 150, 200, and 250 mg/kg body weight in olive oil orally (volume 0.5 ml) after 14 days from the day of adjuvant injection.
• 5.5. After administration of the extract the lysosomal enzyme activity and protein-bound carbohydrate component levels were significantly normalized.
• 6.6. The data obtained clearly indicate that the Semecarpus anacardium is effective at the dose level of 150 mg/kg body weight in adjuvant-induced arthritis in albino Wistar rats.

     

The atypical antidepressant mianserin exhibits agonist activity at κ-opioid receptors

BACKGROUND AND PURPOSE

Antidepressants are known to interact with the opioid system through mechanisms not completely understood. We previously reported that tricyclic antidepressants act as agonists at distinct opioid receptors. Here, we investigated the effect of the atypical antidepressant mianserin at cloned and native opioid receptors.

EXPERIMENTAL APPROACH

Effects of mianserin were examined in CHO cells transfected with human opioid receptors, C6 glioma cells and rat brain membranes by the use of radioligand binding and functional assays including the stimulation of [³⁵S]GTPγS binding and MAPK phosphorylation.

KEY RESULTS

Mianserin displayed 12- and 18-fold higher affinity for κ- than µ- and δ-opioid receptors respectively. In [³⁵S]GTPγS assays, mianserin selectively activated κ-opioid receptors. The agonist activity was antagonized by the selective κ-opioid blocker nor-binaltorphimine (nor-BNI). The mianserin analogue mirtazapine also displayed κ-opioid agonist activity. Mianserin and mirtazapine increased ERK1/2 phosphorylation in CHO cells expressing κ-opioid receptors and C6 cells, and these effects were antagonized by nor-BNI. In rat striatum and nucleus accumbens, mianserin stimulated [35S]GTPγS binding in a nor-BNI-sensitive manner with maximal effects lower than those of the full κ-opioid agonists (–)-U50,488 and dynorphin A. When combined, mianserin antagonized the effects of the full κ-opioid receptor agonists in [³⁵S]GTPγS assays and reduced the stimulation of p38 MAPK and ERK1/2 phosphorylation by dynorphin A.

CONCLUSIONS AND IMPLICATIONS

In different cell systems, mianserin directly activates κ-opioid receptors, displaying partial agonist activity at brain receptors. Thus, this property appears to be a common feature of different classes of antidepressants.     

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.     

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.     

Effect of Tris,HEPES, and TES buffers on binding at μ-, δ-, and κ-opioid sites in guinea pig brain

In homogenates of guinea-pig brain minus cerebellum, the δ-binding of 1.5 nM [³H]-[D-Pen²,D-Pen⁵]enkephalin is little affected by the type and concentration of the three buffers, Tris-HCl, HEPES-KOH, or TES-KOH (10–75 mM). However, the μ-binding of 1 nM [³H][D]A[a²,MePhe⁴,GIy-ol⁵]enkephalin or the κ-binding of 1.5 nM [³H]-U-69,593 is influenced by the choice of buffer. A suitable concentration of buffer for further analyses of opioid binding has been found to be 10 mM. At each site, the effects of MgCl₂ on binding are the same whether 10 mM Tris-HCl, HEPES-KOH, or TES-KOH are used but variations of the effects of NaCl confirm the view that μ- and κ-sites, but not δ-sites, are affected by choice of buffer. Furthermore, under some assay conditions the effects of NaCl and MgCl₂ at the κ-sites of guinea-pig cerebellum differ from their effects in brain minus cerebellum, indicating that these are differences of binding characteristics at the κ-sites of these tissues.     

The effect of chronic treatment with naltrindole,a selective δ-opioid antagonist,on μ-opioid receptor-mediated antinociception in diabetic mice

The effects of chronic treatment with naltrindole (NTI), a selective -opioid receptor antagonist, on the antinociceptive effects of -opioid agonists, such as morphine and [D-Ala²,N-MePhe⁴, Gly-ol⁵]enkephalin (DAMGO) were examined in diabetic mice. Antinociception induced by morphine (10 g, ICV) and DAMGO (0.5 g, ICV) was significantly lower in diabetic mice than in non-diabetic mice. The low sensitivities to the antinociceptive potencies of ICV morphine (10 g) and DAMGO (0.5 g) in diabetic mice were reversed compared with those in saline-treated non-diabetic mice when diabetic mice had been pretreated with NTI (2 mg/kg per day, SC) for 14 days. Naive mice which had been injected with spleen mononuclear cells from saline-treated diabetic mice were less sensitive to DAMGO-induced antinociception. However, adoptive transfer of spleen mononuclear cells from NTI-treated diabetic mice to naive mice had no effect on the recipients' antinociceptive sensitivity to DAMGO. These results suggest that the effect of NTI on the sensitivity to -opioid agonists in diabetic mice may be due to the immunosuppressive effects of NTI.     

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.     

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.

     

The Effect of Hydrophobic Character of Drugs and Helix-coil Transition of κ-Carrageenan on the Polyelectrolyte-drug Interaction

Purpose. The extent of adsorption.of different drug molecules to -carrageenan was investigated in order to evaluate the effect of drug hydrophobicity on the adsorption isotherm. Methods. Dialysis experiments were used to determine the amount of drug adsorbed to the polyelectrolyte. The amount of drug on both sides of the membrane was determined spectrophotometrically after attaining equlibrium. CMC for the drugs were determined by the dye solubilisation method. Results. It is shown that the small differences in structure between the drug molecules used in this study still leads to considerable difference in adsorption properties, especially the onset of adsorption. It was also found that the slope of the adsorption isotherms among the drug molecules followed the same pattern as the CMC values for drugs. The extent of adsorption of drugs to the helix form of -carrageenan was much higher than to the coil form. Conclusions. These results suggest that the adsorption of charged drug molecules to an oppositely charged polymer is effected not only by the coulombic interactions, but also by the hydrophobicity of the drug. Furthermore, the adsorption of drug molecules to -carrageenan in the helix form is higher than for the coil form because of the shorter distance between the charges and the thereby enhanced hydrophobic interaction between bound drug molecules.     

Differential effects of systemically administered nor-binaltorphimine (nor-BNI) on κ-opioid agonists in the mouse writhing assay

The opioid antagonist effects of systemically administered nor-binaltorphimine (nor-BNI) were evaluated against the kappa agonists CI-977, U69,593, U50,488, ethylketocyclazocine (EKC), Mr2034 and bremazocine, the mu agonist morphine and the alkaloid delta agonist BW-373U86 in the acetic acid-induced writhing assay in mice. All eight agonists completely and dose-dependently inhibited writhing. Antagonism of CI-977 was apparent 1 h after administration of 32 mg/kg nor-BNI, peaking after 4 h and was maintained for at least 4 weeks; no antagonist effects of nor-BNI were apparent after 8 weeks. Nor-BNI (32 mg/kg) caused little or no antagonism of morphine or BW-373U86 at 1 h and none at 24 h after nor-BNI administration. Subsequently, dose-effect curves for CI-977, U50,488, U69,593, EKC, Mr2034 and bremazocine were determined 24 h after pretreatment with 3.2, 10 and 32 mg/kg nor-BNI. Pretreatment with 3.2 mg/kg nor-BNI produced significant antagonism of all six kappa agonists, suggesting that their antinociceptive effects were mediated at least in part by nor-BNI-sensitive kappa receptors. At higher doses, nor-BNI dose-depend-ently shifted the agonist dose-effect curves of CI-977, U50,488, U69,593 and bremazocine, but not those of EKC and Mr2034, suggesting that the latter compounds may be producing effects via nor-BNI-insensitive receptors. Mu receptor involvement was demonstrated following a 24 h pretreatment with 32 mg/kg-FNA in combination with nor-BNI, which significantly increased the degree of antagonism of Mr2034 and EKC from that seen with nor-BNI alone. Hence, SC administered nor-BNI selectively antagonized agonist activity mediated through kappaopioid receptors without differentiating between kappa subtypes. Nor-BNI also enabled the mu agonist activity of proposed kappa agonists to be measured.A preliminary report of these findings was made at the International Narcotic/Research/Conference in Keystone, CO, June, 1992. This work was supported by USPHS grant DA 00254. Animals used in these studies were maintained in accordance with the University Committee on Use and Care of Animals, University of Michigan, and guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council (Department of Health, Education and Welfare, Publication No. (NIH) 85-23, revised 1983)     

Antagonism of the morphine-induced Straub tail reaction by κ-opioid receptor activation in mice

The Straub tail reaction (STR) induced by intracerebroventricular injection (ICV) of morphine was significantly antagonized by -funaltrexamine (-FNA, antagonist), given intracerebroventricularly (ICV), but not naltrindole given ICV (NTI, antagonist) or SC norbinaltorphimine given subcutaneously (SC) (nor-BNI, antagonist). When given either SC or ICV the -agonist, U-50,488H markedly suppressed the STR elicited by ICV morphine; these effects were reversed by nor-BNI. These results suggest that the activation of supraspinal receptors can inhibit the ICV morphine-induced STR which results from activation of supraspinal receptors.