Effects Of Ethylketocyclazocine Research Articles

Effects of kappa opioid agonists alone and in combination with cocaine on heart rate and blood pressure in conscious squirrel monkeys

As kappa agonists have been proposed as treatments for cocaine abuse, the cardiovascular effects of the kappa opioid receptor agonists ethylketocyclazocine (EKC) and enadoline were investigated in conscious squirrel monkeys. Both EKC and enadoline increased heart rate with little effect on blood pressure. This effect appeared to be specific for kappa receptors as the mu opioid agonist morphine did not mimic the effects of the kappa agonists. The opioid antagonist naltrexone, at a dose of 1.0 mg/kg, blocked the effect of EKC. An action at both central and peripheral receptors may be responsible for the heart rate increase following kappa agonist treatment. The ganglionic blocker chlorisondamine partially antagonized the effect of EKC on heart rate, suggesting central involvement, while the peripherally-acting agonist ICI 204,448 ((±)-1-[2,3- (Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) also increased heart rate, supporting a peripheral site of action. When given in combination with cocaine, EKC produced effects that were sub-additive, suggesting that the kappa agonists may be used safely as cocaine abuse treatments.

The modulation of placental lactogen release by opioids: a role for extracellular calcium

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 μg/ml streptomycin. 45Ca 2+ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of 45Ca 2+ influx by ethylketocyclazocine (EKC) with an EC 50 of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50, 488 (kappa agonist) had the same stimulatory effect as EKC (221 ± 25% of control). D-Ala 2,NMe-Phe 4,Gly-ol 5)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca 2+ influx (128 ± 5% of control, p > 0.05) whereas D-Ser 2,Leu, Thr 6)-enkephaIin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced 45Ca 2+ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins. The role of extracellular calcium in the regulation of hPL release by kappa opiates was confirmed by experiments in which ionomycin (ionophore) and Bay K 8644 (calcium-channel agonist) mimicked the stimulatory effect of EKC on hPL release. Also, without extracellular Ca 2+, the basal (control) and EKC-stimulated hPL release were significantly lower than with 1.8 mM Ca 2+. Moreover, nifedipine (calcium-channel antagonist) inhibited both basal and EKC-stimulated hPL release. These results suggest that calcium influx is one mechanism mediating opioid-stimulated hPL release by human term trophoblastic cells.

Pharmacological profile of various kappa-agonists at kappa-, mu- and delta-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the rat brain.

1. The potency, relative efficacy and selectivity of a series of kappa-opioid receptor agonists at the mu-, delta- and kappa-opioid receptors mediating inhibition of electrically-induced (radiolabelled) neurotransmitter release from superfused rat brain slices was determined. 2. With regard to their potencies at kappa-receptors mediating inhibition of striatal [3H]-dopamine release, the highest pD2 value (8.7) was found for bremazocine and the lowest (7.1) for U50488; the pD2 values for ethylketocyclazocine (EKC), tifluadom, U69593 and PD117302 were between 8.0 and 8.3. There were no marked differences between the relative efficacies of the kappa-agonists (maximum inhibition being 60-70%). In contrast to the other kappa-agonists, at a concentration of 1 microM, PD117302 caused a significant (25-40%) increase of the spontaneous efflux of tritium. 3. None of the kappa-agonists significantly affected striatal [14C]-acetylcholine (ACh) release, with the exception of a slight inhibitory effect of EKC. The delta-receptor-mediated inhibitory effect of [D-Ala2, D-Leu5]enkephalin (DADLE) on [14C]-ACh release was antagonized in a concentration-dependent manner by bremazocine (0.1 and 1.0 microM) and also partially by EKC (1 microM), but not by the other kappa-agonists. The pA2 value for bremazocine as an antagonist at the delta-receptors involved was 8.0, compared to 7.6 for naloxone. 4. None of the kappa-agonists significantly affected cortical [3H]-noradrenaline (NA) release, with the notable exception of tifluadom, which strongly inhibited release by activating mu-receptors. The mu-receptor-mediated inhibitory effect of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) on [3H]-NA release was antagonized in a concentration-dependent manner by bremazocine and EKC, but not by the other K-agonists. The pA2 value for bremazocine as an antagonist at the mu-receptors involved was 8.2, compared to 8.6 for naloxone. 5. Thus, whereas U69593 and PD1 17302 display high potency and selectivity towards K-opioid receptors, the potent benzomorphan K-agonists bremazocine and EKC also appear to be strong mu-opioid receptor antagonists.

β-Funaltrexamine antagonizes the analgesic effects of mu and kappa agonists in the formalin test

The formalin test assesses the behavioral response of an animal to minor tissue injury-induced pain. Opioid antinociception in this test has been suggested to depend largely on activation of kappa receptors but mu agonists are also potent in reducing pain behavior. The present study used the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), to examine the role of mu receptor activation in this test. beta-FNA given intracranially 4 h before testing fully blocked the effects of morphine and attenuated the effects of ethylketocyclazocine and U50,488H. The results do not support a role for kappa receptors in antinociception in the formalin test. Instead, mu and, possibly, delta receptors are involved.

Estimation of affinities and efficacies for κ‐receptor agonists in guinea‐pig ileum

1. Ethylketocyclazocine (EKC) and U50,488H have been employed widely as kappa-receptor agonists in the study of opioid receptor systems. However, the quantification of their agonism in terms of affinities and relative efficacies has not been investigated. 2. In this study, operational model-fitting was used to analyse the effects of irreversible receptor alkylation by beta-chlornaltrexamine (beta-CNA) on the kappa-receptor mediated effects of EKC and U50,488H in the isolated, coaxially stimulated ileum of the guinea-pig. 3. EKC produced monophasic inhibitory concentration-effect curves which were readily amenable to analysis. In contrast U50,488H produced biphasic curves characterized by a higher potency phase of agonism that was susceptible to antagonism by 16-methylcyprenorphine (RX8008M) and a lower potency phase that was apparently non-opioid in nature. 4. Analysis of the kappa-receptor-mediated effects of both agonists indicated that EKC has fifteen fold higher affinity than U50,488H and that the two agonists possess similar intrinsic efficacies.

Effect of ethylketocyclazocine on acetylcholine release in guinea-pig brain slices

The effect of ethylketocyclazocine (EKC) on 3H-Choline (Ch) efflux and on endogenous acetylcholine (ACh) release from guinea-pig brain slices was studied. The drug inhibited the 3H-Ch efflux at a low concentration (0.1 mumol/l) in thalamus slices, while only at high concentrations (30-100 mumol/l) did EKC induce deep inhibition in the caudate nucleus and slight reduction in the cerebral cortex. Dynorphin (1-13) and morphine (Mo) inhibited the ACh release from thalamus slices as well. Naloxone (Nx) was more effective in antagonizing Mo than EKC. The experiments carried out with the endogenous ACh bioassay technique confirmed the above results. Mr 2266 and Mr 1452, both proposed as preferential k antagonists, per se enhanced 3H-Ch efflux from thalamus slices, while the dextrorotatory isomer Mr 1453, devoid of opioid properties, did not share such action. The role of k-opioid receptors in cholinergic system modulation in the guinea-pig brain is discussed.

Different effects of ethylketocyclazocine on phencyclidine- and N-allylnormetazocine-induced stereotyped behaviors in rats

The effects of ethylketocyclazocine (EKC) on the stereotyped behaviors induced by intraperitoneal injection of phencyclidine (PCP) or N-allylnormetazocine (SKF 10,047) were examined. EKC markedly antagonized PCP-induced stereotyped behaviors such as sniffing, head-weaving, turning and backpedalling. On the other hand, EKC failed to antagonize SKF 10,047-induced stereotyped behaviors, which are PCP-like stereotyped behaviors, except sniffing and head-weaving at 0–15 min after the SKF 10,047 injection. PCP-induced turning and backpedalling were potentiated by pretreatment with SKF 10,047, while PCP-induced sniffing and head-weaving were not. EKC failed to affect the enhancing effect of SKF 10,047 on PCP-induced turning and backpedalling. These results suggest that part of the PCP- and SKF 10,047-induced stereotypy may be mediated by different neuronal mechanisms.

Effects of ethylketocyclazocine on behaviors in mice using multi-dimensional behavioral analyses

The effects of ethylketocyclazocine, a relatively selective kappa opioid agonist on the behaviors in mice were examined by using multi-dimensional behavioral analyses. Within 15 min after the measurements, ethylketocyclazocine (0.03, 0.1 and 0.3 mg/kg) dose-dependently decreased the linear locomotion, rearing, and grooming behaviors in mice. Fifteen to 30 min ethylketocyclazocine (0.3 mg/kg) decreased most of the behaviors in mice. The ethylketocyclazocine (0.3 mg/kg)-induced behavioral effects were antagonized by naloxone (1 and 2 mg/kg). These data suggest that the decreases in the linear locomotion, rearing and grooming behaviors are mediated via kappa opioid system.

Spinal κ-opioid receptor-mediated antinociception is stimulus-specific

The intrathecal injection of a variety of selective κ-opioid receptor ligands did not result in significant inhibition of thermal nociceptive tail flick responses in rats. In contrast, these compounds dose dependently inhibited pressure nociceptive responses. Cross-tolerance studies revealed that the κ-opioid receptor ligands tifluadom, U-50488H and dynorphin-(1–17) act upon a receptor distinguishable from the receptor through which morphine exerts its inhibition of mechanical nociceptive responses. The less selective κ-opiate receptor ligands bremazocine and ethylketocyclazocine (EKC), however, blocked both tail flick and tail pressure nociceptive responses and their effect showed marked cross-tolerance to morphine in the tail flick nociceptive test, but not for the pressure nociceptive responses. We suggest that EKC and bremazocine act upon the spinal κ-opioid receptor to block mechanical nociceptive responses but that the analgesic effect of EKC and bremazocine on thermal nociceptive responses is probably mediated via spinal μ-and/or δ-, and δ-opioid receptors, respectively.

Beta-endorphin-sensitive opioid receptors in the rat tail artery.

Isolated tail arteries of rats were perfused and field-stimulated every 2 min with 2 pulses at 1 Hz. Different opioid peptides depressed the contractile responses to stimulation; their concentration-response curves showed a maximum at about 40% inhibition. The rank order of potency of the peptides was beta-endorphin (IC50 = 97 nmol/l) approximately equal to BAM-22P greater than FK-33824 greater than DAGO greater than [D-Ala2,D-Leu5]-enkephalin greater than or equal to metorphamide greater than dynorphin A-(1-13) approximately equal to [Met5]enkephalin. All these substances have in common a certain activity at opioid mu-receptors, although the enkephalins are preferential delta-, and the dynorphins preferential kappa-agonists. However, the selective delta-agonist [D-Pen2,L-Pen5]enkephalin was ineffective at up to 10 mumol/l, and the kappa-agonists ethylketocyclazocine and U-50488 acted only at concentrations higher than 3 mumol/l. Whereas the effects of beta-endorphin, DAGO and [D-Ala2,D-Leu5]enkephalin could be reduced by the mu-preferential antagonist naloxone, the effects of ethylketocyclazocine and U-50488 were not changed. The delta-selective antagonist ICI 174864 did not influence the action of [D-Ala2,D-Leu5]enkephalin. Naloxone in a concentration (1 mumol/l) which nearly abolished the effect of DAGO 3 mumol/l, slightly enhanced responses to stimulation. Neither beta-endorphin nor DAGO influenced vasoconstriction evoked by the application of noradrenaline or adenosine triphosphate; U-50488 reduced it. In arteries preincubated with [3H]noradrenaline DAGO depressed, whereas naloxone enhanced the tritium overflow and vasoconstriction evoked by field stimulation (0.4 Hz, 24 pulses every 14 min). In addition, naloxone antagonized the effect of DAGO.(ABSTRACT TRUNCATED AT 250 WORDS)

Bidirectional cross-tolerance between methadone ( mu) — and ethylketocyclazocine ( kappa) — tolerant rats

Our laboratory previously reported on unidirectional cross-tolerance between morphine and methadone, both mu opioid agonists, and between morphine and ethylketocyclazocine (EKC), the latter being a relatively selective kappa opioid agonist. Morphine-tolerant rats were found to be non-cross-tolerant to methadone and EKC, but methadone- and EKC-tolerant rats were cross tolerant to morphine. In the present study, we characterized the cross-tolerance between methadone and EKC. A group of female adult Sprague-Dawley rats was made tolerant to methadone by a series of automatic i.v. injections ranging from 0.25 mg/kg per 2 h on the first day to 2.0 mg/kg per 1.5 h on the ninth day. Another group of rats was similarly made tolerant to EKC with doses ranging from 0.5 mg/kg per 2 h on the first day to 4 mg/kg per h on the ninth day. Relatively similar degrees of tolerance development to the EEG and behavioral effects of methadone and EKC were reflected by decreases in durations of action and decreases in opioid-induced EEG power spectral changes. Methadone-tolerant rats were found to be cross-tolerant to the EEG and behavioral effects of EKC, and, similarly, EKC-tolerant rats were found to be crosstolerant to those of methadone. Thus, a bidirectional cross-tolerance between a mu and a kappa agonist was demonstrated. The present results together with those reported earlier indicate that cross-tolerance may not be directly related to the receptor selectivity of the opioids. It is possible that differential physicochemical properties of these opioids may play a more decisive role in the phenomenon of cross-tolerance.

Phencyclidine-induced stereotyped behaviors after injection of ethylketocyclazocine, Mr 2266 and naltrexone in rats

The effects of ethylketocyclazocine (EKC), Mr 2266 and naltrexone on the stereotyped behaviors induced by an intraperitoneal injection of phencyclidine (PCP) were examined. PCP-induced turning, backpedalling, head weaving and sniffing were antagonized by pretreatment with EKC (0.25-4.0 mg/kg). While pretreatment with Mr 2266 (2.5 mg/kg), a kappa selective antagonist, and naltrexone (10 mg/kg), a mu selective antagonist, failed to affect the PCP-induced stereotypy, Mr 2266 antagonized the suppressing effect of EKC on PCP-induced stereotypy. Taken into consideration, this suggests that kappa opioid agonists such as EKC antagonize PCP-induced stereotyped behaviors through a kappa opioid mechanism, and that the mu opioid receptor may not play an important role in the PCP-induced stereotypy in rats.

The sympathetic axons innervating the sinus node of the rabbit possess presynaptic opioid kappa- but not mu- or delta-receptors.

The postganglionic sympathetic nerves of rabbit isolated hearts were stimulated with pulses delivered at 5 Hz and train durations of 1-5 s. Ethylketocyclazocine 0.01-1 mumol/l and fentanyl 1 and 10 mumol/l but not morphine 1 and 10 mumol/l, Met-enkephalin 1 and 4 mumol/l or D-Ala2, D-Leu5-enkephalin 0.5 and 5 mumol/l diminished the stimulation-evoked increase in heart rate. The effect of ethylketocyclazocine 0.1 mumol/l was antagonized by naloxone 1 and 10 mumol/l. In contrast, the effect of fentanyl was not changed by naloxone 10 mumol/l. Ethylketocyclazocine 0.03 and 1 mumol/l did not reduce the tachycardia elicited by exogenous noradrenaline. The results suggest that, under in vitro conditions, only presynaptic opioid kappa- but not mu- or delta-receptors inhibit the release of noradrenaline from the sympathetic neurones innervating the sinus node.

Profile of activity of κ receptor agonists in the rabbit vas deferens

The purpose of this study was to investigate further the κ opioid receptor selectivity of the field-stimulated isolated rabbit vas deferens preparation and to study the profile of a series of κ agonists in this tissue. Agonists acting at μ, δ and σ receptors were without detectable effect in the rabbit vas deferens. But a number of κ agonists, including bremazocine, tifluadom, ethylketocyclazocine, ketocyclazocine, U-50,488 and Win 42,610 all depressed contractions, producing parallel dose-response curves. Mr 2034 generally produced a shallower dose-response curve and achieved a lower maximum effect, thus acting like a partial agonist. The effect of ethylketocyclazocine was not reduced by the irreversible μ antagonist, β-funaltrexamine, confirming that it is not acting via μ receptors. Another group of drugs, including nalorphine, butorphanol and proxorphan, which produce an agonist action via κ receptors in the guinea-pig ileum and mouse vas deferens, were antagonists in the rabbit vas deferens, suggesting that this tissue will only respond to high efficacy κ agonists.

Effects of ethylketocyclazocine on adrenergic transmission in the isolated perfused cat spleen.

The pre- and postsynaptic effects of ethylketocyclazocine (EKC), a putative kappa opiate receptor agonist, on sympathetic adrenergic neurotransmission were investigated using the isolated perfused cat spleen. Perfusion of spleens with EKC (10(-7) and 10(-6)M) produced a modest dose-dependent inhibition of nerve-stimulation-mediated overflow of norepinephrine (NE) and total 3H. In contrast, EKC (10(-4)M) produced an increase in the release of NE, and total 3H overflow. Perfusion with EKC (10(-5) and 10(-4)M) resulted in a dose-dependent inhibition of the postsynaptic response to stimulation-mediated release of NE. Likewise, EKC inhibited splenic contraction due to exogenously administered NE which was not antagonized by naloxone. These results suggest that EKC exerts a direct effect on peripheral adrenergic neurotransmission not mediated via classical kappa opiate receptors.

Ethylketocyclazocine decreases noradrenaline release and blood pressure in the rabbit at a peripheral opioid receptor.

Rabbits were pithed and their sympathetic outflow was stimulated electrically via the pithing rod. Arterial blood pressure, heart rate, the endogenous plasma noradrenaline level, the plasma 3H-noradrenaline clearance and the noradrenaline release rate (the rate of entry of endogenous noradrenaline into the plasma) were determined. Ethylketocyclazocine 0.1 mg kg-1 + 0.02 mg kg-1 h-1 and 1 mg kg-1 + 0.2 mg kg-1 h-1 but not 0.01 mg kg-1 + 0.002 mg kg-1 h-1 decreased blood pressure, the endogenous plasma noradrenaline level and the noradrenaline release rate. The effects of ethylketocyclazocine 1 mg kg-1 + 0.2 mg kg-1 h-1 were antagonized by naloxone 1 mg kg-1 + 0.5 mg kg-1 h-1. Given alone, naloxone caused no change. It is concluded that ethylketocyclazocine inhibits action potential-evoked release of noradrenaline from postganglionic sympathetic neurones, and hence can lower blood pressure, by a peripheral effect, possibly mediated by opioid receptors at the terminal axons.

Analgesic effects of ethylketocyclazocine and morphine in rat and toad

We have previously found rat and toad ( Bufo marinus ) brain to contain inverse ratios of benzomorphan-preferring ( κ σ ) and morphine-preferring (μ) opioid receptor types. The aim of the present study was to compare in vivo pharmacologic activity of a benzomorphan, ethylketocyclazocine (EKC) and morphine sulfate (MS) in rat and toad. Footshock intensity thresholds for eliciting locomotion were determined and dose-response curves for EKC and MS analgesia were obtained. Drugs were injected subcutaneously. In rats (high μ, low κ in brain), both compounds produced analgesia and displayed similar sensitivity to naloxone antagonism. The analgesti effects of EKC and MS may, therefore, be mediated by a common receptor type (μ) in this pain test in rats. In toads (high κ, low μ in brain), MS produced naloxone-reversible analgesia at doses 20-fold higher than were effective in rats. Toads did not display EKC analgesia at doses below those producing motor impairment. Moreover, 50-fold higher doses were required to produce such impairment in toads. Thirty minutes following subcutaneous injection of 3H-EKC, similar concentrations were found in rat and toad brain. Uptake into brain is probably not a factor in the behavioral resistance of toads to EKC.

Effects of a Kappa receptor agonist, ethylketocyclazocine, on water consumption in water-deprived and nondeprived rats in diurnal and nocturnal tests

In 24 hr water-deprived male hooded rats, ethylketocyclazocine (EKC), 0.1–3.0 mg/kg, dose-dependently suppressed water intake. Within the first 30 min access to water, drinking was virtually abolished by 1.0 and 3.0 mg/kg EKC. Significant reductions in the level of water intake were found after 0.1 mg/kg EKC. After 2 hr access to water, the suppressant effect was attenuated indicating some recovery. The antidipsogenic action of EKC in water-deprived rats was comparable in its effect for both daytime and noctural testing. Circadian variation may not be an important modulator of the antidipsogegic action. Naloxone, an opiate receptor antagonist, when administered in a dose of 0.3 mg/kg also significantly reduced drinking in deprived animals. EKC (0.3 mg/kg) and naloxone (0.3 mg/kg) when administered together displayed mutual antagonism. Drinking was at control levels. In nondeprived male rats, EKC exerted some dipsogenic action, most noticeably during diurnal testing. Within 30 min access to water, 0.1 mg/kg EKC significantly elevated the level of water intake. This effect did not occur during nocturnal testing, when the only immediate effect of EKC was a suppression of drinking at 1.0 and 3.0 mg/kg dose levels. After 2 hr access to water, there was a significant peak effect to enhance drinking at the 0.3 mg/kg dose level during the daytime. Effects of EKC during the night were less pronounced. The dipsogenic action of EKC (0.3 and 1.0 mg/kg) in satiated animals during the day was abolished by naloxone and Mr-2266BS, also an opiate receptor antagonist. The results indicate that the effects of EKC on drinking are profoundly affected by the deprivation status of the animal. Further, the phase of the circadian cycle may be an important modulator of the expression of the dipsogenic action of EKC in nondeprived rats.

Opposite central cardiovascular effects of various morphine-like drugs and opiate peptides in the rat

Opiate agonists were administered into the cisterna magna of normotensive urethane-anaesthetized artificially-ventilated rats. Increases in blood pressure and heart rate were produced by fentanyl (0.3-3.0 nmol). [D-ala]2-met-enkephalinamide (17 nmol), and beta-endorphin (2.9 nmol). On the contrary, ethylketocyclazocine (78.7 nmol) and dynorphin (1-13) (62.3 nmol) produced decreases in blood pressure and heart rate. The cardiovascular effects of ethylketocyclazocine and dynorphin (1-13) were antagonized by higher doses of naloxone than that required to counteract the effects of fentanyl. These results suggest that opposite central cardiovascular effects could be induced by activation of various opiate receptors.

Effects of ketocyclazocine and ethylketocyclazocine on electric shock titration

The effects of two putative κ agonists, ketocyclazocine and ethylketocyclazocine, on electric shock titration in squirrel monkeys were determined. Drugs were tested alone, in combination with naloxone and following the development of morphine tolerance. Both ketocyclazocine and ethylketocyclazozine produced biphasic dose-effect curves. Low doses decreased whereas high doses increased the intensity at which monkeys maintained the shock. Naloxone antagonized the effects of ketocyclazocine and ethylketocyclazocine; however, the dose of naloxone required to antagonize their effects was much larger than the dose required to antagonize equivalent effects of morphine. When the effects of ethylketocyclazocine were redetermined in monkeys tolerant to morphine, the ethylketocyclazocine dose-effect curve was shifted to the left whereas the morphine curve was shifted to the right. It was concluded that the effects of the κ agonists can be differentiated from morphine by the shape of their dose-effect curve, the ease with which they are antagonized by naloxone and the lack of cross tolerance to morphine.