Latent inhibition in an insect: the role of aminergic signaling.

Latent inhibition (LI) is a decrement in learning performance that results from the nonreinforced pre-exposure of the to-be-conditioned stimulus, in both vertebrates and invertebrates. In vertebrates, LI development involves dopamine and serotonin; in invertebrates there is yet no information. We studied differential olfactory conditioning of the proboscis extension response in the honeybee Apis mellifera, and we compared LI in individuals treated with antagonists of biogenic amines (dopamine, octopamine, and serotonin). An antagonist of octopamine receptors and two antagonists of serotonin receptors showed LI disruption. We thus provide evidence that serotonin would participate in the regulation of LI in honeybees.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 4. Opioid receptor binding properties of 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogues of cyclazocine and ethylketocycalzocine.

The synthesis and evaluation of a series of aryl-containing N-monosubstituted analogues of the lead compound 8-carboxamidocyclazocine were performed to probe a putative hydrophobic binding pocket of opioid receptors. High binding affinity to mu, kappa, and delta opioid receptors was observed for the 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogue.

Diazabicyclononanones, a potent class of kappa opioid analgesics.

The 1,5-dimethyl 3,7-diaza-3,7-dimethyl-9-oxo-2,4-di-2-pyridine-bicyclo[3.3.1]nonane-1,5-dicarboxylate, HZ2, has a high and selective affinity for the kappa opioid receptor and an antinociceptive activity comparable to morphine. In addition, it is characterized by a long duration of action and a high oral bioavailability. QSAR studies within series of kappa agonists revealed a chair-boat conformation of a double protonated HZ2 characterized by an almost parallel orientation of the C9 carbonyl group and the N7-H group and at least one aromatic ring to be the pharmacophoric arrangement. Structural variations showed that the pyridine rings in 2 and 4 position can be replaced with p-methoxy-, m-hydroxy- and m-fluoro-substituted phenyl rings. However, all other substituents have to be kept the same for a high affinity to the kappa receptor.

Synthesis and opioid receptor affinity of morphinan and benzomorphan derivatives: mixed kappa agonists and mu agonists/antagonists as potential pharmacotherapeutics for cocaine dependence.

This report concerns the synthesis and preliminary pharmacological evaluation of a novel series of kappa agonists related to the morphinan (-)-cyclorphan (3a) and the benzomorphan (-)-cyclazocine (2) as potential agents for the pharmacotherapy of cocaine abuse. Recent evidence suggests that agonists acting at kappa opioid receptors may modulate the activity of dopaminergic neurons and alter the neurochemical and behavioral effects of cocaine. We describe the synthesis and chemical characterization of a series of morphinans 3a-c, structural analogues of cyclorphan [(-)-3-hydroxy-N-cyclopropylmethylmorphinan S(+)-mandelate, 3a], the 10-ketomorphinans 4a,b, and the 8-ketobenzomorphan 1b. Binding experiments demonstrated that the cyclobutyl analogue 3b [(-)-3-hydroxy-N-cyclobutylmethylmorphinan S(+)-mandelate, 3b, MCL-101] of cyclorphan (3a) had a high affinity for mu, delta, and kappa opioid receptors in guinea pig brain membranes. Both 3a,b were approximately 2-fold more selective for the kappa receptor than for the mu receptor. However 3b (the cyclobutyl analogue) was 18-fold more selective for the kappa receptor in comparison to the delta receptor, while cyclorphan (3a) had only 4-fold greater affinity for the kappa receptor in comparison to the delta receptor. These findings were confirmed in the antinociceptive tests (tail-flick and acetic acid writhing) in mice, which demonstrated that cyclorphan (3a) produced antinociception that was mediated by the delta receptor while 3b did not produce agonist or antagonist effects at the delta receptor. Both 3a,b had comparable kappa agonist properties. 3a,b had opposing effects at the mu receptor: 3b was a mu agonist whereas 3a was a mu antagonist.

Differential effects of intrahypothalamic administration of opioids on food intake in naive and tolerant rats.

We investigated the effects of intrahypothalamic administrations of the opioid agonists morphine (MOR) and ketocyclazocine (KCZ) and antagonists naltrexone (NALTX) and Mr2266 on food intake (FI) during light and dark phases of the diurnal cycle, after acute or chronic administration in rats. Acute intralateral hypothalamic (LH) administration of MOR or KCZ (1 microgram/rat) enhanced FI during dark and light phases, respectively, whereas intraventromedial hypothalamic (VMH) injections resulted in moderate hyperphagia during dark phases by both mu and kappa agonists. The receptor specificity was evident from blockade of the responses to MOR or KCZ by the respective antagonists NALTX and Mr2266. After repeated administrations of MOR and KCZ, FI responses to the test dose of these agonists injected in LH were modulated in opposite directions. However, the adaptative changes in FI after intra-VMH injection of KCZ were similar to those seen with MOR. These results are discussed in light of a differential opioid receptor involvement and their possible functional interactions within the hypothalamus during food intake.

Evidence against the participation of mu- and kappa-opioid receptors in the analgesic activity of ketorolac in rats.

The possibility that activation of opioid receptors is involved in the analgesic activity of ketorolac was explored. The analgesic effects of ketorolac, of ketocyclazocine, the prototype kappa-agonist, and of morphine, the prototype mu-agonist, were assayed in the pain-induced functional impairment model in the rat. All three drugs induced a significant analgesic effect in this model. Naloxone was able to antagonize the effects of ketocyclazocine and morphine. However, the effect of ketorolac was not blocked by naloxone, although a high dose, 3.2 mg kg-1, capable of blocking kappa-receptors was used. It is concluded that activation of mu- or kappa-opioid receptors, by either a direct or an indirect mechanism, does not play a role in the analgesic activity of ketorolac.

X-ray molecular structures and theoretical conformational studies of narcotic analgesics alpha-(-)-N-cis-3-chloroallyl-normetazocine, ethylketazocine, and ketazocine.

The X-ray molecular structures of the narcotic analgesics alpha-(-)-2-cis-3-chlorallyl-2'-hydroxy-5,9-dimethyl-6,7-benzomorp han (1) and alpha-(+-)-2-cyclopropylmethyl-2'-hydroxy-5-ethyl-9-methyl-8-oxo-6,7- benzomorphan (ethylketazocine, 2) were determined. The structures and conformations in the crystal were compared and discussed with respect to that of alpha-(+-)-2-cyclopropylmethyl-2'-hydroxy-5,9-dimethyl-8- oxo-6,7-benzomorfan (ketazocine, 3) and those of 15 analogous compounds of the 2'-hydroxy-6,7-benzomorphan series whose structures were previously determined by X-ray analysis. Molecular modeling routines for 1, 2, and 3 produced configurations (N-equatorial) and conformations (distorted chair) of the piperidine ring that were in agreement with those found in the solids. Theoretical studies of the conformations and the rotational energetics of 1, 2, and 3 as cationic species were performed by both the force field (MM2) and the semiquantitative (AM1) methods. The latter method predicted three low energy conformations about N--C(12) and C(12)--C(13) bonds, one of these being more significantly populated (60-68%). The AM1 results were not reproduced by the MM2 method, which predicted four low energy conformations. An interesting common feature of 1, 2, and 3 that was noted with both methods was the restricted interconversion route from the conformational state to another through rotations about the C(12)--C(13) bond. The conformational results were discussed in terms of a working hypothesis for regulation of relative mu and kappa analgesic activities of benzomorphans.

The thyroadrenal axis, food deprivation and retention of a behavioural response.

The effects of adrenalectomy on retention of the immobile response in the Porsolt swimming rat test are equally reversed by dexamethasone, ketocyclazocine and thyroxine; the effects of hypothyroidism are similarly equally well reversed by all three agents, or by adrenalectomy. Animals food deprived for 24 h lose the response, but retain it after 48 h food deprivation; the latter effect is blocked by adrenalectomy. These counterintuitive findings suggest hitherto unrecognized commonalities and/or interactions in the metabolic effects of the thyroid and adrenal glands.

Role of kappa opioid receptors during stress responsiveness in rats.

Effects of kappa opioid agonist, ketocyclazocine (KCZ) and its antagonist, M(r) 2266, were evaluated on some stress responses in rats. KCZ (1 or 10 mg/kg, ip) dose-dependently attenuated cold restraint stress (CRS)-induced gastric ulcer formation. Similar gastric cytoprotection was also seen with KCZ (1 or 10 micrograms/rat, icv). Pretreatment of rats with M(r) 2266 (0.3 mg/kg, ip) clearly antagonized the ulceroprotective effects of both ip and icv KCZ. KCZ effects on the gastric mucosa during CRS were also reduced by naltrexone (5 mg/kg, ip) pretreatment. KCZ (1 or 10 mg/kg, ip) also attenuated the plasma corticosterone response to CRS and these effects were blocked by M(r) 2266 (0.3 mg/kg) pretreatment. These results indicate kappa opioid receptor involvement during stress reactions and also suggest possible opioidergic interactions during CRS.

Analgesic effects of intraventricular and intrathecal injection of morphine and ketocyclazocine in the infant rat.

Little is known of the neural bases of analgesia in immature animals. This experiment examined the effects of intracerebroventricular (i.c.v.) and intrathecal (i.t.) administration of morphine or ketocyclazocine in tests of antinociception in rats aged 3 to 14 days of age. Analgesia tests were conducted using both thermal and mechanical (pressure) noxious stimuli applied to the forepaw, hindpaw or tail. In the 3-day-old morphine-injected i.c.v. produced analgesia in the forepaws when either the mechanical or thermal noxious stimulus was used. There was no effect when the hindpaw or tail was tested. At 10 days of age, when the mechanical stimulus was used, morphine was analgesic in tests on all three appendages but was only effective in the forepaw when the thermal stimulus was used. Morphine was fully effective in all tests with both stimuli at 14 days of age. Ketocyclazocine had no consistent effect when given i.c.v. When injected i.t., morphine produced analgesia in the forepaws in the thermal test at 4 days of age and in all appendages by 10 days. When the mechanical test was used, morphine was effective in all appendages at all ages tested. Ketocyclazocine was analgesic at all appendages for the mechanical stimulus at all ages but was only transiently effective in the thermal test. The results demonstrate differential development of analgesia mediated at different levels of the neural axis and are consistent with the development of descending inhibitory that may mediate analgesia induced by i.c.v. injections of morphine. Neural mechanisms that are involved in the analgesic effects of these drugs against the two types of stimuli are also developmentally distinct.

Effects of acute or chronic opioid agonists and their modulation by diurnal rhythmicity and satiety states on food intake in rats.

The effects of acute or chronic treatment with mu and k opioid agonists were investigated on food intake during light (0-6 hr) and dark (6-24 hr) phases in free fed and fasted rats. In free fed rats, morphine (MOR, 5 mg/kg, ip), a mu-agonist, induced a hyperphagic response during both light and dark phases, whereas ketocyclazocine (KCZ, 1 mg/kg, ip), a k-agonist, enhanced food intake only during the light phase. Chronic MOR (x 7 days) produced a further enhancement of hyperphagia in the light phase and attenuated the dark phase response. Chronic KCZ, however, had opposite effects, i.e. tolerance to light phase hyperphagia and an enhancement in the dark phase response. In fasted rats, neither MOR nor KCZ appreciably enhanced food intake after acute administration but chronic treatment potentiated the acute opioid effects. These results are discussed in light of the role of diurnal rhythmicity, satiety states and receptor (mu and k) specificity/interactions in the opioidergic regulation of food intake.

Effects of acute and chronic ketocyclazocine and its modulation by oxytocin or vasopressin on food intake in rats.

The effects of acute and chronic ketocyclazocine (KCZ, a kappa receptor agonist) and its interactions with oxytocin (OXY) or vasopressin (AVP) were investigated on food intake in free-fed rats. Acute treatment with KCZ (1 mg/kg) produced a generalized hyperphagia during the light phase (0-6 h) without influencing dark phase (6-24 h) food intake. On chronic administration, tolerance developed to hyperphagic effect during light phase, whereas an enhancement in the food intake was seen during dark phase. OXY or AVP (both at 10 micrograms/kg) per se, did not affect the food intake response during either the light or the dark phase, after acute as well as chronic treatment. In the interaction studies, acute AVP or OXY attenuated the hyperphagia of KCZ during the light phase. On chronic treatment, both AVP and OXY blocked (a) the tolerance, and (b) the "reverse tolerance" to the food intake response to KCZ during light and dark phases, respectively. These results are discussed in light of complex opioid-OXY/AVP interactions during food intake in rats.

The forced swimming test: effects of glucose administration on the response to food deprivation and adrenalectomy.

Rats food deprived for 24 h prior to a 15 min swimming test have no difficulty in acquiring the immobile response but showed significantly reduced levels of immobility (40%) on retest compared with controls (70%). This effect of food deprivation was reversed, by glucose (100 mg/kg), dexamethasone (6 micrograms/rat) and ketocyclazocine (25 micrograms/rat). Adrenalectomised animals also acquire but cannot retain the immobile response, however, adrenalectomised rats given 1000 mg/kg glucose within 2 h of the initial swimming test are immobile for 75-85% of the retest period. We interpret these findings as suggesting a complex interplay of endocrine and metabolic factors are necessary for retention of the behavioural response.

Reversal by naloxone of spinal antinociceptive effects of fentanyl, ketocyclazocine and midazolam.

Experiments using measurement of electrical-current threshold as a nociceptive test in the skin of the tail and neck in rats demonstrated that fentanyl, ketocyclazocine and midazolam caused spinally mediated antinociception when the drugs were administered intrathecally via chronically implanted lumbar subarachnoid catheters. The benzodiazepine antagonist flumazenil selectively suppressed the midazolam response, indicating that this benzodiazepine exerted its segmental antinociceptive effect via spinal-cord benzodiazepine receptors. Naloxone blocked the responses to both opioids and also midazolam. The dose of naloxone which suppressed the midazolam response was similar to that required to suppress the response to the kappa-opioid agonist. We suggest that the segmental antinociceptive effects of fentanyl and midazolam are mediated via different pathways; the benzodiazepine exerts its antinociceptive action via a spinal-cord opioid pathway which does not involve mu-receptors.

Pharmacodynamic and pharmacokinetic actions of ketocyclazocine enantiomers in the dog: absence of sigma- or phencyclidine-like activity.

The effects of the optical isomers and the racemic form of ketocyclazocine (KC) were compared with morphine and U-50,488H in the chronic spinal dog. l-KC and dl-KC produced depression of nociceptive reflexes, miosis, relaxation of the nictitating membrane and sedation, whereas d-KC lacked pharmacological activity. Peak plasma levels and distribution phase half-lives for dl-, l- and d-KC were similar, indicating no major dispositional differences between the isomers of KC despite a trend for d-KC to have a longer elimination half-life, slower plasma clearance and a greater apparent volume of distribution than l-KC. Although a relatively low dose of naltrexone (0.01 mg/kg) was sufficient to shift morphine dose-effect curves to the right, this dose of naltrexone was not sufficient to shift the dose-effect curves of dl-KC to the right. A dose of 1 mg/kg of naltrexone was required, consistent with the view that the effects were mediated by kappa opioid receptors. The overall pharmacological profile of l-KC differed from that of the more selective kappa opioid agonist U-50,488H, which produced both stimulatory and sedative effects. Neither l-KC nor U-50,488H produced pharmacological profiles typical of the sigma agonist d-N-allylnormetazocine or phencyclidine. The data suggest that the pharmacological activity of KC resides in the l-enantiomer, that the effects are kappa opioid receptor-mediated and that the binding of d-KC to haloperidol-sensitive sigma receptors does not produce N-allylnormetazocine- or phencyclidine-like actions in the dog.

On the mechanism by which midazolam causes spinally mediated analgesia.

The electrical current thresholds for pain (ECTP) in the skin of the neck and tail were measured in rats with chronically implanted lumbar subarachnoid catheters. The effects of a benzodiazepine antagonist and a gamma-aminobutyric acid (GABA) antagonist on the analgesic effects of equivalent doses of midazolam, fentanyl, and ketocyclazocine were studied. These were the minimum doses producing maximal segmental analgesia when given intrathecally (i.e., they all caused a significant and maximum increase in ECTP in the tail, which was similar for all three drugs, but no significant change in the ECTP in the neck). Flumazenil (Ro 15-1788) administration caused a parallel shift to the right of the dose-response curve for midazolam spinal analgesia. Segmental analgesia following midazolam was also significantly attenuated (P less than 0.05) when the selective GABA antagonist bicuculline was given intrathecally at the same time as midazolam. The highest dose of bicuculline used (50 pmol) caused no significant attenuation of the segmental analgesic effects of either ketocyclazocine or fentanyl. The authors concluded that the segmental analgesia produced by intrathecal midazolam is mediated by the benzodiazepine-GABA receptor complex that is involved in other benzodiazepine actions.

Kappa agonists inhibit gastric emptying but not acid secretion in rhesus monkeys.

Gastric function was evaluated in rhesus monkeys during a continuous, s.c. infusion of three kappa agonists; dynorphin-(1-13), (trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate hydrate) (U50,488H) and ketocyclazocine (KETO). A dye dilution technique was used to determine gastric fractional emptying rate, hydrogen ion, sodium ion and fluid secretion after the intragastric administration of a water meal. All agonists significantly inhibited fractional emptying rate after the water meal. The kappa receptor antagonist, (-)-(1R,5R,9R)-5,9-dimethyl-2-(3-furylmethyl)-2'-hydroxy-6,7-benzomor pha n methanesulfonate, prevented the inhibitory response to dynorphin-(1-13) and partially blocked the effect of KETO, but, at the dose used in the present study, was completely ineffective against the specific kappa agonist, U50,488H. This suggests that dynorphin-(1-13) and U50,488H may not bind to the same kappa receptor isotype. The partial antagonism of KETO by both naloxone and (-)-(1R,5R,9R)-5,9-dimethyl-2-(3-furylmethyl)-2'-hydroxy-6,7-benzomor pha n methanesulfonate is consistent with a kappa/mu effect of this compound. Naloxone, at the dose used in these studies, did not modify the response to U50,488H. In contrast to their inhibitory action on gastric emptying, the kappa agonists, dynorphin-(1-13) and U50,488H, did not alter acid secretion. The suppressive action of KETO on acid secretion may be due to activation of mu receptors. The inhibitory effect of dynorphin-(1-13) on sodium output was blocked by (-)-(1R,5R,9R)-5,9-dimethyl-2-(3-furylmethyl)-2'-hydroxy-6,7-benzomor pha n methanesulfonate, suggesting a role for kappa agonists in the control of nonparietal secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Multidimensional scaling of subjective judgements of drug similarities among ketocyclazocine, morphine, cyclazocine, naloxone and placebo.

Profiles of the subjective and physiologic effects of opioid drugs in man cannot be assigned with precision to specific opioid drug-receptor interactions. We administered a set of training doses of ketocyclazocine, morphine, cyclazocine, naloxone and placebo to 10 drug-using volunteers and obtained similarity judgements between each of 2 test doses of the drugs and a training dose. These data were submitted to multidimensional scaling analysis (INDSCAL) using both neighboring cells estimates and root mean square estimates to estimate missing cells in the data matrices. The results of these analyses are convergent, appear valid and indicate that there are three drug dimensions expressed in this data set: morphine versus placebo and naloxone; cyclazocine and ketocyclazocine versus placebo and naloxone; and ketocyclazocine versus cyclazocine. We interpret this result as supporting evidence that in the set of five drugs studied, three subjective states are induced.

Kappa opioid receptors stimulate phosphoinositide turnover in rat brain.

The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The kappa-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other kappa-agonists Dynorphin-A (1-13) amide, and its protected analog D[Ala]2-dynorphin-A (1-13) amide also produced a significant increase in the formation of [3H]-IP's, whereas the mu-selective agonists [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin and morphine and the delta-selective agonist [D-Pen2,5]-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the kappa-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medulla. The results indicate that brain kappa- but neither mu- nor delta-receptors are coupled to the PI turnover response.

Effects of the opiate antagonists diprenorphine and naloxone and of selected opiate agonists on feeding behavior in guinea pigs.

Opiate-sensitive feeding behavior has now been demonstrated in a number of species. We sought information on which opioid receptors might be involved in the observed feeding behaviors. Guinea pigs are known to have higher concentrations of the opioid kappa receptor than any other laboratory animal, so we compared the feeding suppressive potency of the general opiate antagonist, diprenorphine to that of the relatively more mu-specific antagonist, naloxone in that species. We found that diprenorphine was over twenty times more effective than naloxone in suppressing feeding in guinea pigs, suggesting the importance of receptors other than mu in feeding initiation in the guinea pig. Confirmatory evidence for the role of kappa receptors was sought, but not found, in comparisons of the effectiveness of different types of opiate agonists in promoting feeding in these animals. These agonists suppressed, rather than stimulated feeding. We conclude that no feeding stimulatory effects of opiates can be demonstrated in guinea pigs. This observation may indicate that opioids play little role in the natural regulation of feeding in this species or that opioids result in prolonged sedation during which the animals fail to eat. The greater feeding suppressive potency of diprenorphine, a general opiate antagonist, versus naloxone, a mu-preferential antagonist, indicates that to whatever extent opiates are involved in guinea pig feeding, the opiate effect is probably not a mu receptor effect.