The opiate addiction test: a clinical evaluation of a quick test for physical dependence on opiate drugs.

1. Mydriasis (pupil dilation) in response to conjunctivally applied naloxone hydrochloride has been demonstrated using an innovative electronic binocular pupillometer in 40 opiate dependent patients, on maintenance methadone treatment. 2. No pupillary response to naloxone was seen when an identical procedure was carried out in a control population of 12 healthy volunteers. 3. After a baseline measurement of pupil size, two drops of naloxone hydrochloride were instilled into the conjunctival sac of one eye. Serial binocular pupillometry was then carried out at 5, 10, 15, 20, 25, 30, 35, 40 and 45 min post-instillation. 4. Discriminant analysis between the control and patient groups showed that the false negative rate (error of misclassification to the wrong population) was lowest (20%) at 40 min post-eyedrop instillation, with no false positives in the control group. 5. The study has therefore shown an improvement in the previously reported false negative rate (25%) [1,2], of the conjunctival naloxone test of opiate dependence, with the use of our innovative electronic binocular pupillometer.     

Interactions of Tyr-MIF-1 at opiate receptor sites

Binding of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to mu and delta opiate receptors was compared with other putative opiate antagonist peptides by displacement of iodinated ligands selective for mu (DAGO, FK33824, and morphiceptin) and delta (DPDPE) receptors. Tyr-MIF-1 and ACTH (1-24 and 1-39) inhibited binding of 125I-DAGO with IC50's of about 1 microM. FMRF-NH2 was about an order of magnitude weaker while CCK-8 and MIF-1 failed to inhibit 50% of binding at concentrations up to 100 microM. Morphiceptin, Tyr-MIF-1, and ACTH were less potent but more efficacious than DAGO, FK33824, morphine, or naloxone in inhibiting the binding of 125I-morphiceptin. Tyr-MIF-1 appeared to have a more selective action at opiate receptors than ACTH; in contrast to their effects at 125I-DAGO-labeled sites, morphiceptin and Tyr-MIF-1 inhibited less than 50% of 125I-DPDPE binding at concentrations up to 10 and 50 microM, while ACTH 1-39 and 1-24 inhibited more than 80% of the binding at 2.5 and 5 microM, respectively. The results indicate that at relatively high concentrations Tyr-MIF-1, like ACTH, can affect binding to the opiate receptor, but unlike ACTH, binding of Tyr-MIF-1 appears relatively selective for the mu site.     

Effect of psychotropic drugs on histamine H2-receptors in rat isolated uterus.

The effects of the monoamine oxidase inhibitors iproniazid, nialamide and phenelzine, the neuroleptics haloperidol and thioridazine, and the benzodiazepines diazepam and clonazepam on histamine H2-receptors were assessed on rat isolated uterus. The monoamine oxidase inhibitors showed a slight non-competitive antihistamine H2 activity, while diazepam and clonazepam were devoid of any action. Haloperidol and thioridazine inhibited in a dose-dependent manner the tonic component of KCl induced contraction, while thioridazine under the same conditions exhibited a slight antihistamine H2 activity. These data show that the drugs tested are devoid of or elicited only a slight antihistamine H2 activity at high non-therapeutic concentrations.     

Multiple opiate receptors of brain and spinal cord in opiate addiction.

1. Chronic administration of opiates to rodents results in the development of tolerance to their pharmacological effects. Physical dependence also develops and is shown by the appearance of abstinence syndrome. 2. Opiates produce their effects by acting on three types of opiate receptors, namely mu, delta and kappa. The qualitative and quantitative aspects of the tolerance-dependence and abstinence symptoms observed after chronic administration of agonists acting at mu-, delta- and kappa-opiate receptors appear to differ. 3. Tolerance-dependence on mu-opiate agonists, such as morphine, is associated with down-regulation of mu-opiate receptors in spinal cord and specific areas of the brain but delta- and kappa-opiate receptors are unchanged. During abstinence from mu-opiate agonists, brain and spinal cord mu-, delta- and kappa-opiate receptors are unaffected. 4. Chronic administration of kappa-opiate agonists, such as U-50,488H, results in the development of tolerance to its pharmacological effects and a mild degree of physical dependence. Such changes are associated not only with alterations of delta and kappa opiate receptors in brain and spinal cord, but also primarily with a down-regulation of kappa-opiate receptors in spinal cord and specific brain regions. mu-Opiate receptors are unaffected. 5. Chronic administration of delta-opiate agonists results in down-regulation of brain delta-opiate receptors. 6. It is concluded that tolerance-dependence on mu-, delta- and kappa-opiate receptors is associated with down-regulation of their own type of receptors in the spinal and supraspinal areas. Abstinence, on the other hand, does not alter brain and spinal cord opiate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformationally defined adrenergic agents. 2. Catechol imidazoline derivatives: biological effects at alpha 1 and alpha 2 adrenergic receptors

The synthesis and pharmacology of 2-(5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphthyl)imidazoline (A-54741, 4), a very potent alpha-adrenergic agonist, are described. The change in biological activity resulting from variation of the carbocyclic ring size of 4 from four through seven members (2-5) is presented, as well as an explanation that accounts for this change in activity by considering the "exactness of fit" of these compounds to both the alpha 1- and alpha 2-adrenergic receptors. Compound 4 was found in vitro to be a full agonist with greater potency at the alpha 2 receptor (ED50 norepinephrine (NE)/ED50 4 = 188 +/- 22) than at the alpha 1 receptor (ED50 NE/ED50 4 = 13 +/- 2).     

Hypothalamic regulation of the cardiovascular and respiratory systems: role of specific opiate receptors.

Experiments were designed to evaluate the role of mu and delta opiate receptors in central cardiovascular control in the hypothalamic nucleus preopticus medialis of rats anaesthetized with pentobarbitone. The highly selective mu opiate receptor agonist D-Ala2-MePhe4-Gly5-ol-enkephalin was extremely potent in eliciting hypotension and bradypnoea; tachycardia was elicited by a low dose (0.064 nmol), but not by higher doses (0.64-6.4 nmol). Other selective mu receptor agonists (morphine sulphate, morphiceptin) caused tachycardia at lower doses (0.64, 6.4 nmol), hypotension and bradypnoea after the highest dose (64 nmol). The relatively selective delta receptor agonist D-Ala2-D-Leu5-enkephalin caused profound bradypnoea and hypotension at the high dose (64 nmol), tachycardia after the lowest dose (0.64 nmol), but bradycardia was found during the hypotension induced by the high dose (64 nmol). All of the opiate/opioid effects were reversed by naloxone (0.5 mg kg-1, i.v.). It is concluded that mu receptors may mediate the cardiovascular and respiratory effects of opiates and opioid peptides in the nucleus preopticus medialis of the rat.     

Prenatal cocaine exposure increases the behavioral sensitivity of neonatal rat pups to ligands active at opiate receptors

Offspring of dams given 40 mg/kg cocaine HCl (C40) from gestational day 8–20 (E8–E20), pair-fed dams injected daily with saline (PF), nutritional control dams placed on a 40% cellulose based diet and injected with saline daily (NC), and untreated dams (LC) were examined. Offspring were given morphine (0.0, 0.1, or 0.5 mg/kg SC) on postnatal day 10–11 (P10–11) in Experiment 1, and isolation-induced ultrasonic vocalizations were measured. Planned comparisons indicated that both C40 and NC offspring exhibited a greater sensitivity to the morphine-related decrease in isolation-induced ultrasounds than LC controls. However, the presence of an anesthetized littermate suppressed isolation-induced ultrasounds equally across all groups, with all groups of offspring spending equal amounts of time in physical contact with the littermate. A tail-flick measure of analgesia indicated that PF animals were hyperalgesic relative to the other prenatal treatment groups; however, no differences in sensitivity to morphine were seen across the prenatal groups. In Experiment 2, animals were given the selective δ, [D-Pen²,D-Pen⁵]-enkephalin (DPDPE), and μ, [D-Ala²-NMe-Phe⁴Gly ol]-enkephalin (DAMGO) agonists ICV and ultrasonic vocalizations were recorded. Results indicated that both C40 and NC offspring were more sensitive to the low dose of DAMGO; however, because of the profound suppression of vocalizations seen at both doses of DPDPE, potential differences among the prenatal treatment groups in responsiveness to the δ agonist were difficult to detect. The altered psychopharmacological sensitivity of C40 and NC offspring is unlikely to be related simply to the anorexic effects of the treatments, as the PF offspring did not differ from LC offspring in terms of sensitivity to the agonists. The increased sensitivity of C40 offspring to morphine and DAMGO in terms of isolation-induced ultrasounds in the absence of any alteration in sensitivity to morphine-induced analgesia is consistent with previous findings showing that C40 weanlings exhibit increased binding of [³H]naloxone in forebrain regions such as the nucleus accumbens but no increase in binding in brain stem regions associated with the control of analgesia (13).     

Effect of drugs active at adenosine receptors upon chronic stress-induced hyperalgesia in rats

Hyperalgesia and altered activities of enzymes involved in nucleotide hydrolysis are observed after exposure to repeated restraint in rats. Here, we investigated the effect of an adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine (CPA, 3.35 mg/kg, i.p.), adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.8 mg/kg, i.p.) as well the effect of an adenosine reuptake blocker, dipyridamole (5 mg/kg, i.p.), on nociception in chronically stressed and control rats. We repeatedly submitted rats to restraint for 40 days. Nociception was assessed with a tail-flick apparatus. The control group presented increased tail-flick latencies after administration of CPA and dipyridamole, but this effect was not observed in the stressed group. DPCPX by itself had no effect on nociception. The analgesic effect of CPA and dipyridamole observed in the control group was reverted by DPCPX. These results indicate the involvement of adenosine A(1) receptor in the antinociception observed in control animals and suggest that the pain signaling induced by chronic stress presents a different modulation involving the adenosinergic system.     

Pharmacological profiles of fentanyl analogs at mu, delta and kappa opiate receptors.

Receptor binding assays using [3H]DAGO ([D-Ala2,MePhe4-Gly5-ol]enkephalin) (mu), [3H]DPDPE ([D-Pen2,D-Pen5]enkephalin) (delta) and [3H]U-69593 (kappa) were done in guinea pig whole brain membranes. Agonist activity was determined in norbinaltorphimine or beta-funaltrexamine (beta-FNA) treated guinea pig ileum (mu and kappa, respectively) and beta-FNA-treated mouse vas deferens (delta). The compounds with highest affinity were the most potent at the mu-receptor. The selectivity observed in the binding affinities was also found in in vitro activity. No correlation was found between mu-affinity and selectivity; the highest affinity analog, lofentanil, was found to be among the least selective, while another high affinity analog, R30490, was the most mu-selective. The results show that not all fentanyls are highly mu-selective, and could produce actions through delta- and kappa-opiate receptors.     

Interactions of alpha 2-adrenoceptor antagonists with medetomidine and with ethanol in a holeboard test

The effects of two selective alpha 2-adrenoceptor agonists (clonidine and medetomidine) and antagonists (atipamezole and idazoxan) were examined in the holeboard test. The interactions of the two antagonists with ethanol were also investigated. Atipamezole (0.1-3.0 mg/kg) and idazoxan (0.01-0.3 mg/kg) were without effect on either directed exploration or locomotor activity in the holeboard test, whereas clonidine (0.003-0.1 mg/kg) and medetomedine (0.003-0.1 mg/kg) were sedative. Atipamezole (1-3 mg/kg) and idazoxan (0.3-1.0 mg/kg) reversed the behavioral effects of 0.1 mg/kg of medetomidine. When administered with ethanol (2 g/kg), atipamezole (1-3 mg/kg) showed a significant antagonism of the ethanol-induced reduction in exploratory head-dipping: no change in the locomotor stimulant properties of ethanol was seen. A similar trend was seen for exploration after a combination of idazoxan (1-3 mg/kg) with 2 g/kg ethanol; however, the 3 mg/kg dose attenuated the locomotor stimulant effect. Both antagonists caused a dose-related reduction in the increase in head-dipping seen after administration of 1 g/kg of ethanol, without any effect on the locomotor stimulant effect of this dose. These results suggest mediation of alpha 2-adrenoceptors in some of the behavioral effects of ethanol.     

Presynaptic localization of opiate receptors in the vagal and accessory optic systems: an autoradiographic study.

Various biochemical and autoradiographic studies suggest a close association of opiate receptors with certain groups of primary sensory nerve fibres in the spinal cord and brainstem. The effect of deafferentation on autoradiographically-determined. stereospecific ³H-diprenorphine binding sites, in two afferent systems, the vagus nerve and the accessory optic system, was investigated in the rat. Vagotomies appreciably decreased the density of opiate receptors associated with the intramedullary portion of the vagus nerve, the solitary tract and its nucleus and nucleus ambiguus. Enucleation reduced the density of opiate receptors associated with the midbrain accessory optic tract and its nuclei. These results, plus other autoradiographic and biochemical evidence, suggest a rather specific presynaptic localization of some opiate receptors to small calibre and unmyelinated sensory fibres in some areas of the CNS.     

Activity of spantide I and II at various tachykinin receptors and NK-2 tachykinin receptor subtypes.

We compared the ability of spantide I and II to antagonize tachykinins in monoreceptor bioassays. Both peptides antagonized the response to substance P methylester in the guinea-pig ileum (NK-1 receptor-mediated) with greater affinity than the responses mediated by NK-2 or NK-3 receptors in other bioassays. Spantide II was about 10 times more potent than spantide I as an NK-1 antagonist and also possessed some selectivity for the NK-2 receptor subtype present in the hamster trachea. Spantide II is a suitable tool to assess the role of NK-1 receptors in the central and peripheral nervous system.     

Interactions of ligands at angiotensin II-receptors and imidazoline receptors

Ligands for angiotensin II-(AT)-receptors and imidazoline receptors have structural similarities and influence blood pressure via various mechanisms. The goal of this study was to study the specificity of various ligands by displacement experiments. Antazoline, cimetidine, clonidine, efaroxan, guanabenz, guanethidine, idazoxan, moxonidine and rilmenidine up to a concentration of 100 microM failed to displace the specific binding of [125I]Sar1,Ile8 angiotensin II at the AT1-receptor characterized by losartan (IC50 = 26 +/- 12 nM) in liver homogenate. The same substances up to 100 microM produced no reduction of specific [125I]Sar1,Ile8 angiotensin II binding to the AT2-receptor of phaeochromocytoma cell membranes characterized by PD123319 (IC50 = 20 +/- 5 nM). Displacement experiments at the imidazoline I1-receptors were performed on bovine adrenal medulla membranes using [3H]clonidine after characterization by the I1-ligand clonidine (IC50 = 459 +/- 13 nM) and the I2-ligand idazoxan (IC50 = 3.29 +/- 0.88 microM). The investigated AT-receptor ligands angiotensin II, losartan, EXP 3174 and PD123319 revealed no displacement of [3H]clonidine up to a concentration of 100 microM. The I2-receptor in liver homogenate was characterized by displacement of [3H]idazoxan by cold idazoxan and clonidine (IC50 = 0.37 +/- 0.17 and 68 +/- 31 microM, respectively). The investigated AT-receptor ligands angiotensin II, losartan and PD123319 failed to displace [3H]idazoxan specifically up to 100 microM. Hence, the tested substances showed no cross-reactivity at the corresponding AT- and I-receptors up to 100 microM, a concentration markedly higher than the plasma concentrations achieved after therapeutic application.     

EEG-behavioral dissociation after morphine-and cyclazocine-like drugs in the dog: further evidence for two opiate receptors.

Previous studies have suggested the presence of multiple opioid receptors. In the chronic spinal dog, the μ receptor was postulated to mediate behavioral indifference, whereas the $\text{k}$ receptor was associated with sedation. The effects of morphine, a μ agonist, were compared with those of Win 35,197-2 (ethylketocyclazocine) and ketocyclazocine, $\text{k}$ agonists, in the unrestrained intact dog. Intravenous administration of morphine (0.5 1.0 and 2.0 mg/kg), Win 35,197-2 (0.05 0.1 and 0.2 mg/kg), and ketocyclazocine (1.6 mg/kg) caused similar dissociation of the EEG and behavior. The dissociation was characterized by high voltage delta EEG synchrony primarily in the parietal cortex which accompanied ataxia and catalepsy in nonsleeping postures. Morphine increased total sleep and slow wave sleep, and lowered temperature and heart and respiratory rates. The $\text{k}$ agonists did not increase total sleep but increased temperature and heart and respiratory rates. Vomiting occurred more often after morphine than after the $\text{k}$ agonists. In certain instances, the effects of morphine were completely antagonized by a low dose of naloxone (30μg/kg), whereas 33 times the dose (1 mg/kg) was required to antagonize the effects of Win 35,197-2. The disparate effects of the μ and $\text{k}$ opioid agonists on behavior, sleep and the EEG and their differences in sensitivity to naloxone antagonism extend and support the concept of multiple opiate receptors in the brain. It was concluded, however, that the sedative effects of the opioids are mediated at the μ receptor.     

Pharmacological characterization of rat alpha 2-adrenergic receptors.

We described previously the molecular characterization of a rat alpha 2B-adrenergic receptor and have shown also that the rat genome contains three closely related alpha 2-adrenergic receptor genes. To characterize the ligand-binding properties of these receptor gene products, we expressed the DNAs encoding these receptors individually in COS-1 cells and studied their binding to a wide variety of typical and atypical adrenergic ligands. The receptors displayed high affinity binding to the radioligand [3H] rauwolscine, with equilibrium dissociation constants ranging from 1.4 to 2.8 nM. Kinetic analysis of the binding of [3H]rauwolscine to membranes from transfected cells was in very good agreement with data obtained from saturation analysis. We examined the ability of a number of agents to compete for the binding of [3H]rauwolscine to the alpha 2-adrenergic receptor-transfected membranes. Whereas one of these receptors displayed a pharmacological profile typical of an alpha 2A-adrenergic receptor, the other two receptors showed similar pharmacological properties characteristic of an alpha 2B-adrenergic receptor. The two alpha 2B-like adrenergic receptors differed, however, in the ratios of Ki values for oxymetazoline and prazosin, as well as the Ki ratio of prazosin and yohimbine. In addition, the two alpha 2B-like adrenergic receptors had a 9-fold difference in affinity for chlorpromazine. The pharmacological characterization of the three rat alpha 2-adrenergic receptor gene products is consistent with the known pharmacology of alpha 2-adrenergic receptors, as documented using tissues and cell lines.