Ewa Malatynska

Molecular biology and pharmacology of cloned opioid receptors.

The cloning and expression of DNA for the three major opioid receptor types (μ, δ, and χ) present new research opportunities for the characterization of opioid drugs and their interactions with these receptors. Genomic and cDNA clones for opioid receptors exist for several animal species including mouse, rat, guinea pig, and human. These include clones for all three human opioid receptor types. The receptor proteins consist of about 400 amino acids and have the characteristic seven trans‐membrane domain structure of G‐protein‐coupled receptors. There is about 60% amino acid identity between opioid receptor types and about 90% identity between a receptor type cloned from different animal species. All opioid receptor types mediate the inhibition of adenylyl cyclase in response to agonist binding. Radioligand binding and functional studies using the cloned receptors tend to support current conclusions on opioid drug receptor selectivity and activity. Investigations of opioid receptor chimeras and single amino acid mutants are providing information on the ligand recognition sites of these receptors and essential support for the development of computational opioid receptor models. A molecular model of the human δ opioid receptor is included in this review.—Knapp, R. J., Malatynska, E., Collins, N., Fang, L., Wang, J. Y., Hruby, V. J., Roeske, W. R., Yamamura, H. I. Molecular biology and pharmacology of cloned opioid receptors. FASEB J. 9, 516–525 (1995)

Antidepressant activity of memory-enhancing drugs in the reduction of submissive behavior model

The present study tests the activity of nootropic drugs in a behavioral test linked to depression. This test measures the reduction of submissive behavior in a competition test as the relative success of two food-restricted rats to gain access to a feeder. Nootropic drugs tested include piracetam (2-oxo-1-pyrrolidineacetamide), aniracetam (1-(4-methoxybenzoyl)-2-pyrrolidinone), the Ampakine, Ampalex, 1-(quinoxalin-6-ylcarbonyl)piperidine, and analogs were compared to the antidepressants, fluoxetine ((+/-)-N-methyl-gamma-(4-[trifluoromethyl]phenoxy)-benzenepropanamine) and desimpramine (5H-dibenz[b,f]azepine-5-propanamine, 10,11-dihydro-N-methyl-, monohydrochloride), while the anxiolytic diazepam (7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2(1H)-one) served as a control. Drugs were given intraperitoneally for 3 weeks. The antidepressant and nootropic drugs reduced submissive behavior over time. The effect was dose dependent as measured for fluoxetine and Ampakines. The reduction of submissive behavior by Ampakines gradually faded after cessation of treatment and had a more rapid onset of activity (during the 1st week of treatment) than fluoxetine (after 2 weeks). The results suggest that Ampakines may have antidepressant activity. The potential of depression treatment with memory-enhancing drugs is hypothesized and the link between cognition and depression is discussed.

Central GABAergic Systems and Depressive Illness

Clinical depression and other mood disorders are relatively common mental illnesses but therapy for a substantial number of patients is unsatisfactory. For many years clinicians and neuroscientists believed that the evidence pointed toward alterations in brain monoamine function as the underlying cause of depression. This point of view is still valid. Indeed, much of current drug therapy appears to be targeted at central monoamine function. Other results, though, indicate that GABAergic mechanisms also might play a role in depression. Such indications stem from both direct and indirect evidence. Direct evidence has been gathered in the clinic from brain scans or postmortem brain samples, and cerebrospinal fluid (CSF) and serum analysis in depressed patients. Indirect evidence comes from interaction of antidepressant drugs with GABAergic system as assessed by in vivo and in vitro studies in animals. Most of the data from direct and indirect studies are consistent with GABA involvement in depression.

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.

Antidepressants and seizure-interactions at the GABA-receptor chloride-ionophore complex

Convulsive seizures are a potential side effect of antidepressant drug treatment and can be produced by all classes of antidepressants. It is also known that some convulsant and anticonvulsant drug actions are mediated by the GABA-receptor chloride-ionophore complex. Drugs acting at this complex appear to induce convulsions by inhibiting chloride conductance through the associated chloride channel. Using the method of GABA-stimulated 36Cl-uptake by rat cerebral cortical vesicles, we show that some antidepressant drugs (imipramine, amitryptyline, and mianserine) can inhibit the GABA-receptor chloride uptake, and that the degree of chloride channel inhibition by these drugs correlates with the frequency of convulsive seizures induced by them.

Reduction of Submissive Behavior in Rats: A Test for Antidepressant Drug Activity

Randomly paired rats were food deprived overnight and placed in an apparatus compelling them to compete for a food reward. About half of these pairs developed a dominant-submissive relationship measured as a significant difference in time spent on the feeder by each rat. This relationship developed over a 2-week period and remained stable for at least the next 5 weeks. Treatment of the submissive subjects, for at least 2 weeks, with imipramine, desipramine, or fluoxetine (10 mg/kg) significantly reduced submissive behavior. The effect faded after cessation of treatment with desipramine. Fluoxetine was further tested at 2.5- and 5-mg/kg doses and showed a dose-dependent reduction of submissive behavior. Treatment of submissive rats with the anxiolytic diazepam (1 mg/kg) was ineffective. The prevalence of dominant-submissive relationships and the effect of desipramine and imipramine on submissive behavior were gender independent. The predictive, face, and construct validity of the behavioral test is discussed.

Human δ opioid receptor: A stable cell line for functional studies of opioids

A human delta opioid receptor cDNA clone (pREP10/hDOR) was transfected into Chinese hamster ovary (CHO) cells. The stable cell line expressed a high density of delta opioid receptors (137,000 +/- 21,600 receptors/cell). DPDPE inhibited 90% of the forskolin-stimulated cAMP accumulation in these cells with high potency (EC50 = 1.3 nM). This effect of DPDPE was antagonized by naltrindole. The pseudo-pA2 value (Ke) of 155 pM for naltrindole is consistent with that measured for delta receptor antagonism in the mouse vas deferens. This is the first detailed characterization of DPDPE activity on forskolin-stimulated cAMP accumulation mediated through a human delta opioid receptor. The data support the use of the recombinant cell line for functional studies of opioid drugs.

The GABAA Receptor Complex as a Target for Fluoxetine Action

The clinically important antidepressant fluoxetine is established as a selective serotonin reuptake inhibitor. This study demonstrates that fluoxetine also interacts with the GABAA receptor complex. At concentrations above 10 μM fluoxetine inhibited the binding of both [3H]GABA (IC50 = 2 mM) and [3H]flunitrazepam (IC50 = 132 μM ) to the GABAA receptor complex in brain cortical membranes. Low fluoxetine concentrations (1 nM) enhanced GABA-stimulated Cl− uptake by a rat cerebral cortical vesicular preparation. At higher concentrations (100 μM and 1 mM), however, fluoxetine inhibited GABA-stimulated Cl− uptake, an effect related to a reduction in Emax. These observations might assist in an explanation of the basis of the antidepressant action of fluoxetine.

Properties of TAN-67, a nonpeptide δ-opioid receptor agonist, at cloned human δ-and μ-opioid receptors

Abstract 2-methyl-4a α(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a α-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67) is a nonpeptidic δ-opioid receptor agonist. This report describes its receptor binding affinity and agonist potency at human and mouse δ and μ-opioid receptors. The binding affinities of TAN-67 and the cyclic enkephalin analog, [D-Pen2, 4′-Cl-Phe4, D -Pen5]enkephalin (pCl-DPDPE) were measured by the radioligand binding inhibition studies at mouse and human variants of the δ and μ-opioid receptor using [3H]Naltrindole and [3H] D -Phe- Cys  Tyr  D  Trp  Orn  Thr  Pen -Thr-NH2, respectively. TAN-67 showed high binding affinity (Ki = 0.647 nM) at the human δ-opioid receptor and high δ-opioid receptor binding selectivity (> 1000-fold) relative to the human μ-opioid receptor. TAN-67 also showed high potency (EC50 = 1.72 nM) for the inhibition of forskolin-stimulated cAMP accumulation at human δ-opioid receptors expressed by intact Chinese hamster ovary cells but low potency (EC50 = 1520 nM) at human μ-opioid receptors expressed by intact B82 mouse fibroblast cells. The results show that TAN-67 has similar binding affinities, selectivity and potencies as pCl-DPDPE at human δ and μ-opioid receptors. These results combined with the nonpeptidic structure of TAN-67 suggest that this compound has therapeutic potential as a δ-opioid receptor agonist.

δ-Opioid receptor: the third extracellular loop determines naltrindole selectivity

Human delta/mu-opioid receptor chimeras were constructed to determine the role of the second and third extracellular loops in alkaloid ligand selectivity. Exchanging the third extracellular loop of the delta-opioid receptor with that of the mu-opioid receptor dramatically decreased the affinity of naltrindole, but not that of morphine. The results suggest that different domains of the opioid receptor are involved in the binding of naltrindole and morphine.