Aaron Janowsky

The N-methyl-d-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications

The N-methyl-D-aspartate (NMDA) receptor is an example of a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits. NMDA receptors are composed of seven known subunits (NR1, NR2A-D, NR3A-B). The present review focuses on the NR2B subunit of the receptor. Over the last several years, an increasing number of reports have demonstrated the importance of the NR2B subunit in a variety of synaptic signaling events and protein-protein interactions. The NR2B subunit has been implicated in modulating functions such as learning, memory processing, pain perception, and feeding behaviors, as well as being involved in a number of human disorders. The following review provides a summary of recent findings regarding the structural features, localization, functional properties, and regulation of the NR2B subunit. The review concludes with a section discussing the role of NR2B in human diseases.

Action of mianserin and zimelidine on the norepinephrine receptor coupled adenylate cyclase system in brain: Subsensitivity without reduction in β-adrenergic receptor binding

The present experiments were undertaken to ascertain whether non-tricyclic, clinically effective, antidepressant drugs such as the tetracyclic mianserin and the serotonin uptake inhibitor, zimelidine, cause alterations of central noradrenergic receptor systems similar to those elicited by classical tricyclic antidepressants, MAO inhibitors and ECT. Like desipramine (DMI), mianserin and zimelidine caused, after chronic administration, a significant reduction in the sensitivity of the cyclic AMP response to (R)-norepinephrine while a single dose of the drugs did not affect the neurohormonal response. The basal levels of the nucleotide were not changed by the antidepressant drugs following acute or chronic administration. While the noradrenergic subsensitivity caused by DMI was linked to a reduction in the Bmax value of 3H-dihydroalprenolol binding, the subsensitivity caused by mianserin and zimelidine was not associated with a decreased density of β-adrenergic receptors. In accord with these data on β-adrenergic receptor binding, the responsivenss of the adrenergic cyclic AMP generating system to the β-adrenergic agonist, (R)-isoproterenol, was significantly reduced following chronic treatment with DMI but not with mianserin or zimelidine. The results provide further evidence for the theory that the delayed therapeutic action of different prototypes of antidepressant treatments may be related to the delayed change in noradrenergic receptor function and that a reduction in the density of β-adrenergic receptors is only one mechanism by which the sensitivity of the system is regulated.

Characterization of Sodium‐Dependent [3H]GBR‐12935 Binding in Brain: A Radioligand for Selective Labelling of the Dopamine Transport Complex

Abstract: High‐affinity and saturable binding sites for the diphenyl‐substituted piperazine derivative [3H]GBR‐12935 have been characterized in crude synaptosomal membranes prepared from rat brain. The specific binding of [3H]GBR‐12935 is sodium‐dependent and is unevenly distributed among various brain regions, with the highest concentration of binding sites being found in the corpus striatum and nucleus accumbens. Sodium‐dependent [3H]GBR‐12935 binding in all other brain areas was 10% or less of the binding found in the striatum. The affinity of [3H]GBR‐12935 for binding sites in the striatum is increased in the presence of Na+ but other cations, including K+, Ca2+, or Mg2+, inhibit specific binding. There is an excellent correlation (r= 0.96, p < 0.01) between the potencies of a series of drugs in inhibiting [3H]GBR‐12935 binding to striatal membranes and their potencies in inhibiting [3H]3,4‐dihydroxyphenylethylamine ([3H]dopamine) uptake in synaptosomes. Agonists and antagonists of other neurotransmitter receptor or drug recognition sites have little or no effect on specific [3H]GBR‐12935 binding to striatal membranes. In addition, prior intracerebroventricular administration of 6‐hydroxydopamine results in a decrease in the number of specific [3H]GBR‐12935 binding sites in the striatum. These data indicate that [3H]GBR‐12935 is a selective radioligand of the presynaptic dopamine transport complex in brain.

[3H]GBR-12935 binding to the dopamine transporter is decreased in the caudate nucleus in Parkinson's disease

The specific binding of [3H]GBR‐12935 to membranes prepared from human caudate nucleus is saturable (Bmax 1.36 ± 0.18 pmol/mg protein), sodium dependent, and of high affinity (KD 2.34 ± 0.18 nM). Freezing of tissue from rat brain, or refrigeration followed by freezing, results in a small but significant (20%) decrease in specific [3H]GBR‐12935 binding when compared to the binding observed in fresh (nonfrozen) tissue, and this decrease may account, in part, for the differences in specific binding between rat and human brain membranes. Despite small differences in binding site density between fresh and frozen tissue there is a good correlation (r= 0.98; p < 0.01) between the potencies of a series of drugs in displacing specific [3H]GBR‐12935 binding to human caudate membranes and rat striatum as well as in inhibiting dopamine uptake in rat striatal synaptosomes (r= 0.96; p < 0.01). The specific binding of [3H]GBR‐12935 to membranes prepared from the caudate nuclei of patients with Parkinsons disease is decreased compared to membranes prepared from age‐and sex‐matched controls. These data suggest that [3H]GBR‐12935 binds in a sodium‐dependent fashion to the dopamine transport complex in human brain and that specific binding is decreased by a pathological degeneration of dopaminergic neurons to the caudate nucleus.

Regulation of recognition and action function of the norepinephrine (NE) receptor-coupled adenylate cyclase system in brain: Implications for the therapy of depression

Abstract In this paper, the characteristics and the regulation of the norepinephrine (NE) receptor-coupled adenylate cyclase system with its β-adrenoceptor sub-population in brain are reviewed. While α-adrenoceptors in brain are not clearly coupled in an inhibitory fashion to adenylate cyclase, gb-adrenoceptors and a non-β-population of NE receptors in brain tissue are coupled to adenylate cyclase. The NE receptor-coupled adenylate cyclase system displays remarkable plasticity. Both supersensitivity and subsensitivity of the NE-sensitive adenylate cyclase system are of the “homologous” type. Most, if not all, clinically effective antidepressant treatments, including ECT, cause upon chronic administration subsensitivity of the system accompanied by a decrease in the density of β-adrenoceptors. The formation of the NE receptor complex is a prerequisite for the regulation of both the sensitivity of the system and the number of its β-adrenoceptor population. In addition, serotonergic neuronal input is co-required for the down-regulation of β-adrenoceptors by DMI-like drugs, but not for the up-regulation by propranolol. In the absence of serotonergie neuronal input, β-adrenoceptors display characteristics of “uncoupled” receptors. Steroid hormones (adrenocorticoids, sex steroids) have also been shown to alter the biological responsiveness of the system and/or the density of the β-adrenoceptor population, and β-adrenoceptor-mediated phospholipid methylation has been implicated in the regulation of the NE signal transfer through the membrane. The regulation of the number of NE membrane receptors and the efficacy of their coupling to adenylate cyclase represent physiologically important steps in the transfer of the NE signal through the membrane. Consequently, the changes induced by antidepressants at the level of membrane receptor regulation are altering the intensity of the signal transfer and appear to represent a therapeutically relevant biochemical action.

The niacin skin flush abnormality in schizophrenia: a quantitative dose-response study

Niacin dilates cutaneous blood vessels, resulting in a pronounced skin flush in most people. The flush response to niacin is attenuated in schizophrenia, but the quantification and physiological mechanism of this abnormality have not been described in detail. It is not clear whether the mechanism involves changes in the pharmacological sensitivity to niacin, or whether there is a reduced ability of the vasculature to dilate adequately in subjects with schizophrenia. We address this question in the present study by characterizing the dose-response relationship between topically applied alpha-methylnicotinate (AMN) and cutaneous blood flow changes, which were quantified by laser Doppler flowmetry. The dose-response curve was shifted to the right in subjects with schizophrenia. The EC(50) value of AMN was significantly increased in the schizophrenia group (mean: 1.66 mM; 95% confidence interval: 1.04-2.65 mM) as compared to the control group (mean: 0.38 mM; 95% confidence interval: 0.263-0.547 mM). The blood flow responses to higher AMN doses were lower in the schizophrenics; however, there was no statistically significant difference in the extrapolated maximal blood flow response value (F(max)) between the two groups. The results suggest that the skin flush abnormality in schizophrenia primarily reflects reduced pharmacological sensitivity to niacin rather than an inadequate cutaneous vasodilatory response to the stimulus. Since vasodilatation in response to niacin requires the release of prostaglandins, the data from this study suggest that schizophrenia is associated with abnormalities in enzymes, receptors, or signal transduction mechanisms that affect the synthesis, release, or response to vasodilatory prostaglandins.

Effects of subchronic clozapine and haloperidol on striatal glutamatergic synapses

Abstract: Subchronic treatment with haloperidol increases the number of asymmetric glutamate synapses associated with a perforated postsynaptic density in the striatum. To characterize these synaptic changes further, the effects of subchronic (28 days) administration of an atypical antipsychotic, clozapine (30 mg/kg, s.c.), or a typical antipsychotic, haloperidol (0.5 mg/kg, s.c.), on the binding of [3H]MK‐801 to the NMDA receptor‐linked ion channel complex and on the in situ hybridization of riboprobes for NMDAR2A and 2B subunits and splice variants of the NMDAR1 subunit were examined in striatal preparations from rats. The density of striatal glutamate immunogold labeling associated with nerve terminals of all asymmetric synapses and the immunoreactivity of those asymmetric synapses associated with a perforated postsynaptic density were also examined by electron microscopy. Subchronic neuroleptic administration had no effect on [3H]MK‐801 binding to striatal membrane preparations. Both drugs increased glutamate immunogold labeling in nerve terminals of all asymmetric synapses, but only haloperidol increased the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated postsynaptic density. Whereas subchronic administration of clozapine, but not haloperidol, resulted in a significant increase in the hybridization of a riboprobe that labels all splice variants of the NMDAR1 subunit, both drugs significantly decreased the abundance of NMDAR1 subunit mRNA containing a 63‐base insert. Neither drug altered mRNA for the 2A subunit, but clozapine significantly increased hybridization of a probe for the 2B subunit. The data suggest that some neuroleptic effects may be mediated by glutamatergic systems and that typical and atypical antipsychotics can have varying effects on the density of glutamate in presynaptic terminals and on the expression of specific NMDA receptor splice variant mRNAs. Alternatively, NMDAR1 subunit splice variants may differentially respond to interactions with glutamate.

Regulation of NMDA Receptor Subunits and Nitric Oxide Synthase Expression During Cocaine Withdrawal

Abstract: The present study characterized the effects of withdrawal from cocaine on the expression of NMDA receptor subunits (NR1, NR2B) and neuronal nitric oxide synthase. FosB induction was measured to confirm that repeated cocaine exposure influenced protein expression, as previously reported. Administration of cocaine followed by 24 h, 72 h, or 14 days of withdrawal resulted in alterations of NR1 and NR2B subunits and neuronal nitric oxide synthase expression as measured by immunohistochemical labeling of rat brain sections. Optical density analyses revealed significant up‐regulation of NR1 in the ventral tegmental area at 72 h and 14 days of withdrawal. Structure‐specific and withdrawal time‐dependent alterations in NR2B expression were also found. After 24 h of withdrawal, cocaine‐induced decreases in NR2B expression were observed in the nucleus accumbens shell, whereas increases in NR2B expression were found in medial cortical areas. Two weeks of withdrawal from cocaine caused an ∼50% increase in NR2B subunit expression in regions of the cortex, neostriatum, and nucleus accumbens. In contrast, cocaine‐induced up‐regulation of neuronal nitric oxide synthase was transient and evident in cortical areas only at 24 h after the last drug injection. The results suggest that region‐specific changes in interactions among proteins associated with the NMDA receptor complex may underlie neuronal adaptations following repeated cocaine administration.

Subsensitivity of the norepinephrine receptor-coupled adenylate cyclase system in brain: Effects of Nisoxetine versus fluoxetine

Chronic administration of the norepinephrine (NE) reuptake inhibitor nisoxetine reduced the sensitivity of the NE receptor-coupled adenylate cyclase system in rat cortex. This subsensitivity, unlike that caused by desipramine (DMI), was not related to a reduction in specific beta-adrenergic receptor binding. The specific serotonin reuptake inhibitor fluoxetine had no effect on either neurohormonal sensitivity or 3H-dihydroalprenolol binding.

Characterization of the Antinociceptive Actions of Bicifadine in Models of Acute, Persistent, and Chronic Pain

Bicifadine (1-p-tolyl-3-azabicyclo[3.1.0]hexane) inhibits monoamine neurotransmitter uptake by recombinant human transporters in vitro with a relative potency of norepinephrine > serotonin > dopamine (≈1:2:17). This in vitro profile is supported by microdialysis studies in freely moving rats, where bicifadine (20 mg/kg i.p.) increased extrasynaptic norepinephrine and serotonin levels in the prefrontal cortex, norepinephrine levels in the locus coeruleus, and dopamine levels in the striatum. Orally administered bicifadine is an effective antinociceptive in several models of acute, persistent, and chronic pain. Bicifadine potently suppressed pain responses in both the Randall-Selitto and kaolin models of acute inflammatory pain and in the phenyl-p-quinone-induced and colonic distension models of persistent visceral pain. Unlike many transport inhibitors, bicifadine was potent and completely efficacious in both phases of the formalin test in both rats and mice. Bicifadine also normalized the nociceptive threshold in the complete Freunds adjuvant model of persistent inflammatory pain and suppressed mechanical and thermal hyperalgesia and mechanical allodynia in the spinal nerve ligation model of chronic neuropathic pain. Mechanical hyperalgesia was also reduced by bicifadine in the streptozotocin model of neuropathic pain. Administration of the D2 receptor antagonist (-)-sulpiride reduced the effects of bicifadine in the mechanical hyperalgesia assessment in rats with spinal nerve ligations. These results indicate that bicifadine is a functional triple reuptake inhibitor with antinociceptive and antiallodynic activity in acute, persistent, and chronic pain models, with activation of dopaminergic pathways contributing to its antihyperalgesic actions.