Elevated levels of endocannabinoids and CB1 receptor-mediated G-protein signaling in the prefrontal cortex of alcoholic suicide victims.

BACKGROUND: Alcoholism is often comorbid with mood disorders and suicide. We recently reported an upregulation of CB(1) receptor-mediated signaling in the dorsolateral prefrontal cortex (DLPFC) of subjects with major depression who died by suicide. In the present study, we sought to determine whether the changes in depressed suicides would also be present in alcoholic suicides and whether the endocannabinoid (EC) system plays a role in suicide in alcoholism. METHODS: The density of CB(1) receptor and its mediated [(35)S]GTP gamma S signaling were measured in the DLPFC of alcoholic suicides (AS) (n = 11) and chronic alcoholics (CA) (n = 11). The levels of ECs were measured by a liquid chromatograph/mass spectrometry. RESULTS: The CB(1) receptor density was higher in AS compared with the CA group in the DLPFC. Western blot analysis confirmed a greater immunoreactivity of the CB(1) receptor in AS. The CB(1) receptor-mediated [(35)S]GTP gamma S binding indicated a greater signaling in AS. Higher levels of N-arachidonyl ethanolamide and 2-arachidonylglycerol were observed in the DLPFC of AS. CONCLUSIONS: The elevated levels of ECs, CB(1) receptors, and CB(1) receptor-mediated [(35)S]GTP gamma S binding strongly suggest a hyperactivity of endocannabinoidergic signaling in AS. EC system may be a novel therapeutic target for the treatment of suicidal behavior.     

mu-opioid receptor and alpha2-adrenoceptor agonist stimulation of [35S]GTPgammaS binding to G-proteins in postmortem brains of opioid addicts

Repeated opioid administration has been associated in human brain with unaltered density of mu-opioid receptors (agonist radioligand binding sites and immunodetected receptor protein). These receptors are coupled to Gi/Go-proteins, which are increased in brain of heroin addicts. To assess the activity of G-proteins and their coupling to receptors after chronic opioid abuse, [35S]GTPgammaS binding was quantified in postmortem prefrontal cortices of 15 opioid-dependent subjects and 15 matched controls. The stimulation of [35S]GTPgammaS binding by the mu-opioid receptor agonist DAMGO or the alpha2-adrenoceptor agonist UK14304 was used as a functional measure of the status of the receptor-G-protein coupling. [35S]GTPgammaS binding basal values were similar in opioid addicts (819+/-83 fmol mg-1 of protein) and controls (918+/-106 fmol mg(-1) of protein). In opioid addicts, [35S]GTPgammaS binding stimulation by DAMGO showed a maximal effect (62+/-8%) and a potency (EC50 = 1.09+/-0.26 microM) that did not differ from the maximal effect (60+/-12%) and potency (EC50 = 2.01+/-0.58 microM) in controls. In opioid addicts, [35S]GTPgammaS binding stimulation by UK14304 was not different in maximal effect (28+/-3%) from controls (32+/-8%), but the potency of the agonist was decreased (EC50 = 4.36+/-1.81 microM) when compared with controls (EC50 = 0.41+/-0.15 microM). The results provide a direct evidence of an apparent normal functional activity of brain mu-opioid receptors (Gi/Go-protein coupling) during the opioid dependence process in humans. The data also demonstrate a functional uncoupling of alpha2-adrenoceptors from G-proteins, which indicates a heterologous desensitization of these receptors. This finding could represent an adaptive mechanism against the decreased noradrenergic activity induced by the chronic presence of opioid drugs.     

Neurotransmitter receptor-mediated activation of G-proteins in brains of suicide victims with mood disorders: selective supersensitivity of alpha(2A)-adrenoceptors

Abnormalities in the density of neuroreceptors that regulate norepinephrine and serotonin release have been repeatedly reported in brains of suicide victims with mood disorders. Recently, the modulation of the [(35)S]GTPgammaS binding to G-proteins has been introduced as a suitable measure of receptor activity in postmortem human brain. The present study sought to evaluate the function of several G-protein coupled receptors in postmortem brain of suicide victims with mood disorders. Concentration-response curves of the [(35)S]GTPgammaS binding stimulation by selective agonists of alpha(2)-adrenoceptors, 5-HT(1A) serotonin, mu-opioid, GABA(B), and cholinergic muscarinic receptors were performed in frontal cortical membranes from 28 suicide victims with major depression or bipolar disorder and 28 subjects who were matched for gender, age and postmortem delay. The receptor-independent [(35)S]GTPgammaS binding stimulation by mastoparan and the G-protein density were also examined. The alpha(2A)-adrenoceptor-mediated stimulation of [(35)S]GTPgammaS binding with the agonist UK14304 displayed a 4.6-fold greater sensitivity in suicide victims than in controls, without changes in the maximal stimulation. No significant differences were found in parameters of 5-HT(1A) serotonin receptor and other receptor-mediated [(35)S]GTPgammaS binding stimulations. The receptor-independent activation of G-proteins was similar in both groups. Immunoreactive densities of G(alphai1/2)-, G(alphai3)-, G(alphao)-, and G(alphas)-proteins did not differ between suicide victims and controls. In conclusion, alpha(2A)-adrenoceptor sensitivity is increased in the frontal cortex of suicide victims with mood disorders. This receptor supersensitivity is not related to an increased amount or enhanced intrinsic activity of G-proteins. The new finding provides functional support to the involvement of alpha(2)-adrenoceptors in the pathogenesis of mood disorders.     

Subchronic haloperidol increases CB(1) receptor binding and G protein coupling in discrete regions of the basal ganglia

The present study was designed to test whether chronic neuroleptic treatment, which is known to alter both expression and density of dopamine D(2) receptors in striatal regions, has effects upon function and binding level of the cannabinoid CB(1) receptor in the basal ganglia by using receptor autoradiography. As predicted, subchronic haloperidol treatment resulted in increased binding of (3)H-raclopride and quinpirole-induced guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) in the striatum when compared to that measured in control animals. This increased D(2) receptor binding and function after 3 days washout was normalized after a 2-week washout period. Effect of haloperidol treatment was studied for CB(1) receptor binding and CP55,940-stimulated [(35)S]GTPgammaS in the striatum, globus pallidus, and substantia nigra. (3)[H]CP55,940 binding levels were found in rank order from highest to lowest in substantia nigra > globus pallidus > striatum. Furthermore, subchronic haloperidol treatment resulted in elevated binding levels of (3)[H]CP55,940 in the striatum and the substantia nigra and CB(1) receptor-stimulated [(35)S]GTPgammaS bindings in the substantia nigra after 3 days washout. These increased binding levels were normalized at 1-4 weeks after termination of haloperidol treatment. Haloperidol treatment had no significant effect on CB(1) receptor or [(35)S]GTPgammaS binding levels in globus pallidus. The results help to elucidate the underlying biochemical mechanism of CB(1) receptor supersensitivity after haloperidol treatment.     

Binding of a tritiated inverse agonist to cannabinoid CB(1) receptors is increased in patients with schizophrenia

This study sought to determine whether cannabinoid-1 (CB(1)) receptor binding was altered in the postmortem dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia (schizophrenia; n=47) compared to controls (n=43). The CB(1) receptor inverse agonist radioligand [(3)H]MePPEP was used to measure specific binding to CB(1) receptors. The specific binding of [(3)H]MePPEP to CB(1) receptors was 20% higher in patients with schizophrenia than in controls. Power analyses suggested that 53 subjects per group would be needed to detect a similar difference in vivo with positron emission tomography (PET) and the structurally related inverse agonist radioligand [(18)F]FMPEP-d(2) (80% statistical power, p<0.05).     

Cannabinoid receptor agonist-stimulated [35S]guanosine triphosphate gammaS binding in the brain of C57BL/6 and DBA/2 mice

The two inbred strains of mice C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-avoiding) mice have been shown to differ significantly in their preference for alcohol (EtOH). We have previously demonstrated the differences in the density and the affinity of cannabinoid (CB1) receptors in the brains of the two inbred C57BL/6 and DBA/2 mouse strains. In the present study, we investigated the CB1 receptor agonist-stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding in plasma membranes (PM) from C57BL/6 and DBA/2 mice. The results indicate that the net CP55,940-stimulated [(35)S]GTPgammaS binding was increased with increasing concentrations of CB1 receptor agonists and GDP. The net CB1 receptor agonist (WIN55,212-2 or HU-210 or CP55,940)-stimulated [(35)S]GTPgammaS binding was reduced significantly (-10% to -12%, P < 0.05) in PM from DBA/2 mice; no significant differences were observed in basal [(35)S]GTPgammaS binding among these strains. Nonlinear regression analysis of net CP55,940-stimulated [(35)S]GTPgammaS binding showed that the B(max) of cannabinoid agonist-stimulated binding was significantly reduced (-24%) in DBA/2 mice (B(max) = 12.43 +/- 0.64 for C57BL/6 and 9.46 +/- 0.98 pmol/mg protein for DBA/2; P < 0.05) without any significant changes in the G protein affinity. The pharmacological specificity of CP55,940-stimulated [(35)S]GTPgammaS binding was examined with CB1 receptor antagonist SR141716A, and these studies indicated that CP55,940-stimulated [(35)S]GTPgammaS binding was blocked by SR141716A, with a decrease in the IC(50) values in the PM from the DBA/2 mouse strain. These results suggest that a signal transduction pathway(s) downstream from the CB1 receptor system may play an important role in controlling the voluntary EtOH consumption by these strains of mice.     

Cocaine alters mu but not delta or kappa opioid receptor-stimulated in situ [35S]GTPgammaS binding in rat brain

Chronic cocaine administration produces alterations in mu and kappa opioid receptor density as well as striatal and accumbens opioid-regulated adenylyl cyclase activity, suggesting a psychostimulant responsive interaction between opioidergic and dopaminergic systems. Stimulation of G-protein-coupled opioid receptors inhibits adenylyl cyclase production of cyclic AMP. The present study employed in situ [(35)S]GTPgammaS binding to measure opioid receptor-stimulated activation of G-proteins in response to acute and chronic cocaine exposure. Male Fischer rats received acute (1 or 3 days) or chronic (14 days) binge pattern cocaine administration. Three and 14 days of cocaine injections resulted in greater increases in the ability of the mu receptor agonist DAMGO to stimulate [(35)S]GTPgammaS binding in both the core and the shell of the nucleus accumbens, all regions of the caudate putamen and the cingulate cortex compared with saline-matched controls. The greatest increases in DAMGO-stimulated [(35)S]GTPgammaS binding were observed in the dorsal areas of the caudate putamen in animals that received 14 days of cocaine. No significant changes in delta (DPDPE), or kappa (dynorphin A(1-17)) receptor-stimulated [(35)S]GTPgammaS binding were found in any brain region in response to cocaine administration. These results demonstrate that binge pattern cocaine administration induce changes in mu but not delta or kappa opioid receptor-mediated G-protein activity. This study provides support for the hypothesis that the addictive properties of both psychostimulants and opiates may share common neurochemical signaling substrates.     

Serotonin-1A autoreceptor binding in the dorsal raphe nucleus of depressed suicides

Serotonergic dysfunction is present in mood disorders and suicide. Brainstem 5-HT1A somatodendritic autoreceptors regulate serotonin neuron firing but studies of autoreceptor binding in the dorsal raphe nucleus (DRN) in depressed suicides report conflicting results. We sought to determine: (1) the anatomical distribution of 5-HT1A receptor binding in the DRN in depressed suicides and psychiatrically normal controls; and (2) whether sex differences in 5-HT1A binding in the DRN contribute to differences between depressed suicides and controls. Previously collected quantitative receptor autoradiograms of [3H]8-hydroxy-2-(di-n-propyl)aminotetralin (3H-8-OH-DPAT) in postmortem tissue sections containing the DRN from drug-free suicide victims (n=10) and matched controls (n=10) were analyzed. Less total receptor binding (fmol/mg tissuexmm3) was observed in the entire DRN in depressed suicides compared with controls (p<0.05). Group differences along the rostrocaudal extent of the DRN were observed for cross-sectional 5-HT(1A) binding (fmol/mg tissue) and receptor binding (fmol/mgxmm3, p<0.05). Cross-sectional 5-HT1A DRN binding in depressed suicides compared with controls was higher rostrally and lower caudally. The differences between depressed suicides and controls were present in males and females, although females had more binding than males. Less autoreceptor binding in the DRN of depressed suicides may represent a homeostatic response to less serotonin release, increasing serotonin neuron firing. More autoreceptor binding in rostral DRN might contribute to deficient serotonin release in ventromedial prefrontal cortex by lower neuronal firing.     

Selective heterologous regulation of 5-HT1A receptor-stimulated 35S GTPgammaS binding in the anterior cingulate cortex as a result of 5-HT2 receptor activation

Previous studies have shown that administration of the 5-HT(2) receptor agonist DOI to rats results in the heterologous desensitization of 5-HT(1A) receptor-mediated behavioral and neuroendocrine responses [Neuropsychopharmacology 19 (1998) 354; J. Neurosci. 21 (2001) 7919]. We hypothesized that the basis for these changes in 5-HT(1A) receptor function may involve changes in the capacity of the 5-HT(1A) receptor to activate G proteins. We examined the effect of chronic administration of DOI on the regulation of 5-HT(1A) receptor function at the level of receptor-G protein interaction using quantitative autoradiography of [(35)S]GTPgammaS binding stimulated by the 5-HT(1A) receptor agonist (+/-)8-OH-DPAT (1 microM). Repeated administration of DOI (1 mg/kg, s.c. once daily for 8 days) resulted in a marked down-regulation in 5-HT(2A) binding sites, as labeled by the antagonist radioligand [(3)H]ketanserin, throughout the cerebral cortex. Chronic DOI treatment also resulted in a significant and selective attenuation of 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in the anterior cingulate cortex (vehicle-treated: 74+/-7.7% above basal; DOI-treated: 43+/-4.6% above basal). Interestingly, 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding was not altered in the dorsal or median raphe, or in the limbic structures and other cortical regions examined. The decrease in 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in anterior cingulate cortex was not due to a decrease in 5-HT(1A) receptor number, indicating that the capacity of the 5-HT(1A) receptor to activate G proteins is attenuated in this cortical area following repeated DOI treatment. The heterologous regulation of 5-HT(1A) receptor function by chronic 5-HT(2) receptor activation in the anterior cingulate cortex raises interesting questions as to how the regulatory interaction between these serotonin receptor subtypes influences cognition, memory and emotion.     

Autoradiographic analysis of [3H]ketanserin binding in the human brain postmortem: effect of suicide

In vitro quantitative autoradiography of 5-HT2 receptors, using [3H]ketanserin as a ligand, was performed on 24 human brains postmortem. Twelve brains were donated by suicide victims and 12 by matched controls. We found a characteristic decline in 5-HT2 receptors with age in several brain regions of the control group. This age dependence of ketanserin binding was not present in some of these brain regions of the suicide group. We also found a significant but anatomically selective reduction in the density of ketanserin binding sites in the young suicide group, compared to age-matched controls. This reduction was evident in portions of the prefrontal cortex. Homogenate binding assays on prefrontal cortex samples from a large group of suicides (n = 20) and controls (n = 23) showed that the difference in age dependence of ketanserin binding and the reduced binding in the young suicide group were explained by differences in Bmax values. No differences were observed in Kd. Sex, presence of alcohol and time from death to autopsy did not affect ketanserin binding, in our sample, as measured by either autoradiography or homogenate binding assay.     

In vitro autoradiography of serotonin 5-HT(2A/2C) receptor-activated G protein: guanosine-5'-(gamma-[(35)S]thio)triphosphate binding in rat brain

Agonist activation of G protein-coupled receptors induces an increase in the binding of guanosine 5'-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS); this increase in binding has been used as a tool to investigate receptor interaction with the heterotrimer guanine nucleotide-binding regulatory protein (G protein). The present study uses agonist-stimulated [(35)S]GTPgammaS binding to characterize serotonin 5-HT(2A/2C) receptors in rat brain membrane fractions and demonstrate the anatomical localization of the receptors by in vitro autoradiography on slide-mounted sections. The stimulatory effect of the agonist [1-(2,5-dimethoxy-4-iodophenyl)]-2 aminopropane (DOI) is compared to that of serotonin (5-HT). Autoradiography revealed a similar localization of DOI- and 5-HT-stimulated binding of [(35)S]GTPgammaS in distinct areas of prefrontal and parietal cortex, consistent with previously reported 5-HT(2A) receptor distribution. Specific binding was demonstrated in the frontal and parietal cortex, medial prefrontal, and cingular and orbital-insular areas as well as in the hippocampal formation, septal areas, the nucleus accumbens, and the choroid plexus. MDL 100105, a specific 5-HT(2A) antagonist, and ketanserin, an antagonist of 5-HT(2A/2C) receptors, blocked DOI stimulation in all labeled areas, whereas 5-HT stimulation was only partially blocked (70-80%). A small but significant inhibition was observed with the specific antagonist of 5-HT(2C/2B), SB 206553. This autoradiographic technique provides a useful tool for measuring in situ changes in specific receptor-Gq protein coupling in anatomically discrete brain regions, under physiological and pathological conditions.     

Autoradiographic analysis of dopamine receptor-stimulated [(35)S]GTPgammaS binding in rat striatum

Autoradiographic analysis of [(35)S]GTPgammaS binding was used to investigate functional activation of dopamine receptors in rat striatum. Dopamine-stimulated [(35)S]GTPgammaS binding was observed with a maximal increase of 38% over basal activity. A similar stimulatory response was obtained with the D(2) agonist quinpirole, but not SKF-238393, a D(1) agonist. The effect of dopamine was blocked by the D(2) antagonist raclopride, but was unaffected by SCH-23990, a D(1) antagonist. There appeared to be a differential distribution of dopamine-stimulated [(35)S]GTPgammaS binding, with the lowest activity obtained in the medial portion of the caudal striatum. These results demonstrate, using an autoradiographic approach, (i) that dopamine stimulated [(35)S]GTPgammaS binding in the rat striatum occurs through activation of D(2) receptors, and (ii) that the effects of dopamine activation vary in different areas of the rat striatum.     

Cannabinoid system in the budgerigar brain

Cannabinoid receptor density and cannabinoid receptor-mediated G protein stimulation were studied by autoradiographic techniques throughout the budgerigar (Melopsittacus undulatus) brain. The maximal CB(1) receptor density value (using [(3)H]CP55,940 as radioligand) was found in the molecular layer of the cerebellum (Mol), and high binding values were observed in the nucleus taeniae amygdalae (TnA), nucleus preopticus medialis, and nucleus pretectalis. The highest net-stimulated [(35)S]GTPgammaS binding values induced by the selective CB(1) receptor agonist WIN55,212-2 were observed in the nucleus paramedianus internus thalami, and high values of [(35)S]GTPgammaS binding were observed in the TnA, Mol, arcopallium dorsale and arcopallium intermedium. The distribution data suggest that in the budgerigar, as previously indicated in mammals, cannabinoid receptors may be related to the control of several brain functions in the motor system, memory, visual system, and reproductive behavior. The discrepancies between the cannabinoid receptor densities and the cannabinoid receptor-mediated stimulation found in several budgerigar brain nuclei support the hypothesis, previously described for mammals, of the existence of different G(i/o) protein populations able to associate with the cannabinoid receptors, depending on the brain structure, and could reflect the relative importance that cannabinoid transmission could exerts in each cerebral area.     

Dopamine D1 and D2 receptor binding sites in brain samples from depressed suicides and controls

Dopamine D₁ and D₂ receptors were measured (by saturation binding of [³H]SCH23390 and [³H]raclopride) in caudate, putamen and nucleus accumbens, obtained at post-mortem from suicide victims with a firm retrospective diagnosis of depression and matched controls. There were no differences in the number or affinity of D₁ or D₂ receptors between suicides who had been free of antidepressants for at least three months prior to death, and controls. Increased numbers and decreased affinity of D₂ receptors were however found in each brain region of antidepressant-treated suicides. We argue that these increases are related to concurrent treatment with neuroleptics rather than a direct effect of antidepressants. Increased numbers of D₁ receptors in antidepressant-treated suicides were seen only in nucleus accumbens. This increase could not be clearly attributed to neuroleptics and may be related to antidepressant treatment.     

Brain 5-HT uptake sites, labelled with [3H]paroxetine, in antidepressant-free depressed suicides

Brain serotonin (5-HT) uptake sites were quantitated, by saturation binding of [3H]paroxetine, in 10 brain regions from 22 suicide victims and 20 control subjects. Suicide victims were restricted to those subjects in whom a firm retrospective diagnosis of depression was established and who had not recently been prescribed antidepressant drugs. The Kd and Bmax of [3H]paroxetine did not differ significantly between controls and depressed suicides in any of the brain regions. In putamen, Bmax values of suicides who died non-violently were lower than controls, whereas those who died by violent methods did not differ from controls. No significant differences between violent or non-violent suicides and their matched controls were found in other brain areas. These results offer little support for the view that suicide/depression is associated with an abnormality in 5-HT uptake.     

Functional coupling of the nociceptin/orphanin FQ receptor in dog brain membranes

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg(-1) protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [leucyl-(3)H]N/OFQ(1-17)OH ([leucyl-(3)H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 microM GTPgammaS. Data from standard [(35)S]GTPgammaS binding and immunoprecipitation (G(alphai1-3)) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO(hNOP)) cells. N/OFQ displaced [leucyl-(3)H]N/OFQ binding with pK(i) and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPgammaS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [(35)S]GTPgammaS binding with pEC(50) and E(max) values of 8.21+/-0.17 and 1.17+/-0.01, respectively (in CHO(hNOP), pEC(50) and E(max) values were 8.47+/-0.01 and 7.01+/-0.63). N/OFQ stimulated [(35)S]GTPgammaS binding in dog and CHO(hNOP) cell membranes could be immunoprecipitated with an anti-G(alphai1-3) antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 microM [Nphe(1)]N/OFQ(1-13)NH(2) and 1 microM [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) (partial agonist) yielding pK(B) values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO(hNOP), a pK(B) for J-113397 of 8.33+/-0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.     

Dopamine D2 receptor-mediated G protein activation assessed by agonist-stimulated [35S]guanosine 5'-O-(gamma-thiotriphosphate) binding in rat striatal membranes

In order to investigate the functional interaction between the native dopamine receptors and their coupled guanine nucleotide-binding regulatory (G) proteins, dopamine-stimulated [(35)S]guanosine 5'-O-(gamma-thiotriphosphate) ([(35)S]GTPgammaS) binding was pharmacologically characterized in rat striatal membranes. Following optimizing the experimental conditions as to the concentrations of GDP, MgCl(2) and NaCl in the assay medium, the agonist and antagonist properties for a series of dopamine receptor ligands were determined mainly under the standard assay condition. The pharmacological profile of this response clearly indicated the involvement of dopamine D(2)-like receptors, but not of dopamine D(1)-like receptors. Among the types of dopamine D(2)-like receptors, dopamine D(2) receptors most likely appeared to be involved in dopamine-stimulated [(35)S]GTPgammaS binding in rat striatal membranes, because the affinities of agonists and antagonists determined in the present study were significantly correlated with those reported in the previous literature only for dopamine D(2) receptors, but not for dopamine D(3) or D(4) types. Though the concentration-dependent inhibition curves of dopamine-stimulated [(35)S]GTPgammaS binding by spiperone and S(-)-raclopride were apparently biphasic, the origin of the low-affinity minor components was not fully determined. The antiparkinsonian drugs with the properties of dopamine receptor agonism were shown to behave as stimulants with varied affinities and relative efficacies in the current assay system. On the other hand, neither phencyclidine (PCP) nor ketamine stimulated the specific [(35)S]GTPgammaS binding, in contrast with the previous report demonstrating that these two N-methyl-D-aspartic acid (NMDA) receptor antagonists behaved as agonists at human dopamine D(2) receptors expressed in Chinese hamster ovary (CHO) cells. These results provide important information about the functional activation of G proteins coupled with dopamine D(2) receptors as well as agonist actions of various compounds at native dopamine D(2) receptors, which are potentially involved in pathophysiology and pharmacotherapy of neuropsychiatric diseases such as Parkinson's disease, schizophrenia and depression.     

Cholesterol-dependent modulation of type 1 cannabinoid receptors in nerve cells

Type 1 cannabinoid receptors (CB1R) are G-protein-coupled receptors that mediate several actions of the endocannabinoid anandamide (N-arachidonoylethanolamine; AEA) in the central nervous system. Here we show that cholesterol enrichment of rat C6 glioma cell membranes reduces by approximately twofold the binding efficiency (i.e., the ratio between maximum binding and dissociation constant) of CB1R and that activation of CB1R by AEA leads to approximately twofold lower [(35)S]GTPgammaS binding in cholesterol-treated cells than in controls. In addition, we show that CB1R-dependent signaling via adenylate cyclase and p42/p44 mitogen-activated protein kinase is almost halved by cholesterol enrichment. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by cholesterol, whereas the catalytic efficiency (i.e., the ratio between maximal velocity and Michaelis-Menten constant) of the AEA membrane transporter AMT is almost doubled compared with control cells. These data demonstrate that, among the proteins of the "endocannabinoid system," only CB1R and AMT critically depend on membrane cholesterol content. This observation may have important implications for the role of CB1R in protecting nerve cells against (endo)cannabinoid-induced apoptosis.     

Reduced tyrosine hydroxylase immunoreactivity in locus coeruleus of suicide victims.

Antibodies against tyrosine hydroxylase (TH, the rate-limiting enzyme in norepinephrine synthesis) and dopamine beta-hydroxylase (DBH, the last enzyme in the synthesis) were used for immunohistochemical staining of human brain locus coeruleus sections, obtained postmortem from suicide victims and matched controls. Stain density over individual cells was quantified by a computerized, video-camera-based image analysis system. Mean stain density for TH was significantly lower (by about 30%) in the locus coeruleus of suicide victims. There was no difference between suicides and controls in DBH immunoreactivity or in the number of TH immunoreactive cells. Reduced TH availability, either genetically or environmentally determined, may contribute to the noradrenergic insufficiency postulated to occur in depression and the increased beta-adrenergic receptor concentrations observed in prefrontal cortex of suicide victims.