Regulation of melatonin 1a receptor signaling and trafficking by asparagine-124.

Melatonin is a pineal hormone that regulates seasonal reproduction and has been used to treat circadian rhythm disorders. The melatonin 1a receptor is a seven- transmembrane domain receptor that signals predominately via pertussis toxin-sensitive G-proteins. Point mutations were created at residue N124 in cytoplasmic domain II of the receptor and the mutant receptors were expressed in a neurohormonal cell line. The acidic N124D- and E-substituted receptors had high-affinity (125)I-melatonin binding and a subcellular localization similar to the neutral N124N wild-type receptor. Melatonin efficacy for the inhibition of cAMP by N124D and E mutations was significantly decreased. N124D and E mutations strongly compromised melatonin efficacy and potency for inhibition of K(+)-induced intracellular Ca(++) fluxes and eliminated control of spontaneous calcium fluxes. However, these substitutions did not appear to affect activation of Kir3 potassium channels. The hydrophobic N124L and N124A or basic N124K mutations failed to bind (125)I-melatonin and appeared to aggregate or traffic improperly. N124A and N124K receptors were retained in the Golgi. Therefore, mutants at N124 separated into two sets: the first bound (125)I-melatonin with high affinity and trafficked normally, but with reduced inhibitory coupling to adenylyl cyclase and Ca(++) channels. The second set lacked melatonin binding and exhibited severe trafficking defects. In summary, asparagine-124 controls melatonin receptor function as evidenced by changes in melatonin binding, control of cAMP levels, and regulation of ion channel activity. Asparagine-124 also has a unique structural effect controlling receptor distribution within the cell.

Comparative molecular field analysis-based prediction of drug affinities at recombinant D1A dopamine receptors.

Determination of quantitative structure-activity relationship (QSAR) for affinity at particular dopamine (DA) receptors has become an even greater priority with the cloning of five DA receptor subtypes. The use of agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in QSAR presents a unique opportunity for accuracy and precision in measurement of biological values. Bound conformations of 11 agonists (for which both affinity data at the recombinant D1A DA receptor and stereochemical configurations were available) were determined by alignment with a template compound, SKF38393, which shows high affinity and selectivity for D1A receptors and is fairly rigid in structure. These aligned structures suggested a 3-point pharmacophore map (one cationic nitrogen and two electronegative centers) of the D1A DA receptor. This map shows both similarities and differences when compared with a previously reported D2 DA receptor pharmacophore map based on biological data from rat brain and with a recently published map of the native D1 DA receptor using several semirigid compounds. Log(1/K(d)) values at recombinant D1A DA receptors were used as the target property for a CoMFA (comparative molecular field analysis) of the 11 aligned structures. The resulting CoMFA model yielded a cross-validated r(2)(q(2)) value of 0.829 and a simple r(2) = 0.96. In contrast, when a CoMFA model was developed for 10 of these compounds using striatal D1 K(d) values, the q(2) value was reduced to 0.178. These results are consistent with the idea that drug affinity data obtained from clonal cells expressing recombinant receptors may be superior to that obtained using heterogeneous mixtures of native receptors prepared from brain membranes. The predictive utility of the CoMFA model was evaluated using several high-affinity dopamine agonists and m- and p-tyramine, two compounds with a single hydroxyl group on the aromatic ring. Predictions were fairly accurate for all compounds but the two tyramines.

CoMFA-based prediction of agonist affinities at recombinant D1 vs D2 dopamine receptors.

We have previously shown that using agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in three-dimensional quantitative structure-activity relationship studies (3D-QSAR) presents a unique opportunity for accuracy and precision in measurement. Thus, a comparison of affinity's structural determinants for a set of compounds at two different recombinant dopamine receptors represents an attainable goal for 3D-QSAR. A molecular database of bound conformations of 16 structurally diverse agonists was established by alignment with a high-affinity template compound for the D1 receptor, 3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-1H-benzazepin. A second molecular database of the bound conformations of the same compounds was established against a second template for the D2 receptor, bromocriptine. These aligned structures suggested three-point pharmacophore maps (one cationic nitrogen and two electronegative centers) for the two dopamine receptors, which differed primarily in the height of the nitrogen above the plane of the catechol ring and in the nature of the hydrogen-bonding region. The ln(1/KL) values for the low-affinity agonist binding conformation at recombinant D1 and D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA (comparative molecular field analysis) of the 16 aligned structures. The resulting CoMFA models yielded cross-validated R2 (q2) values (standard error of prediction) of 0. 879 (1.471, with five principal components) and 0.834 (1.652, with five principal components) for D1 and D2 affinity, respectively. The simple R2 values (standard error of the estimate) were 0.994 (0.323) and 0.999 (0.116), respectively, for D1 and D2 receptor. F values were 341 and 2465 for D1 and D2 models, respectively, with 5 and 10 df. The predictive utility of the CoMFA model was evaluated at both receptors using the dopamine agonists, apomorphine and 7-OH-DPAT. Predictions of KL were accurate at both receptors. Flexible 3D searches of several chemical databases (NCI, MDDR, CMC, ACD, and Maybridge) were done using basic pharmacophore models at each receptor to determine the similarity of hit lists between the two models. The D1 and D2 models yielded different lists of lead compounds. Several of the lead compounds closely resembled high-affinity training set compounds. Finally, homology modeling of agonist binding to the D2 receptor revealed some consistencies and inconsistencies with the CoMFA-derived D2 model and provided a possible rationale for features of the D2 CoMFA contour map. Together these results suggest that CoMFA-homology based models may provide useful insights concerning differential agonist-receptor interactions at related receptors. The results also suggest that comparisons of CoMFA models for two structurally related receptors may be a fruitful approach for differential QSAR.

CoMFA-based prediction of agonist affinities at recombinant wild type versus serine to alanine point mutated D2 dopamine receptors.

Agonist affinity changes dramatically as a result of serine to alanine mutations (S193A, S194A, and S197A) within the fifth transmembrane region of D2 dopamine receptors and other receptors for monoamine neurotransmitters. However, agonist 2D-structure does not predict which drugs will be sensitive to which point mutations. Modeling drug-receptor interactions at the 3D level offers considerably more promise in this regard. In particular, a comparison of the same test set of agonists across receptors differing minimally (point mutations) offers promise to enhance the understanding of the structural bases for drug-receptor interactions. We have previously shown that comparative molecular field analysis (CoMFA) can be applied to comparisons of affinity at recombinant D1 and D2 dopamine receptors for the same set of agonists, a differential QSAR. Here, we predicted agonist K(L) for the same set of agonists at wild type D2 vs S193A, S194A, and S197A receptors using CoMFA. Each model used bromocriptine as the template. ln(1/K(L)) values for the low-affinity agonist binding conformation at recombinant wild type and mutant D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA of the 16 aligned agonist structures. The resulting CoMFA models yielded cross-validated R(2) (q(2)) values ranging from 0.835 to 0.864 and simple R(2) values ranging from 0.999 to 1.000. Predictions of test compound affinities at WT and each mutant receptor were close to measured affinity values. This finding confirmed the predictive ability of the models and their differences from one another. The results strongly support the idea that CoMFA models of the same training set of compounds applied to WT vs mutant receptors can accurately predict differences in drug affinity at each. Furthermore, in a "proof of principle", two different templates were used to derive the CoMFA model for the WT and S193A mutant receptors. Pergolide was chosen as an alternate template because it showed a significant increase in affinity as a result of the S193A mutation. In this instance both the bromocriptine- and pergolide-based CoMFA models were similar to one another but different from those for the WT receptor using bromocriptine- or pergolide- as templates. The pergolide-based S193A model was more strikingly different from that of the WT receptor than was the bromocriptine-based S193A model. This suggests that a "dual-template" approach to differential CoMFA may have special value in elucidating key differences across related receptor types and in determining important elements of the drug-receptor interaction.

Effects of chronic administration of agonists and antagonists on the density of beta-adrenergic receptors.

It is frequently hypothesized that drug-induced alterations in the density of beta-adrenergic receptors underlie tolerance to and physical dependence on agonists and antagonists at beta-adrenergic receptors. Two approaches to determining the effect of treatment with drugs on the density of beta-adrenergic receptors are described. In the first, the density of beta-adrenergic receptors was measured on leukocytes taken from human subjects during and after drug treatment. Treatment with the antagonist propranolol caused an increase in the density of beta-adrenergic receptors on leukocytes, whereas treatment with the agonists terbutaline and ephedrine, or pindolol, an antagonist with intrinsic sympathomimetic activity, caused a decrease in the density of beta-adrenergic receptors. In the second approach, the effect of agonists on the density of beta-adrenergic receptors on C6 glioma cells in culture was determined. Incubation with the full agonist isoproterenol decreased the density of both beta 1- and beta 2-adrenergic receptors. In contrast, incubation with pindolol or celiprolol, also an antagonist with intrinsic sympathomimetic activity, selectively decreased the density of beta 2-adrenergic receptors. Pindolol and celiprolol may be useful in situations in which selective stimulation of beta 2-adrenergic receptors and blockade of beta 1-adrenergic receptors is desirable.

Interaction of beta-adrenergic receptors with the inhibitory guanine nucleotide-binding protein of adenylate cyclase in membranes prepared from cyc- S49 lymphoma cells.

beta-Adrenergic receptors on membranes prepared from L6 myoblasts, wild-type S49 lymphoma cells, and an adenylate cyclase-deficient variant (cyc-) of S49 lymphoma cells bind the agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) with high affinity. In each case the agonist [3H]HBI is associated with a larger complex than is the antagonist [125I]iodopindolol, and the binding of [3H]HBI can be inhibited by GTP. These observations suggest that there is an agonist-dependent association of the receptor with a guanine nucleotide-binding protein. The goal of the present experiments was to investigate the possibility that an interaction of beta-adrenergic receptors with the inhibitory guanine nucleotide-binding protein of adenylate cyclase was responsible for these observations. Treatment of S49 cells with pertussis toxin decreased the extent of pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41,000-dalton protein, measured in vitro, and decreased the inhibition of adenylate cyclase activity observed in the presence of somatostatin or analogues of GTP. Isoproterenol-stimulated adenylate cyclase activity was potentiated following treatment of wild-type S49 cells and L6 myoblasts with pertussis toxin. Although the ability of receptors on membranes prepared from L6 myoblasts to bind the agonist [3H]HBI was not affected by treatment of cells with pertussis toxin, treatment of cyc- S49 cells with pertussis toxin markedly decreased the ability of receptors to bind [3H]HBI. The observed inhibition of the binding of the agonist [3H]HBI to beta-adrenergic receptors on membranes prepared from cyc- S49 cells after treatment with pertussis toxin could be explained by an interaction between beta-adrenergic receptors and the inhibitory guanine nucleotide-binding protein. Such an interaction may represent a mechanism through which stimulation of the activity of adenylate cyclase by beta-adrenergic receptors can be regulated or through which beta-adrenergic receptors can affect the activity of cyclic AMP-independent cellular processes.

LSD and structural analogs: pharmacological evaluation at D1 dopamine receptors.

The hallucinogenic effects of lysergic acid diethylamide (LSD) have been attributed primarily to actions at serotonin receptors. A number of studies conducted in the 1970s indicated that LSD also has activity at dopamine (DA) receptors. These latter studies are difficult to interpret, however, because they were completed before the recognition of two pharmacologically distinct DA receptor subtypes, D1 and D2. The availability of subtype-selective ligands (e.g., the D1 antagonist SCH23390) and clonal cell lines expressing a homogeneous receptor population now permits an assessment of the contributions of DA receptor subtypes to the DA-mediated effects of LSD. The present study investigated the binding and functional properties of LSD and several lysergamide and analogs at dopamine D1 and D2 receptors. Several of these compounds have been reported previously to bind with high affinity to serotonin 5HT2 (i.e., 3H-ketanserin) sites in the rat frontal cortex (K0.5 5-30 nM). All tested compounds also competed for both D1-like (3H-SCH 23390) and D2-like (3H-spiperone plus unlabeled ketanserin) DA receptors in rat striatum, with profiles indicative of agonists (nH < 1.0). The affinity of LSD and analogs for D2 like receptors was similar to their affinity for 5HT2 sites. The affinity for D1 like receptors was slightly lower (2- to 3-fold), although LSD and several analogs bound to D1 receptors with affinity similar to the prototypical D1 partial agonist SKF38393 (K0.5 ca. 25 nM). A second series of experiments tested the binding and functional properties of LSD and selected analogs in C-6 glioma cells expressing the rhesus macaque D1A receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Short- and long-term heterologous sensitization of adenylate cyclase by D4 dopamine receptors.

The D4 dopamine receptor, a member of the D2-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D4 receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D4.2, D4.4, or D4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D4 receptor variants. D4 dopamine receptor-mediated sensitization was blocked by the D4 antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for Gi/Go proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D4 receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of Gialpha without altering the abundance of Gsalpha, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D4 receptor may involve mechanisms that do not contribute to short-term sensitization.

Computer-assisted video analysis of [3H]spiroperidol binding autoradiographs.

A computer-based video image processing system is described that quantifies the binding of the neuroleptic drug, [3H]spiroperidol, to rat forebrain sections. Adjacent sections were incubated in buffer containing [3H]spiroperidol or [3H]spiroperidol plus 1 microM (+)-butaclamol and exposed to tritium-sensitive film to produce autoradiographs of total binding or non-butaclamol-displaced ('non-specific') binding. The image processing system digitized each autoradiograph, attenuated geometric distortion and unevenness of background illumination, and reassigned the digitized image intensities (gray values) to be a linear function of fmol [3H]spiroperidol bound per mg protein by using a calibration curve generated from 3H-containing tissue standards. An image of butaclamol-displaced ('specific') [3H]spiroperidol binding was produced by subtracting the linearized image of non-specific binding from the superimposed image of total binding. An image of percent specific [3H]spiroperidol binding was obtained by dividing the image of specific binding by the superimposed image of total binding. The computer-derived images, which could be displayed in gray tones or pseudocolor-coded, revealed that the greatest amounts of specific [3H]spiroperidol binding (1000-3000 fmol/mg protein; 60-80% specifically bound) were located in layers 5A and 5C of the neocortex, the claustrum, and in the caudate-putamen, where a lateral-to-medial binding gradient occurred. [3H]Spiroperidol was bound to a lesser extent (400-1000 fmol/mg protein; 31-51% specifically bound) to the medial nucleus accumbens septi or olfactory tubercle and without measurable specificity to the lateral septum, anterior commissure, corpus callosum, or superficial neocortex. These procedures are particularly useful for the quantitative and visual analysis of autoradiographs in which [3H]ligand binding is associated with more than a single site.

Denervation accelerates the reappearance of neostriatal D-2 receptors after irreversible receptor blockade.

The effect of denervation on the turnover of striatal dopaminergic D-2 receptors was examined by determining the rate of receptor reappearance in vivo after administration of the irreversible receptor antagonist, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to rats that received prior unilateral intracerebral injection of 6-hydroxydopamine (6-OHDA). Initial experiments confirmed that EEDQ (10 mg/kg i.p.) induces a severe, prolonged blockade of D-2 receptors. Recovery of [3H]spiroperidol binding occurred at a rate of approximately 9% of control binding per day. 6-OHDA injection into the ascending dopaminergic projection 3 or 5 days prior to EEDQ administration revealed that denervation had no effect on the rate of D-2 receptor recovery during the first post-operative week. By 4-5 weeks postoperatively, however, denervation enhanced the rate of recovery of [3H]spiroperidol binding. These results are consistent with our finding that, when homogenized tissue preparations are used, steady-state receptor density does not change within the first postoperative week but increases by 3-4 weeks after the injury. Saturation analysis determined that both EEDQ and denervation altered the density of binding sites, whereas neither treatment significantly affected the affinity of the receptor for [3H]spiroperidol. By 4-5 weeks postoperatively, the receptor degradation rate constant in the denervated striatum (0.0054/h) was equal to that in the intact striatum (0.0052/h). Thus, only the receptor reappearance rate was elevated in the denervated striatum (3.8 fmol/mg protein/h) relative to the intact striatum (2.8 fmol/mg protein/h).

Recovery of function after mesotelencephalic dopaminergic injury in senescence.

After intracerebral 6-hydroxydopamine injections that extensively damage the ascending mesotelencephalic dopaminergic projection, young adult rats develop severe sensorimotor deficits, including an inability to orient toward somatosensory stimuli. Many rats with such damage recover their localization of touch, and this recovery depends on the survival of a small proportion of the neostriatal dopaminergic terminals. Behavioral recovery appears to be mediated by an increased dopamine synthesis and release within surviving terminals and an increased responsiveness to dopamine of neostriatal neurons. The ability of aged rodents to increase activity at neostriatal dopaminergic synapses following partial injury to these neurons is remarkably intact, despite the reduced basal level of transmission at these synapses in senescence. First, 27-28-month-old rats recovered somatosensory localization after injury as readily as did 4-5-month-old animals that had equivalent neostriatal dopamine depletions. Second, old and young rats developed a similar degree of behavioral supersensitivity to apomorphine (0.25 mg/kg, i.p.) after unilateral 6-hydroxydopamine injections, as measured by contralateral turning. Third, injury to these neurons produced an equivalent increase in [3H]spiroperidol binding in the neostriatum of old and young rats at 5-7 weeks. Old rats suffered more extensive neostriatal dopamine depletions after intracerebral 6-hydroxydopamine injections than did young adult animals, particularly when small quantities (2 micrograms) of the neurotoxin were injected. This enhanced susceptibility to 6-hydroxydopamine of dopaminergic neurons in old rats could not be attributed to age differences in the placement of the injection cannula or the extent of the non-specific damage at the injection site. The implications of this enhanced susceptibility to the neurotoxin are discussed.

Plasticity of neostriatal dopamine receptors after nigrostriatal injury: relationship to recovery of sensorimotor functions and behavioral supersensitivity.

Rats given unilateral injections of 6-OHDA along the course of the mesotelencephalic dopaminergic projection show impairments in contralateral sensorimotor functions from which they often recover. Such rats also display an enhanced sensitivity to DA receptor stimulants, e.g. apomorphine, as revealed by contralateral turning, and an increased binding of neuroleptic compounds (e.g. [3H]spiroperidol) to the denervated striatum. This research examines the relationship of these receptor changes to both behavioral supersensitivity and recovery of sensorimotor functions by quantifying the time course of each phenomenon after injury. The supersensitivity to apomorphine and the behavioral recovery developed with a similar time course after injury, being evident within 1.5-3 days and reaching nearly maximal levels by 2 weeks postoperatively. A significant increase in in vivo [3H]spiroperidol binding to the denervated striatum occurred by 4 days postoperatively, and the magnitude of this change increased linearly during the first postoperative month. In contrast, the in vitro binding of this ligand to membranes of the denervated striatum was not increased until 3 weeks after the lesion. The results suggest that a proliferation of DA receptors may contribute to the pharmacological supersensitivity and the recovery of function, and that these early receptor changes may be revealed with greater sensitivity using in vivo binding techniques.

The crossed mesostriatal projection: neurochemistry and developmental response to lesion.

The projections of substantia nigra and ventral tegmental area (SN-VTA) neurons to either the ipsilateral or contralateral caudate-putamen (CP) were studied in intact and 6-hydroxydopamine-treated rats, using both retrograde transport of fluorescent dyes and measurements of dopamine concentration. After unilateral CP injections of nuclear yellow or granular blue in intact 6- or 30-day-old rats, approximately 1% of the retrogradely labeled cells were located in the contralateral SN-VTA. In 6- or 30-day-old rats which had been injected with 6-hydroxydopamine in the left lateral hypothalamic area (LHA) at 1 day of age, the extent of cell labeling of both the uncrossed projection in the right hemisphere and the crossed projection originating in the left SN-VTA was normal. However, the number of labeled cells of the uncrossed pathway of the left side and the crossed pathway originating in the right SN-VTA were markedly decreased by the lesion, indicating that the crossed nigrostriatal cells decussate before they reach the LHA. The 6-hydroxydopamine-induced decreases in SN-VTA labeling of both the crossed and uncrossed pathway closely matched the degree of dopamine depletion in the lesioned CP at 6 days of age and at 30 days of age. A partial recovery of both measures occurred between days 6 and 30. Thus, retrograde labeling of SN-VTA cells and at 30 days of age. A partial recovery of both measures occurred between days 6 and 30. Thus, retrograde labeling of SN-VTA cells with fluorescent dyes provided a quantitative measure of the dopaminergic innervation of the neostriatum in both the uncrossed and crossed pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic lithium administration alters behavioral recovery from nigrostriatal injury: effects on neostriatal [3H]spiroperidol binding sites.

Unilateral damage to the mesotelencephalic dopaminergic projection of the rat produces impairments in sensorimotor functions, including an inability to localize contralateral somatosensory stimuli. Many rats with 6 hydroxydopamine injections along this pathway show a gradual improvement in somatosensory localization during the first post-operative month. One mechanism that may contribute to this behavioral recovery is the proliferation of dopamine receptor sites in the affected neostriatum. The role of these binding site changes in the recovery was tested by chronic administration of lithium to rats. Rats given lithium in their drinking water for 4 weeks after the 6-hydroxydopamine injection showed a greatly attenuated recovery of sensorimotor functions, compared to brain-damaged rats drinking unadulterated water. When rats that were given lithium for the first four postoperative weeks were subsequently given unadulterated water to drink, they recovered normally. Lithium treatment did not prevent the augmentation of [3H]spiroperidol binding in the neostriatum ipsilateral to the lesion, relative to the contralateral control neostriatum, at 4 or 8 weeks post-operatively. Thus, lithium treatment dissociates behavioral recovery after nigrostriatal injury from the lesion-induced relative proliferation of neostriatal [3H]spiroperidol binding sites. However, the lithium treatment decreased the absolute amount of specific [3H]spiroperidol binding to both the ipsilateral and contralateral neostriata. The significance of these findings for the neural mechanism underlying this recovery sequence is discussed.

Quantitative analysis of [3H]spiroperidol binding to rat forebrain sections: plasticity of neostriatal dopamine receptors after nigrostriatal injury.

The binding of [3H]spiroperidol to rat coronal sections in vitro was investigated using two procedures: swabbing studies, in which the tissue sections are wiped from the microscope slides after incubation in the presence of [3H]spiroperidol, and autoradiographic studies, in which the autoradiographic negatives are analyzed using computer-assisted densitometry. In the swabbing studies, the pharmacological and kinetic properties of butaclamol-displaceable binding were investigated, and the following results suggest that [3H]spiroperidol binds specifically to only a single site within the basal forebrain of tissue sections and that the site is the dopamine D-2 receptor. The pseudo-first order and first order plots for the rate of association to and dissociation from tissue sections appeared to be linear. Dopamine antagonists, such as haloperidol and butaclamol, were much more effective than dopamine agonists or the serotonin S-2 ligand, ketanserin, in inhibiting [3H]spiroperidol binding. The ability of dopamine agonists to inhibit [3H]spiroperidol binding was markedly reduced by the guanine nucleotide, Gpp(NH)p. Saturation analysis of specific [3H]spiroperidol binding revealed a Kd and Bmax of 0.93 nM and 447 fmol/mg protein, and a Hill coefficient of 1.05. The findings are also compatible with the possibility that [3H]spiroperidol binds to several sites that have identical affinities for this ligand. Densitometric studies were used to assess the effect of lesions on [3H]spiroperidol binding in the neostriatum. Intrastriatal injection of kainic acid substantially reduced 1 microM (+)--butaclamol-displaceable binding, indicating that the receptors are in large part on intrinsic striatal neurons. Neostriatal [3H]spiroperidol binding was investigated 7 days after destruction of the mesotelencephalic dopamine system by the ventral tegmental injection of 6-hydroxydopamine. As determined by saturation analysis, the average values for Kd and Bmax were 0.66 nM and 1212 fmol/mg protein in the intact striatum, and 0.82 nM and 1504 fmol/mg in the denervated striatum. The finding of a significant 23.8% increase in receptor density by the end of the first postoperative week, a period during which behavioral supersensitivity to apomorphine increases rapidly, supports the hypothesis that a proliferation of D-2 receptors underlies the behavioral manifestations of denervation supersensitivity.

Hexahydrobenzo[a]phenanthridines: novel dopamine D3 receptor ligands.

We report that certain substituted hexahydrobenzo[a]phenanthridines are novel high affinity ligands selective for the dopamine D3 receptor. These data demonstrate that substitutions on the heterocyclic nitrogen and the pendant phenyl ring of this nucleus cause a marked increase in both affinity and selectivity for dopamine D3 vs. D2 receptors. Thus, these compounds represent important new tools to study the pharmacology of dopamine D3 receptors, and may also provide an opportunity for the synthesis of new radioligands for dopamine D3 receptors.

The effects of denervation and chronic haloperidol treatment on neostriatal dopamine receptor density are not additive in the rat.

The effects of combined denervation and chronic haloperidol treatment on neostriatal D-2 receptor density were examined, and a lack of additivity was found. In the first experiment, haloperidol treatment for 14 days initiated on day 4 after the unilateral intracerebral injection of 6-hydroxydopamine (6-OHDA) abolished the lesion-induced asymmetry of [3H]spiroperidol binding by elevating the density of sites in the intact striatum. These results were replicated in the second experiment, in which the drug treatment was begun 4-5 weeks after 6-OHDA. In both experiments the apomorphine-induced rotation did not differ significantly between lesioned rats treated with haloperidol or its vehicle.

Normalizing dopamine D2 receptor-mediated responses in D2 null mutant mice by virus-mediated receptor restoration: comparing D2L and D2S.

D2 receptor null mutant (Drd2(-/-)) mice have altered responses to the rewarding and locomotor effects of psychostimulant drugs, which is evidence of a necessary role for D2 receptors in these behaviors. Furthermore, work with mice that constitutively express only the D2 receptor short form (D2S), as a result of genetic deletion of the long form (D2L), provides the basis for a current model in which D2L is thought to be the postsynaptic D2 receptor on medium spiny neurons in the basal forebrain, and D2S the autoreceptor that regulates the activity of dopamine neurons and dopamine synthesis and release. Because constitutive genetic deletion of the D2 or D2L receptor may cause compensatory changes that influence functional outcomes, our approach is to identify aspects of the abnormal phenotype of a Drd2(-/-) mouse that can be normalized by virus-mediated D2 receptor expression. Drd2(-/-) mice are deficient in basal and methamphetamine-induced locomotor activation and lack D2 receptor agonist-induced activation of G protein-regulated inward rectifying potassium channels (GIRKs) in dopaminergic neurons. Here we show that virus-mediated expression of D2L in the nucleus accumbens significantly restored methamphetamine-induced locomotor activation, but not basal locomotor activity, compared to mice receiving the control virus. It also restored the effect of methamphetamine to decrease time spent in the center of the activity chamber in female but not male Drd2(-/-) mice. Furthermore, the effect of expression of D2S was indistinguishable from D2L. Similarly, virus-mediated expression of either D2S or D2L in substantia nigra neurons restored D2 agonist-induced activation of GIRKs. In this acute expression system, the alternatively spliced forms of the D2 receptor appear to be equally capable of acting as postsynaptic receptors and autoreceptors.

Regulation of dopamine D2 receptors by sodium and pH.

The role of Na+ and H+ in the regulation of D2 receptor affinity for ligands was studied to determine the molecular mechanisms of this phenomenon. The potency of substituted benzamide derivatives and agonists at D2 receptors depended on the concentration of Na+ and H+, whereas the potency of other antagonists was relatively unaltered by changes in pH or Na+ concentration. The potency of agonists was generally decreased in the presence of NaCl or lowered pH. For example, in the absence of sodium the affinity of D2 receptors for dopamine was decreased 17-fold by lowering of the pH from 8.0 to pH 6.8. Addition of NaCl caused 2-4-fold decreases in affinity for most agonists. The affinity of the receptors for two substituted benzamide derivatives, on the other hand, was reduced 6-44-fold by elevated concentrations of H+ but was enhanced 7-24-fold in the presence of Na+. The regulation by H+ of the potency of dopamine was selective for D2 receptors, because binding of dopamine to neostriatal D1 receptors was unaffected by changes in pH. Decreasing of the pH from 8.0 or 7.3 to 6.8 facilitated the dissociation of the substituted benzamide ligand [125I]epidepride from D2 receptors but inhibited dissociation of [3H]spiperone. Furthermore, the presence of NaCl or lowered pH slowed inactivation of D2 receptors by N-ethylmaleimide. Together, these data suggest that the conformation of D2 receptors is regulated by both Na+ and H+. The affinity of D2 receptors for agonists and substituted benzamide antagonists varies according to the conformational state of the receptors, whereas other antagonists bind to both forms with approximately equal potency. Amiloride is a compound that interacts with many sodium-binding macromolecules. At equilibrium, amiloride inhibited the binding of [3H]spiperone and [125I]epidepride in a manner suggesting a more complex interaction than simple competitive inhibition. The rate of dissociation of both radioligands was enhanced by amiloride, as would be expected for allosteric inhibition of binding. The sensitivity of D2 receptors to pH, sodium, and amiloride may be a reflection of the ability of D2 receptors to modulate Na+/H+ exchange.