Hong-Chang Guan

Human brain D1 and D2 dopamine receptors in schizophrenia, Alzheimer's, Parkinson's, and Huntington's diseases

Because dopamine D2 receptors are known to be elevated in schizophrenic brain striata, this study examined whether a similar dopamine receptor elevation occurred in other diseases including neuroleptic-treated Alzheimers and Huntingtons diseases. The average D1 density in postmortem striata from Alzheimers patients was 17.6 +/- 0.1 pmol/g, similar to an age-matched control density of 16.6 +/- 0.4 pmol/g. The average D1 density in schizophrenia patients was 19.0 +/- 0.6 pmol/g, similar to the age-matched control density of 17.9 +/- 0.6 pmol/g. In Parkinsons disease patients, however, the D1 receptor density was elevated, with values of 22.8 +/- 1.2 pmol/g (in patients not receiving L-DOPA) and 19.6 +/- 1.5 pmol/g (in patients receiving L-DOPA) compared to the age-matched control density of 16.0 +/- 0.4 pmol/g. The D2 receptors in Alzheimers striata averaged 13.4 +/- 0.6 pmol/g (in patients who had not received neuroleptics), almost identical to the control density of 12.7 +/- 0.3 pmol/g. The average D2 density in neuroleptic-treated Alzheimers striata was 16.7 +/- 0.7 pmol/g, an elevation of 31%, the individual values of which had a normal distribution. In Parkinsons disease patients, the D2 densities were elevated in tissues from patients not receiving L-DOPA (19.9 +/- 1.5 pmol/g in putamen and 14.8 +/- 1.2 pmol/g in striatum) compared to the age-matched control values of 13.0 +/- 0.4 pmol/g and 12.6 +/- 0.3 pmol/g, respectively. In Huntingtons disease patients, the D2 density averaged 7.5 +/- 0.4 pmol/g in patients who had not received neuroleptics, but was 10.3 +/- 0.6 pmol/g in those who had. Although all of the D1 and D2 densities in each of the above diseases and subgroups revealed a normal distribution pattern, the D2 densities in schizophrenia displayed a bimodal distribution pattern, with 48 striata having a mode at 14 pmol/g, and the other 44 striata having a mode at 26 pmol/g. Thus, compared to the neuroleptic-induced and unimodal elevations in D2 of 31% in Alzheimers disease and 37% in Huntingtons disease, the schizophrenic striata with a mode of 26 pmol/g (105% above control) appear to contain more D2 receptors than can be accounted for by the neuroleptic administration alone.

Comparative pharmacological and functional analysis of the human dopamine D4.2 and D4.10 receptor variants.

The human dopamine D4 receptor is a D2-like receptor which is a target for most common neuroleptics. Previous investigations have shown that this receptor displays a large polymorphic variation in the third intracellular loop involving a variable number of direct imperfect tandem repeats (VNTR) of 16 amino acids. The shortest and longest repeat variants reported to date contain two and 10 repeat units (D4.2 and D4.10). No major pharmacological differences have been reported for the most common variants of this receptor (D4.2, D4.4 and D4.7), although the D4.7 was reported by us to display a slightly lower potency for dopamine in functional assays. Direct pharmacological and functional comparison of the longest and shortest variants in this study suggest no major discrepancies in pharmacological or functional profile between both receptors. Both receptors display, on average, a 15-fold and 90-fold lower potency for epinephrine and norepinephrine, respectively, compared with dopamine. We observed small increases in functional potency and affinity for dopamine and quinpirole at the D4.10 receptor variant compared with the D4.2 receptor. Our data indicate that there is no direct relationship between the length of the polymorphism and changes in pharmacology or functional activity. These findings are a suitable caution against the arbitrary pooling of D4 receptor VNTR genotypes in genetic studies, based on length.

The cloned dopamine D2 receptor reveals different densities for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography

Since [3H]emonapride ([3H]YM-09151-2), a benzamide neuroleptic, consistently detects more dopamine D2 receptors than [3H]spiperone in the same tissue, we tested whether this property was inherent in the cloned dopamine D2 receptor. We found that the density of dopamine D2 receptors labelled by [3H]emonapride was 1.5-fold to 2-fold (mean of 1.8-fold) higher than the density of dopamine D2 receptors labelled by [3H]spiperone in cells expressing cloned dopamine D2 receptors (either the short form (from rat) or the long form (from human)), matching similar findings in anterior pituitary tissue (rat or pig) or in post-mortem human caudate nucleus tissue. The situation was similar for another benzamide, [3H]raclopride, which revealed 1.3-fold to 1.8-fold (mean of 1.5-fold) more binding sites than that for [3H]spiperone in cell membranes containing cloned dopamine D2 receptors. The apparently different dopamine D2 receptor densities revealed by these two types of 3H-ligands (i.e. [3H]spiperone and the [3H]benzamides), therefore, arise from an inherent property of the dopamine D2 receptor protein. These findings for the cloned dopamine D2 receptor, therefore, partly explain the higher dopamine D2 receptor density measured in human brain (by positron emission tomography) when using radioactive raclopride compared to results using radioactive methylspiperone.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine D2High receptors stimulated by phencyclidines, lysergic acid diethylamide, salvinorin A, and modafinil.

Although it is commonly stated that phencyclidine is an antagonist at ionotropic glutamate receptors, there has been little measure of its potency on other receptors in brain tissue. Although we previously reported that phencyclidine stimulated cloned‐dopamine D2Long and D2Short receptors, others reported that phencyclidine did not stimulate D2 receptors in homogenates of rat brain striatum. This study, therefore, examined whether phencyclidine and other hallucinogens and psychostimulants could stimulate the incorporation of [35S]GTP‐γ‐S into D2 receptors in homogenates of rat brain striatum, using the same conditions as previously used to study the cloned D2 receptors. Using 10 μM dopamine to define 100% stimulation, phencyclidine elicited a maximum incorporation of 46% in rat striata, with a half‐maximum concentration of 70 nM for phencyclidine, when compared with 80 nM for dopamine, 89 nM for salvinorin A (48 nM for D2Long), 105 nM for lysergic acid diethylamide (LSD), 120 nM for R‐modafinil, 710 nM for dizocilpine, 1030 nM for ketamine, and >10,000 nM for S‐modafinil. These compounds also inhibited the binding of the D2‐selective ligand [3H]domperidone. The incorporation was inhibited by the presence of 200 μM guanylylimidodiphosphate and also by D2 blockade, using 10 μM S‐sulpiride, but not by D1 blockade with 10 μM SCH23390. Hypertonic buffer containing 150 mM NaCl inhibited the stimulation by phencyclidine, which may explain negative results by others. It is concluded that phencyclidine and other psychostimulants and hallucinogens can stimulate dopamine D2 receptors at concentrations related to their behavioral actions. Synapse 63:698–704, 2009.

Schizophrenia: elevation of dopamine D4-like sites, using [3H]nemonapride and [125I]epidepride

We here report a three-fold elevation of dopamine D4-like sites in schizophrenia, using [3H]nemonapride to measure dopamine D2 and D3 receptors and D4-like sites, and using [125I]epidepride to measure D2 and D3 sites in ten control and nine schizophrenia post-mortem brain putamen tissues. This result differs from a recent report which did not detect significant D4-like sites in control or schizophrenia putamen (Reynolds and Mason, 1995, Eur. J. Pharmacol. 281, R5). The present finding agrees with other reports wherein an elevation in D4-like sites was found in schizophrenia, using [3H]nemonapride for D2, D3 and D4-like sites, but [3H]raclopride for D2 and D3 sites. The nature of these D4-like sites is not known.

Long-term haloperidol elevates dopamine D4 receptors by 2-fold in rats.

Chronic treatment of rats with neuroleptics results in elevated numbers of dopamine D2-like receptors. The present study was done to determine whether neuroleptics altered the density of one type of dopamine D2-like receptors, namely the dopamine D4 receptor. We here describe the effect of a one-month treatment with haloperidol on dopamine D4 receptor mRNA and protein levels in rat striatum. Endogenous levels of dopamine D4 receptor mRNA in rat striatum are very low and, therefore, reverse transcription and subsequent amplification were used for quantification. Dopamine D4 receptor density was, because of the absence of a dopamine D4 receptor specific [3H]ligand, determined by the difference between the number of binding sites for [3H]nemonapride and [3H]raclopride. Scatchard analysis of [3H]nemonapride and [3H]raclopride binding show that treatment for one month with halperidol elevates the density of dopamine D4 receptors in rat striatum by approximately 2-fold, whereas dopamine D2 and D3 receptors together show a 19% higher receptor density. Dopamine D4 receptor mRNA was also approximately increased by 2-fold.

Phencyclidine and glutamate agonist LY379268 stimulate dopamine D2High receptors: D2 basis for schizophrenia

It has previously been reported that the glutamate ionotropic antagonist phencyclidine directly inhibits the release of prolactin in anterior pituitary cells in culture, suggesting that phencyclidine has a dopamine (DA)‐like action on prolactin‐releasing cells. It has also been reported that the glutamate metabotropic agonist LY379268 can stimulate the incorporation of [35S]GTP‐γ‐S into DA D2Long receptors. The present study was done to examine whether such glutamatergic drugs had similar actions on the DA D2Short receptor. The present results show that phencyclidine, ketamine, and LY379268 also stimulated the incorporation of [35S]GTP‐γ‐S into D2Short receptors. The proportion of D2Long and D2Short receptors existing in the high‐affinity state were both markedly reduced by NaCl. While phencyclidine and LY379268 each stimulated the incorporation of GTP‐γ‐S into D2Long and D2Short receptors, this stimulation was reduced by NaCl, with D2Short being much more sensitive than D2Long to the inhibition by NaCl. The binding of phencyclidine and LY379268 to D2High receptors in vivo was directly confirmed by the i.v. injection of phencyclidine and LY379268 in which 50% inhibited the binding of [3H]PHNO to the striatum ex vivo at 0.25 and 1.5 mg/kg, respectively. The results confirm that glutamate agonists and antagonists have a significant affinity for DA D2High receptors. The psychotogenic action of phencyclidine may stem from a combination or synergistic action of glutamate receptor antagonism and DA D2 agonism. In addition, the antipsychotic clinical action of LY379268 congeners such as LY404039 may be related to a combined or synergistic action of glutamate receptor stimulation together with a partial DA agonist action that reduces endogenous DA neurotransmission. Synapse 62:819–828, 2008.

Glutamate agonist LY404,039 for treating schizophrenia has affinity for the dopamine D2High receptor

The glutamate agonist LY404,039 has been used to treat schizophrenia. Because all currently used antipsychotics act on dopamine receptors, it was decided to examine whether this glutamate agonist also had an affinity for dopamine D2 receptors in vitro. The present data show that LY404,039 inhibited the binding of [3H]domperidone and [3H](+)PHNO by 15.5 ± 1.5% to the high‐affinity state, D2High, of cloned dopamine D2Long receptors and rat striatal tissue with dissociation constants of between 8.2 and 12.6 nM. This high‐affinity component of LY404,039 on the binding of [3H]domperidone was inhibited by the presence of guanine nucleotide, indicating an agonist action of the drug at D2High. LY404,039 also stimulated the incorporation of [35S]GTP‐γ‐S into D2Long receptors (EC50% = 80 ± 15 nM) over the same range of concentrations as occurred for the inhibition of [3H]domperidone by LY404,039 at D2High (IC50%High = 50 ± 10 nM). A possible clinical antipsychotic action of LY404,039 may depend on the combined stimulation of glutamate receptors and a partial dopamine agonist action that would interfere with neurotransmission at D2High receptors. Synapse 63:935–939, 2009.

Glutamate agonists for treating schizophrenia have affinity for dopamine D2High and D3 receptors.

Although the glutamate agonist LY 404,039 has been used to treat schizophrenia, its closest congener LY 379,268 has an affinity for both glutamate and dopamine (DA) D2High receptors. Considering that all antipsychotics act on dopamine receptors, and considering that another laboratory reported that LY 379,268 did not have any affinity for the D2High receptor, it was necessary to examine whether such glutamate agonists have an affinity for D2 and D3 dopamine receptors in vitro. The present data show that 50–200 nM LY 379,268 inhibited the binding of [3H]domperidone and [3H](+)PHNO to cloned dopamine D2 receptors consistently and reproducibly by 16% with dissociation constants of 2.1 and 2.5 nM at D2High, respectively. In addition, LY 379,268 inhibited the binding of [3H]domperidone and [3H](+)PHNO to cloned dopamine D3 receptors with dissociation constants of 130 and 10 nM, respectively. LY 379,268 also inhibited the binding of [3H]domperidone to rat striata with a dissociation constant of 22 nM, predicting a clinical antipsychotic dose of 80–100 mg/day. LY 379,268 appears to act as an agonist at D2High and as an antagonist at D3, because guanine nucleotide eliminated the competition at D2High but had no effect on the competition at D3. The findings indicate that this type of glutamate agonist, LY 379,268, has a significant affinity for D2High and D3 receptors. Synapse 63:705–709, 2009.

Promoter-independent regulation of cell-specific dopamine receptor expression

Here we describe the construction of recombinant adenoviruses expressing dopamine D2 and D4 receptors, and their ability to mediate high levels of heterologous expression in a variety of cell types in vitro and in vivo for at least 7 days post infection. These experiments demonstrated that maximum receptor expression is achieved generally within 24 h and remains constant thereafter. Maximum expression levels were highly variable between cell lines and dependent on infection efficiency and promoter strength. Correction for these two variables revealed differences in relative expression levels between cell lines varying by two orders of magnitude. Our results indicate that in addition to gene transcription, post‐transcriptional mechanisms play a dominant role in determining dopamine receptor levels in this system.