Carla Ulpian

Dopamine Receptor Parameters Detected by [3H]Spiperone Depend on Tissue Concentration: Analysis and Examples

The binding of lipophilic radioligands to homogenized tissue was investigated with the help of a simple, two‐component model: a specific component reflects binding to a single and uniform population of sites; a nonspecific component reflects partitioning into the membrane and the entrapment of some drug present in the aqueous phase prior to separation of the pariculate fraction. The results indicate that the capacity and the affinity of the receptor may be underestimated when the data are analyzed in terms of total rather than free radioligand. Errors in capacity arise when for a significant fraction of the radioligand access to the receptor is blocked by an unlabelled drug and this appears as nonspecific binding. This is most likely to occur when the partition coefficient is such that the free radioligand is located pre‐dominantly in the particulate phase. Errors in affinity reflect the tendency of the membrane to reduce the free concentration of a lipophilic drug in the aqueous phase. A further complication arises when a significant fraction of the total radioligand binds to the receptor. [3H]Spiperone binds to dopamine D2 receptors with a dissociation constant of about 50 pM and partitions into the particulate phase of brain homogenates with a membrane/buffer partition coefficient of 410. As expected, both capacity and affinity can appear to depend on the concentration of tissue used in the assay. If the partition coefficient is known, corrected estimates of both parameters can be obtained knowing only the total concentration of radioligand; if the partition coefficient is not known, the free concentration of radioligand in the aqueous phase must be measured independently. The former procedure requires that the aqueous and particulate components of the system be separated by centrifugation; with filtration, the removal of an indeterminate amount of radioligand from the membrane during washing precludes any correction based on the partition coefficient. For the specific example of [3H]spiperone in human brain, the artifacts become negligible at concentrations of protein below 0.1 mg/ml of incubate. The capacity per unit of original tissue is best determined using unwashed preparations, since about 30% of the total protein and a comparable percentage of the receptors are lost on washing.

Dopamine D4 receptor variant in Africans, D4Valine194Glycine, is insensitive to dopamine and clozapine: Report of a homozygous individual

The D4Valine194Glycine receptor is a variant of the dopamine D4 receptor and is found in 12.5% of the Afro-Caribbean population. Glycine replaces valine at a position one amino acid away from a serine which is critical for the attachment of dopamine. To determine whether this mutation had an effect on the properties of the dopamine D4 receptor, we constructed this variant and tested the sensitivity of the expressed protein with various drugs. We found that the variant receptor was two orders of magnitude less sensitive to dopamine, clozapine and olanzapine. The variant receptor was insensitive to guanine nucleotide, indicating the absence of a high-affinity state or functional state. The one 15-year-old individual found homozygous for this variant also had sickle cell disease. The patient revealed an overall pattern of low weight and no axillary or pubic hair.

Dopamine D1 and D2 Receptor Selectivities of Agonists and Antagonists

The selectivities of various dopamine agonists and antagonists for dopamine D1 and D2 receptors were obtained by comparing their relative dissociation constants for inhibiting the binding of [3H]SCH 23390 at D1 receptors (calf caudate nucleus) and at D2 receptors (pig anterior pituitary tissue). The most selective agonists were SK&F 38393 (for D1) and (+)-PHNO (for D2), while the most selective antagonists were SCH 23390 (for D1) and raclopride or eticlopride (for D2).

Neuroleptics have identical potencies in human brain limbic and putamen regions.

In order to examine whether some neuroleptic drugs were specifically more potent on human limbic dopamine receptors than on striatal dopamine receptors, we tested the potency of eight neuroleptics on their ability to inhibit the binding of [3H]spiperone to D2 dopamine receptors in human putamen and nucleus accumbens. Each of the neuroleptics had an identical potency in both tissues, the IC50 values being 0.2 nM for spiperone, 2.5 nM for haloperidol, 2.6 nM for trifluperidol, 5 nM for fluphenazine, 20 nM for thioridazine, 25 nM for chlorpromazine, 100 nM for metoclopramide and 300 nM for clozapine. There is no evidence, therefore, for the concept of a limbic-specific dopamine receptor antagonist.

Schizophrenia: Normal Sequence in the Dopamine D2 Receptor Region that Couples to G-Proteins. DNA Polymorphisms in D2

Because dopamine (DA) D2 receptors are a target in neuroleptic therapy and have been found to be elevated in schizophrenia, the human DA D2 receptor gene was examined for possible abnormalities in schizophrenia. Moreover, since D2 receptors in psychosis have a reduced coupling to D1 receptors, the cytoplasmic third loop of D2 was chosen for deoxyribonucleic acid (DNA) sequencing, since this region is essential for coupling to G-proteins. This region also contains exon 5, which is expressed in the long form of D2, but not in the short form of D2. In eight schizophrenia cases, this region had normal exon sequences (exons 4, 5 and 6), and normal sequences at its intron-exon junctions. However, exon 6 contained three DNA polymorphic base changes, and introns 4 and 5 revealed three missing bases and two polymorphic base changes, none of which would be expected to alter the D2 receptor protein in schizophrenia.

Schizophrenia: elevated mRNA for dopamine D2Longer receptors in frontal cortex

Because dopamine D2 receptors are the primary targets for antipsychotic drugs, including clozapine and quetiapine, and because some studies have found D2 receptors to be elevated in schizophrenia, we examined the mRNA of three forms of the D2 receptor, particularly the new form of the dopamine D2 receptor, D2(Longer), in post-mortem brains from patients who died with schizophrenia. Using quantitative competitive RT-PCR (reverse transcriptase-polymerase chain reaction), the D2(Longer) mRNA was higher in the frontal cortex, compared to control tissues. The mRNA concentration of D2(Long) and D2(Short) was also higher in the frontal cortex, compared to control tissues. Although most of the schizophrenia patients had received different antipsychotic drugs for varying periods of time, the mRNA of D2(Longer), as well as that for D2(Long) and D2(Short), in such medicated tissues was similar to that in a frontal cortex tissue from a patient who had reliably never received antipsychotic drugs. It is possible, therefore, that the elevation of the mRNAs for D2(Longer), D2(Long) and D2(Short) in the frontal cortex may be related to the disease of schizophrenia itself.

Schizophrenia : dopamine D1 receptor sequence is normal, but has DNA polymorphisms

Genes that regulate dopamine (DA) receptors may underlie the overactive DA system in schizophrenia. Since it is known that there is an abnormally reduced or absent regulation of the DA D2 receptor by the DA D1 receptor in the postmortem schizophrenia brain, the human DA D1 receptor gene was sequenced fom genomic deoxyribonucleic acid (DNA) of seven schizophrenic individuals. The tissues from two schizophrenics had previously been found to have a reduced link between DA D1 and D2 receptors. The D1 receptor genes were amplified by the polymerase chain reaction, subcloned, and sequenced. Although three DNA polymorphisms were found, the deduced amino acid sequence of the DA D1 receptor was normal in these tissues.

Functional States of Dopamine Receptors

Dopamine receptors can exist in either a high-affinity state or in a low-affinity state for dopamine agonists. This is based on the observation that guanine nucleotides can lower the affinity of the receptors for dopamine agonists (Zahniser and Molinoff, 1978; Creese et al., 1979a,b).

A serotonin-4 receptor-like pseudogene in humans

During a search for new G-protein-linked receptors for dopamine and serotonin, we found a serotonin-4 receptor-like pseudogene. This receptor-like pseudogene is intronless, contains an in-frame stop codon following transmembrane-3, and has two one-nucleotide insertions between transmembrane-5 and -6 regions which alter the reading frame. The predicted amino acid sequence of the human pseudogene is about 35% identical with that of the rat serotonin-4 receptor.

A human serotonin-7 receptor pseudogene

Although the serotonin-7 receptor was cloned several years ago, its localization in brain tissues remains confusing because of the existence of a related expressed pseudogene, the sequence of which has not hitherto been reported. During the course of searching for related receptor genes, we also searched for this pseudogene to determine its sequence. Human genomic DNA was screened for dopamine and serotonin receptor-like genes, using the polymerase chain reaction method and degenerate oligonucleotide primers based on the similar sequences in the transmembrane-6 and -7 regions of the serotonin-5A, the serotonin-7, and the dopamine D2, D3 and D4 receptors. This resulted in one of the clones having a 115 bp fragment, of which 89% of the bases were identical to the transmembrane-6 and -7 regions of the serotonin-7 receptor sequence. The fragment was radiolabelled and used to screen a human fetal brain cDNA library. A novel cDNA clone of 1326 bp was isolated. Based on the nucleotide sequence, 88% of the bases in this sequence of the pseudogene are identical to the human serotonin-7 receptor coding sequence. However, compared to the serotonin-7 receptor DNA sequence, the pseudogene sequence has nucleotide deletions and insertions, resulting in frame-shifts and stop codons. It was concluded that this sequence represented a pseudogene related to the serotonin-7 receptor gene.