The dopamine hypothesis of schizophrenia was examined by measuring the density of dopamine receptors in the postmortem brains of 81 control subjects and 59 schizophrenics from four different countries. The densities of dopamine receptors in the tissues from the schizophrenic patients had a bimodal distribution in the caudate nucleus, putamen, and nucleus accumbens. One mode occurred 25 percent above the control density, and a second mode occurred at a density 2.3 times that of the control density for all three regions. Although almost all the patients had been medicated with neuroleptics, the two modes had the same dissociation constant for the labeled ligand used, suggesting that the neuroleptic doses were similar for the two populations of schizophrenics. The results thus provide direct evidence for two distinct categories of schizophrenia.

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 particulate 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 predominantly 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 receptor types D1 and D2 can oppose or enhance each other's actions for electrical, biochemical, and psychomotor effects. We report a D1-D2 interaction in homogenized tissue as revealed by ligand binding. D2 agonists The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D2 receptors in the high-affinity state. Thus, the Dj-D2 link may be mediated by guanine nucleotide-binding protein components. The link may underlie D1-D2 interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata.

Since the high-affinity state of dopamine D2 receptors may be abnormal in psychomotor diseases, it is desirable to develop a radioactive agonist to label this high-affinity site for possible clinical diagnostic use. (+)PHNO is a selective D2 agonist used to treat Parkinson's disease. We prepared [3H](+)PHNO from allyl-des-propyl(+)PHNO. In binding to dopamine receptors in homogenates of canine brain striata, [3H](+)PHNO had a dissociation constant of 0.35 nM in the absence of NaCl, and 0.56 nM in the presence of NaCl. Dopamine agonists and antagonists inhibited the binding of [3H](+)PHNO at drug concentrations similar to those inhibiting other [3H]ligands at D2 receptors, but not similar to those acting at D4 receptors. Approximately 90% of the total [3H](+)PHNO binding was specific. Guanilylimidodiphosphate markedly inhibited [3H](+)PHNO binding, suggesting that [3H](+)PHNO was binding primarily to the high-affinity state of dopamine D2 receptors rather than to D3 receptors. The density of the [3H](+)PHNO binding sites was equal to that of [3H]emonapride (or [3H]YM-09151-2), both densities of which were 1.5- to 2-fold higher than that of [3H]spiperone, compatible with the idea that [3H](+)PHNO binds to monomers of D2, while [3H]spiperone binds to dimers of D2. Although [3H](+)PHNO has good selectivity and affinity for the high-affinity state of D2, the [3H]ligand was sensitive to endogenous dopamine, since washing the tissue lowered the dissociation constant. For future in vivo labelling of D2 by an agonist, therefore, it will be essential to search for a related [3H]ligand with an even lower dissociation constant.

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