Quantitative determination of dopamine receptor subtypes not linked to activation of adenylate cyclase in rat striatum.

Abstract

It is unlikely that either of the two classes of striatal sites are receptors for serotonin. The approach described will make it possible to assess the effects of physiological or pharmacological manipulations on the densities or properties of subtypes of dopamine receptors.The striatum is thought to contain multiple subtypes of dopamine receptors. Kebabian et aL (1) characterized a dopaminesensitive adenylate cyclase activity in the striatum. In vitro binding assays using the butyrophenones [3H]haloperidol and [3H]spiroperidol also revealed the presence of dopamine receptors in this brain region (2-4). However, the properties and distribution of the binding sites for butyrophenones differed from those expected based on studies of dopamine-sensitive adenylate cyclase activity (5-7). A classification scheme was developed such that the subtypes ofdopamine receptors whose effects involved activation of adenylate cyclase were called D-1 receptors and receptors not linked to stimulation of the enzyme were called D-2 receptors (8).Guanine nucleotides decreased the affinity of [3H]spiroperidol binding sites in the striatum for agonists (9). By analogy to other receptor systems that are linked to activation of adenylate cyclase (10-12), we suggested that some of the sites labeled by[3H]spiroperidol were also linked to stimulation ofthis enzyme. METHODSThe striatum or frontal cortex from male Sprague-Dawley rats was homogenized in 35 ml of 20 mM Hepes, pH 7.5/154 mM NaCl/5 mM EDTA. After centrifugation (20,000 X g for 10 min at 4°C) pellets were resuspended in 20 mM Hepes, pH 7.5/ 154 mM NaCI, incubated at 37°C for 15 min, and recentrifuged. Striatal membranes were resuspended in 20 mM Hepes/154 mM NaCl at a concentration of 2-4 mg of tissue per ml for studies of [3H]domperidone binding, 1-2 mg/ml for studies of [3H]spiroperidol binding in the striatum, and 4 mg/ml for studies of [3H]spiroperidol binding in frontal cortex.[3H]Domperidone (59.7 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels), [3H]spiroperidol (29.9 Ci/mmol), drugs, and GTP (final concentration, 0.3 mM) were diluted in 2.6 mM ascorbic acid containing 20 ,ug of bovine serum albumin per ml.Binding reactions were carried out for 45 min at 37°C and then terminated by the addition of 10 ml ofice-cold 10 mM Tris, pH 7.5/154 mM NaCl. Samples were filtered through glass fiber filters (Schleicher & Schuell,no. 30) and washed with an additional 10 ml of the same buffer. Specific binding was defined as the difference between the amount of radioligand bound in the presence and absence of 2 ,uM (+) butaclamol. Protein content was determined by the method ofBradford (20).The untransformed data obtained in studies of the binding of [3H]domperidone were analyzed by nonlinear regression analysis using the computer modeling program SAAM27. The data were compared to model curves describing one, two, and three sites, and F-test analysis was used to determine the most appropriate model (21). Regression analysis of linear Scatchard, Hill, and Hofstee plots was carried out by the leas...

Dopamine release and metabolism in the rat frontal cortex, nucleus accumbens, and striatum: a comparison of acute clozapine and haloperidol

“…For example, the putative atypical neuroleptic, CGS 10746B, was shown to reduce 3-MT concentration in the ST (Alter et al, 1986). Similarly, the selective dopamine D1-receptor antagonist, SCH 23390 (Setler et al, 1978;Huff & Molinoff, 1982;Hytell, 1983), which shares many pharmacological features with CLZ (Lorio et al, 1983;Coward et al, 1989;Farde et al, 1989), has no effect on ST 3-MT accumulation (Boyar & Altar, 1987;Saller & Salama, 1986). The discordance between our findings and those of Saller & Salama, 1986, may result from methodological differences, e.g.…”

Solubilization and Characterization of Striatal Dopamine Receptors

“…The affinities of DA antagonists for E3H]Spi sites are slightly increased by GTP in the CHAPSO-solubilized preparations. This finding suggests that the subclass of D-2 DA receptors, which is negatively coupled to adenylate cyclase (Frey et al, 1982;Huff and Molinoff, 1982), was extracted by CHAPSO from the striatal membranes, or that GTP reciprocally modulates the conversion by agonists and antagonists of the low-and high-affinity forms of the receptor. It is noteworthy that the reciprocal modulation by agonist and antagonist of two different forms of the receptor under the influence of guanine nucleotides was suggested for the membrane-bound DA receptor in the porcine anterior pituitary (De Lean et al, 1982).…”

“…In addition to the D-1 and D-2 DA receptor binding sites, the existence in the brain of other putative DA receptor binding sites, namely D-3 and D-4 sites, was reported (Titeler et al, 1979;Sokoloff et al, 1980). Radioligand binding studies have also revealed the existence of two subtypes of D-2 DA receptors, one of which is affected by guanine nucleotides (Huff and Molinoff, 1982). However, it is not established whether these multiple binding sites represent distinct molecular entities or different states of the same DA receptor.…”

High‐and Low‐Affinity States of Striatal D2 Receptors Are Not Affected by 6‐Hydroxydopamine or Chronic Haloperidol Treatment

“…However that study did not differentiate between binding to D, and 5-HT2 sites. Using tissue from frontal cortex, the K D of spiperone for 5-HT2 sites has been estimated to be 1.0 x M (Huff and Molinoff, 1982). Thus if striatal and frontal cortical 5-HT2 sites have equal affinities for spiperone, then 6 x M [3H]spiperone should have labelled a significant portion of the striata15-HT2 sites and this may have accounted for the multiphasic nature of the binding.…”