Effect of prolactin and estradiol on rat striatal dopamine receptors

Chronic estrogen treatment has been found to increase the level of rat striatal dopamine receptors. Since it is well known that estrogen treatment increases circulating prolactin levels, we have investigated the possibility that the stimulatory effect of estrogens on dopamine receptors is exerted via prolactin. Ovariectomized female or intact male rats were implanted with three adenohypophyses under the kidney capsule or treated with 17 β-estradiol (10 μg, twice daily) for 2 weeks. In animals of both sexes, the pituitary-implanted and estradiol-treated rats showed higher levels of [³H]spiperone binding to striatal dopamine receptors. This effect of estradiol or pituitary implants on dopamine receptors was further investigated in ovariectomized rats. The pituitary-implanted and estradiol-treated rats had elevated plasma prolactin levels and an increased density of striatal dopamine receptors without alteration of their affinity. The role of the pituitary in the effect of estradiol was next investigated using hypophysectomized female rats treated with 17 β-estradiol (10 μg, twice daily), o-prolactin (500 μg, twice daily) or bearing three anterior pituitary implants. The implants as well as the treatment with estradiol or prolactin increased the level of striatal dopamine receptors in hypophysectomized rats while, as expected, the estradiol-treated animals did not have elevated plasma prolactin levels. The present data indicate that high prolactin levels lead, as observed with chronic estradiol treatment, to an increased density of striatal dopamine receptors. However, the effect of estradiol may not be explained exclusively by increased prolactin levels since a similar stimulatory effect is observed in hypophysectomized animals.     

Chronic neuroleptic treatment in rats produces persisting changes in GABAA and dopamine D-2, but not dopamine D-1 receptors

The effects of continuous treatment with haloperidol (HAL) or fluphenazine (FLU) for 10 months on dopamine and GABA receptors in the rat brain was examined using in vitro autoradiography. Rats treated with HAL, but not FLU, showed an increase in D-2 receptor binding in the caudate-putamen as revealed by [3H]spiperone. Labeling of D-1 receptors by [3H]SCH23390 revealed no changes in either drug-treated group. Both drug-treated groups, however, exhibited a significant increase in [3H]muscimol binding in substantia nigra, pars reticulata (SNR). These dopaminergic-GABAergic receptor alterations may be related to previously reported changes in oral movement activity seen in these neuroleptic-treated animals.     

Distinct target size of dopamine D-1 and D-2 receptors in rat striatum

Frozen rat striatal tissue was exposed to 10 MeV electrons from a linear accelerator. Based on the theory of target size analysis, the molecular weights of dopamine D-1 receptors (labelled by 3H-piflutixol) and dopamine D-2 receptors (labelled by 3H-spiroperidol) were 79,500 daltons and 136,700 daltons, respectively. The size of the dopamine-stimulated adenylate cyclase was 202,000 daltons. The estimated molecular sizes were deduced by reference to proteins with known molecular weights which were irradiated in parallel. The results showed that the molecular entities for 3H-piflutixol binding and 3H-spiroperidol binding were not identical. The present results do not allow conclusions as to whether D-1 and D-2 receptors are two distinct proteins in the membrane, or whether the receptors are located on the same protein. In the latter case the binding of 3H-spiroperidol needs the presence of a second molecule.     

6-Hydroxydopamine-induced degeneration of nigral dopamine neurons: differential effect on nigral and striatal D-1 dopamine receptors

Dopamine-sensitive adenylate cyclase and 3H-SCH 23390 binding parameters were measured in the rat substantia nigra and striatum 15 days after the injection of 6-hydroxydopamine into the medial forebrain bundle. The activity of nigral dopamine-sensitive adenylate cyclase and the binding of 3H-SCH 23390 to rat nigral D-1 dopamine receptors were markedly decreased after the lesion. On the contrary, 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway enhanced both adenylate cyclase activity and the density of 3H-SCH 23390 binding sites in striatal membrane preparations. The changes in 3H-SCH 23390 binding found in both nigral and striatal membrane preparations were associated with changes in the total number of binding sites with no modifications in their apparent affinity. The results indicate that: within the substantia nigra a fraction (30%) of D-1 dopamine receptors coupled to the adenylate cyclase is located on cell bodies and/or dendrites of dopaminergic neurons; striatal D-1 dopamine receptors are tonically innervated by nigrostriatal afferent fibers.     

Tamoxifen counteracts estradiol induced effects on striatal and hypophyseal dopamine receptors

We investigated the ability of Tamoxifen (TAM), an antiestrogen drug, to counteract the modifications induced by estrogens on dopamine (DA) receptors on striatum and on adenohypophysis of ovex female rats. Subacute treatment with 17 beta-estradiol (E2) at both low (0.1 micrograms/kg) and high (20 micrograms/kg) doses confirmed its ability to increase the number of striatal 3H-Spiperone (3H-SPI) binding sites in a dose dependent manner. By contrast in the pituitary, only high doses of estrogen were effective in reducing the number of DA receptors. We treated ovex female rats for 15 days with TAM alone or associated with E2, to see if these estrogenic effects could be suppressed by an antiestrogenic drug. TAM did not affect the number of striatal DA receptors, but significantly increased the adenohypophyseal DA binding sites, without varying their affinity. No changes were observed in pituitary and striatal DA receptor density, even when TAM was injected in association with estradiol. In conclusion: TAM is able to counteract the effects estrogens have on DA receptors. However there is some evidence that it could influence the pituitary DA systems independently of its antiestrogenic activity.     

Attenuation of estradiol on the reduction of striatal dopamine by amphetamine in ovariectomized rats

Amphetamine (AMPH) is a highly addictive drug of abuse which exhibits toxicity to dopaminergic neurons in long‐term abusers. Estrogen seems to show neuroprotection in dopamine (DA) deficit caused by AMPH. The present study was to investigate the effects of estradiol on the levels of striatal DA in ovariectomized (Ovx) rats treated with or without AMPH. Female rats were Ovx for 2 weeks before administration of AMPH (5 mg/kg/day, i.p.) with or without 17β‐estradiol benzoate (EB) (25 µg/kg/day, s.c.) for 7 days. The striatal tissues were collected, homogenized with DA mobile phase, and centrifuged. The concentrations of DA in the supernatants were detected by HPLC. The protein expressions of dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT‐2), and tyrosine hydroxylase (TH) were analyzed by Western blotting. The results indicated that AMPH could attenuate DA level significantly in striatum (P < 0.01). Comparing to control groups, administration of either EB or EB plus AMPH increased DA level (P < 0.01). The protein expression of striatal DAT was significant greater (P < 0.01) in rats treated with AMPH plus EB than AMPH treated animals. These results suggest that the DA levels in striatum can be enhanced by EB via an increase of DAT expression following administration of AMPH. J. Cell. Biochem. 108: 1318–1324, 2009. © 2009 Wiley‐Liss, Inc.     

A novel affinity purification of D-1 dopamine receptors from rat striatum

When rat striatal membranes were pretreated with the sulfhydryl (-SH) modifying reagent, N-ethylmaleimide (NEM) in the presence of the D-1-specific agonist, SKF R-38393, the D-1 dopamine receptor was completely protected from NEM-mediated inactivation. The D-1 receptors, solubilized from these membranes with 1% sodium cholate in the presence of phospholipids, bound with high efficiency (greater than 90%) to mercury-agarose columns. The bound receptors were eluted from the affinity column with a -SH reducing agent, beta-mercaptoethanol. Upon removal of beta-mercaptoethanol from the eluted fractions by inclusion chromatography, the receptor was reconstituted into phospholipid vesicles and assayed for ligand binding activity. The affinity purified receptor exhibited saturable and specific binding of the D-1-specific ligand 125I-SCH 23982, with a Kd of 1.6 nM comparable to that measured in intact membranes and solubilized extracts. The binding capacity of these receptors for 125I-SCH 23982 was 11,000 pmol/mg protein, representing greater than an 8000-fold purification over the starting membrane preparation. The purity of the affinity eluted receptors was estimated to be 78%. The purified receptors retained the pharmacological properties of membrane-bound receptors, including the ability to distinguish between active and inactive enantiomers of specific dopaminergic antagonists. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining revealed the presence of two major polypeptides of 74 and 54 kDa. These two polypeptides were absent in those affinity eluted fractions which did not display 125I-SCH 23982-binding activity and also were not detected in preparations obtained from membranes which were NEM-treated in the absence of D-1-specific agonist. The molecular weights of these polypeptides were similar to those of membrane-bound D-1 receptors, when labeled with a D-1-specific photo-affinity ligand, 125I-8-hydroxy-3-methyl-1-(4-azidphenyl)-2,3,4,5-tetrahydro-1H-3-b enzazepine. These two polypeptides may represent glycosylated and deglycosylated forms of the D-1 dopamine receptor.     

Effects of zotepine, chlorpromazine and haloperidol on D-1, D-2, D-3 and D-4 subtypes of dopamine receptors in rat striatal and bovine caudate nucleus membranes

Inhibitory effects of zotepine (Zot) on D-1, D-2, D-3 and D-4 subtypes of dopamine (DA) receptors were investigated in crude synaptic membranes of rat striatum and bovine caudate nucleus and compared to those of chlorpromazine (CPZ) and haloperidol (HAL). From the IC₅₀-values of Zot, CPZ and HAL, the K-values of each drug are estimated as follows: 34.4, 152 and 244 nM (D-1, ³H-labeled cis-flupenthixol binding (1.0 nM) to rat membranes); 37.4, 7.1 and 2.4 nM (D-2, [³H]spiperone (Spi) binding (0.5 nM) to rat membranes in the presence of 0.1 μM ketanserin); 73.1, 15.2 and 22.4 nM (D-3, ³H-labeled N-propylapomorphine (NPA) binding (0.29 nM) to bovine membranes in the presence of 0.1 μM Spi); 9.5, 65.3 and 3.1 nM (D-4, [³H]NPA binding (0.29 nM) bovine membranes in the presence of 25 nM DA), respectively. Zot binds with higher affinity to D-4 but lower affinity to D-3 than to other subtypes. It is also presumed that Zot binds to D-1 with high affinity and D-2 and D-3 with low affinity compared to CPZ and HAL.     

Interactions of novel dopaminergic ligands with D-1 and D-2 dopamine receptors

The interactions of three novel dopaminergic ligands, SKF38393, SKF82526 and SKF83742, with D-1 and D-2 dopamine (DA) receptors have been investigated using radioligand binding techniques and computer modeling procedures. Using the bovine anterior pituitary D-2 DA receptor system, SKF38393 and SKF82526 behave as agonists demonstrating biphasic agonist/3H-antagonist competition curves. For both drugs, the high affinity phase comprised 30% of the total displacement curve. Such findings are atypical as previously tested classical dopamine agonists demonstrated high and low affinity displacement phases in equal proportions. Such behavior exhibited by the SKF agonists may be related to their activity as partial agonists. In contrast, SKF83742 behaves as an antagonist exhibiting homogeneous monophasic competition curves. Similar results are obtained in the rat striatal membrane D-2 DA receptor system. Both SKF38393 and SKF82526 also demonstrate shallow biphasic displacement curves on rat striatal D-1 receptors labeled with 3H-flupentixol whereas SKF83742/3H-flupentixol curves are uniphasic. Of all the ligands, only SKF38393 clearly demonstrates higher affinity for 3H-flupentixol labeled D-1 receptors.     

Behavioral recovery after irreversible inactivation of D-1 and D-2 dopamine receptors

Irreversible inactivation of both D-1 and D-2 dopamine (DA) receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in complete loss of stereotypy response to R-(-)-N-propylnorapomorphine (NPA; 0.1-1.0 mg/kg, s.c.) 24 hr later. Stereotyped sniffing recovered much more rapidly than oral behaviors. The D-2 antagonist sulpiride (200 mg/kg) and the putatively nonselective antagonist cis-flupenthixol (2 mg/kg), administered prior to EEDQ, prevented the loss of NPA-induced sniffing but only partially protected against loss of oral behaviors 24 hr later. Complete protection of both behaviors was seen after pretreatment with a combination of sulpiride and the selective D-1 antagonist SCH 23390 (1 mg/kg); pretreatment with the selective D-1 antagonist SCH 23390 alone, however, did not modify the rate of recovery of either behavioral response. The results suggest that either different populations of DA receptors mediate expression of these behaviors or stimulation of a small fraction of the total DA receptor pool may be sufficient to elicit sniffing but not oral responses. Furthermore, maintaining a normal complement of D-2 rather than D-1 receptors appears to be a critical determinant for the elicitation of these behaviors.     

Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist [3H]SK&F R-83566

The potent and D-1 versus D-2 selective dopamine receptor antagonist, SK&F R-83566, was radiolabelled with tritium and was used as a radioligand for examination of D-1 receptors in rat striatum. Binding of the radioligand was stereoselective, saturable and reversible. In homogenates of rat striatum, nonspecific binding of the radioligand was less than 5% of total binding, the KD was 1.1 +/- 0.2 nM and the Bmax was 1130 +/- 70 fmoles/mg protein. Results of competition binding analyses yielded a pharmacological profile that was characteristic of dopamine D-1 receptor interaction. Competition studies of dopamine agonists against the potent antagonist radioligand indicated multiple affinities of agonist binding to the D-1 receptor. Displacement was best fit to a two-site model of ligand binding and high and low affinities were subject to regulation by guanine, but not adenine, nucleotides. Antagonist binding was not complex and was unaffected by guanine nucleotides. The role of monovalent cations in regulating D-1 receptor binding was evaluated by comparing effects of Na+, Li+, and K+ on binding of the antagonist [3H]SK&F R-83566 and the agonist [3H]fenoldopam (SK&F 82526). Whereas agonist binding was reduced in a concentration dependent fashion by monovalent cations with a ranking of potency Li+ greater than Na+ greater than K+, antagonist binding was enhanced by the cation Na+ but little affected by Li+ or K+. This effect of relatively low concentrations of Na+ to decrease agonist binding and increase antagonist binding suggests similarities between the D-1 receptor which is positively-coupled to adenylate cyclase and other receptors, e.g. alpha 2 adrenergic receptors, which are negatively-coupled to adenylate cyclase.     

Prolactin increases the density of striatal dopamine receptors in normal and hypophysectomized male rats

Administration of prolactin to adult male rats, by s.c. injection, significantly increases the density of the striatal dopamine (DA) receptors, without altering the apparent affinity of the receptors for [³H]spiroperidol. Larger doses of prolactin are required to increase the density of the striatal DA receptors in hypophysectomized rates compared to normal rats. These results suggest that prolactin might be the common mediator of the increase in striatal DA receptor density produced by either estrogen or haloperidol administration. Monitoring and/or altering prolactin levels might be informative in neurologic or psychiatric disorders involving striatal DA neurotransmission.     

Chronic neuroleptic treatment specifically alters the number of dopamine receptors in rat brain

Trifluoperazine dihydrochloride (2.8–4.0 mg/kg/day) was administered continuously to rats in drinking water for six months. Animals killed at this time exhibited an increase in the number of dopamine receptors in the striatum and mesolimbic area, with a corresponding decrease in affinity (increase in the dissociation constant) for ³H-spiperone binding. In frontal cortex, ³H-spiperone binding to 5-HT receptors indicated no apparent change in numbers of receptors, but a slight increase in the dissociation constant. There was no obvious alteration in ³H-apomorphine binding in the striatum and mesolimbic area, but the individual results were very variable. The number and binding affinity of muscarinic receptors in striatum, mesolimbic area and cerebral cortex as identified by ³H-dexetemide were unchanged. Nor was there any alternation in the number or binding affinity of H-1 receptors identified by ³H-mepyramine, or of α-noradrenergic receptors identified by ³H-WB 4101, in cerebral cortex. The number and binding affinity of GABA receptors in the cerebellum identified by ³H-muscimol also was not altered.Chronic neuroleptic administration to rats appears to alter specifically the number of cerebral dopamine receptors.     

D-2 dopamine receptors in the frontal cortex of rat and human

D-2 dopamine receptors and serotonin receptors in the frontal cortex of rat and human were labelled with 3H-spiroperidol. The D-2 receptors were then distinguished in 4 ways. Dissociation of spiroperidol was biphasic, indicating two populations of sites. Cinanserin in competition with 3H-spiroperidol exhibited high (75%) and low (25%) affinity sites. Dopamine and LY 141865 in competition with 1.25 nM 3H-spiroperidol exhibited high (20-25%) and low (80-75%) affinity sites in the absence of cinanserin, while in the presence of 300 nM cinanserin only the high affinity sites remained. Lesioning of the dopaminergic meso-cortical pathway increased the number of cinanserin-resistant sites by 26%. Thus 3H-spiroperidol binding in the presence of cinanserin can be used to selectively label D-2 receptors in the frontal cortex.     

Evidence for dopamine D-2 receptors on cholinergic interneurons in the rat caudate-putamen

The aziridinium ion of ethylcholine (AF64A) is a neurotoxin that has demonstrated selectivity for cholinergic neurons. Unilateral stereotaxic injection of AF64A into the caudate-putamen of rats, resulted in a decrease in dopamine D-2 receptors as evidenced by a decrease in [3H]-sulpiride binding. Dopamine D-1 receptors, labeled with [3H]-SCH 23390, were unchanged. The efficacy of the lesion was demonstrated by the reduction of Na+-dependent high affinity choline uptake sites labeled with [3H]-hemicholinium-3. These data indicate that a population of D-2 receptors are postsynaptic on cholinergic interneurons within the striatum of rat brain.     

Solubilization of the D-1 dopamine receptor from rat striatum

The D-1 dopamine receptor was extracted from rat striatal membranes with 0.7% sodium cholate and 1 M NaCl. Pretreatment of the membranes with a D-1 specific agonist, inclusion of crude phospholipids in the solubilization buffer, and subsequent removal of the detergent led to a maximal extraction of 48% of the receptor binding sites. The D-1 antagonist, [125I]SCH 23982, bound to single class of sites with a Kd of 1.8 nM and a Bmax of 1.65 pmol/mg protein. The solubilized receptors retained the ability to discriminate between active and inactive enantiomers of agonists and antagonists selective for the D-1 receptor.     

Purification of bovine striatal dopamine D-2 receptor by affinity chromatography

Bovine striatal dopamine D-2 receptor has been purified approximately 2000-fold by affinity chromatography. The receptor, solubilized with cholic acid and sodium chloride, was adsorbed on haloperidol-linked Sepharose CL-6B and eluted with spiroperidol. The adsorption of receptor to the affinity matrix was biospecific as preincubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor. The process also displayed stereoselectivity with respect to (+)- and (-)-butaclamol. Nondopaminergic agents such as mianserin and propranolol failed to exhibit any effect on the adsorption process. Elution of the receptor was also biospecific, as dopaminergic drugs were most effective (spiroperidol greater than haloperidol greater than dopamine) in eluting the bound receptor; whereas other agents, e.g. propranolol, mianserin, and acetic acid, were only slightly effective. One-cycle affinity purification resulted in a recovery of 12% of the original membrane-bound dopamine D-2 receptor with a specific activity of 169,600 fmol/mg of protein as assayed with [3H]spiroperidol binding. The order of potency of D-2 agonists (N-propylnorapomorphine greater than NO434 greater than apomorphine greater than dopamine) and antagonists (spiroperidol greater than (+)-butaclamol greater than domperidone) with the purified preparation was found to be similar to that of the solubilized dopamine D-2 receptor.     

Evidence for the presence of both D-1 and D-2 dopamine receptors in human esophagus.

Clinical and pharmacological evidence suggested that dopamine is involved in the control of esophageal motility. The present study was designed to determine whether or not dopamine receptors are present in human esophagus. With this aim we measured adenylate cyclase activity as a biochemical index of dopamine receptor function in esophageal specimens taken from five patients during surgery for upper esophageal carcinoma. The selective D-1 agonist fenoldopam stimulated cAMP formation in the lower esophageal sphincter, but not in the esophageal body; this effect was prevented by the selective D-1 antagonist SCH 23390 and by d-butaclamol. Bromocriptine, a selective D-2 stimulator, inhibited adenylate cyclase activity in the lower esophageal sphincter, an effect blocked by the D-2 antagonist (-)sulpiride. No effects of bromocriptine were found in the esophageal body. These data indicate that both D-1 and D-2 receptors are present in the lower esophageal sphincter, but not in esophageal body and emphasize the role of dopamine in the regulation of esophageal function.