Spiroperidol Binding Research Articles

Neurotransmitter receptors in brain regions of acrylamide-treated rats. I: Effects of a single exposure to acrylamide

A single oral dose of acrylamide (25 or 50 mg/kg) increased the level of striatal 3H-spiroperidol binding in six week old male rats. This enhanced dopamine receptor activity was specific since treatment caused no significant changes in glycine, serotonin, and muscarinic cholinergic binding. At the highest acrylamide dose tested (100 mg/kg), elevations of the medullary glycine and frontal cortical serotonin receptors were also found. Pretreatment of animals with a blocker of hepatic mixed function oxidase (SKF 525a) or a thiol blocker (methylmercuric chloride) prevented the acrylamide-induced elevation of striatal spiroperidol binding, indicating that the causative agent was a secondary metabolite of acrylamide. Apomorphine-induced motility was significantly attenuated by 24 hr predosing with acrylamide, suggesting a change in the sensitivity of the dopamine receptor. The behavioral relevance of observed biochemical changes was thus shown by the altered response of treated animals to apomorphine.

The effects of acrylamide treatment upon the dopamine receptor

The binding of tritiated spiroperidol to striatal membranes prepared from acrylamide-treated male 6-week-old rats and matched controls has been studied. This binding has high-affinity characteristics and is stereospecific and reversible. Equilibrium was attained within 15 min at 37°C. The extent of binding was much more pronounced in the striatum than in any other brain region and was not detectable within the cerebellum or spinal cord. Regional distribution of binding and competition studies with other pharmacologic agents suggested a correspondence between the location of ligand-membrane complex formation and the dopamine receptor. Twenty-four hours after a single oral administration of acrylamide there was a significant increase in [ 3H]spiroperidol binding at all acrylamide doses tested (50, 100, and 200 mg/kg body weight). However, there was no significant change in striatal dopamine levels of treated animals. Kinetic analysis of animals treated with 100 mg acrylamide/kg suggested increased affinity of receptors of treated animals toward the labeled ligand. Receptor density was only slightly elevated in experimental animals. Effects were also studied in rats that had received 10, 20, or 30 mg/kg acrylamide daily for 10 days. Twenty-four hours after the last dose there was a major increase in spiroperidol binding in treated animals. Scatchard plot analysis again revealed that this change was largely attributable to a change in the dissociation constant of the binding interaction but also there was an increase in the overall number of receptor sites. Normal values were restored within 8 days after cessation of dosing. This illustrates that, under the conditions of this experiment, the effect of acrylamide on a central neurotransmitter system is reversible.

Effect of L-prolyl-L-leucyl-glycinamide (PLG) on neuroleptic-induced catalepsy and dopamine/neuroleptic receptor bindings

The mechanism of action subserving the potential anti-Parkinsonian properties of L-prolyl-L-leucyl-glycinamide (PLG) was investigated in behavioural and neurochemical models of dopaminergic function in the rat. Acute administration of PLG (20 and 40 mg kg-1 SC) failed to alter appreciably the intensity of the cataleptic response elicited by haloperidol (3 mg kg-1 IP). By contrast, chronic PLG treatment (20, 40 and 80 mg kg-1 SC twice daily for five days) significantly attenuated haloperidol-induced catalepsy. The effect of PLG on in vitro dopamine/neuroleptic receptor binding in rat striatum as differentially labelled by apomorphine and spiroperidol was also examined. PLG selectively enhanced the affinity of the specific binding of agonist [3H] apomorphine to dopamine receptors in the striatum, but had no effect on [3H] spiroperidol binding. The behavioural and biochemical results obtained in the present study raise the possibility that PLG may facilitate nigro-striatal dopaminergic neurotransmission through interacting with a unique PLG receptor functionally coupled to the dopamine receptor-adenylate cyclase complex.

Effects of antidepressant drugs on different receptors in the brain

Radioligand receptor binding techniques were used to characterize the effects of different structural types of antidepressant drugs on neurotransmitter receptors. The tricyclic antidepressants more or less potently inhibited the binding to rat brain preparations of several different radiolabelled ligands ([ 3H]WB4101, [ 3H]QNB, [ 3H]-d-LSD, [ 3H]mepyramine). The potency of the nontricyclic antidepressants varied greatly. Mianserin, potently displaced [ 3H]mepyramine, [ 3H]d-LSD and [ 3H]WB4101 while it was very weak on [ 3H]QNB-binding. Nomifensine and the specific 5-HT uptake inhibitors zimelidine and alaproclate had very low affinity for these receptors. All the antidepressants tested were practically devoid of activity on [ 3H]DHA binding, [ 3H]spiroperidol binding, [ 3H]flunitrazepam binding, [ 3H]muscimol binding and [ 3H]naloxone binding. The implications of these findings for biogenic amine theories of affective disorders are discussed.

Effect of acute and chronic tri-iodothyronine (T 3) administration to rats on central 5-HT and dopamine-mediated behavioural responses and related brain biochemistry

The effects of both acute and chronic T 3 administration (100 μg/kg, s.c.) on behavioural responses in the rat mediated by 5-hydroxytryptamine (5-HT) and dopamine (DA) were examined 24 hr after the last injection of hormone at a time when serum T3 levels had almost returned to control values. Concomitant changes in the biochemistry of the 5-HT and DA systems were also examined. Administration of T 3 produced complex changes in both central DA and 5-HT systems. Both acute and chronic T 3 treatment enhanced the hyperactivity response to tranylcypromine (TCP) plus L-DOPA, with chronic treatment giving a much greater enhancement. Paradoxically, the accumulation of brain DA at 20 and 30 min after L-DOPA in chronically treated rats was less than in the controls. In order to study possible T 3 effects at the DA postsynaptic receptor, behavioural responses to the DA agonist apomorphine were measured. The activity responses were slightly attenuated in chronically treated rats only. Furthermore, the cataleptogenic effect of haloperidol (0.3 mg/kg) was significantly potentiated in chronically T3-treated rats only. Because striatal [ 3H]-spiroperidol binding was unaltered in chronically treated rats, it is thought that T 3 may produce an altered sensitivity postsynaptic to striatal DA receptors, or a change in activity of a modulatory neuronal system. It was found that 24 hr after either acute or chronic T 3 injection, the hyperactivity response to TCP + L-tryptophan ( L-TP) was markedly enhanced [350% (chronic) and 220% (acute) increase above control activity]. A similar result was obtained for the 5-HT agonists (quipazine and 5-MeODMT). There was no change in whole brain 5-HT or TP accumulation measured 75 min following TP injection in the chronically-treated animals. This, together with enhanced 5-HT postsynaptic agonist responses, may be due to primary changes occurring at or beyond DA neurones, which appear to be interposed between 5-HT neurones and the site expressing the behavioural response.

Effects of acrylamide given during gestation on dopamine receptor binding in rat pups

Acrylamide (20 mg kg ) was administered by gavage to time-pregnant Fischer 344 rats daily from day 7 to day 16 of gestation. Acrylamide did not affect the number, size, or body weights of litters obtained but did decrease the [ 3H]spiroperidol binding in striatal membranes of 2-week-old pups. This effect could not be seen at 3 weeks of age. Scatchard analysis showed that acrylamide changed the affinity as well as the number of dopamine receptor sites. There were no signs of maternal toxicity in dosed mothers as evaluated by their body weights or general appearance.

3H]spiroperidol binding in normal and denervated carotid bodies

Specific dopamine receptors were studied in freshly dissected, unhomogenized rabbit carotid bodies incubated in [ 3H]spiroperidol. Total binding and non-specific binding were determined in the absence and presence of 0.2 μM (+)-butaclamol, respectively. Specific binding in normal carotid bodies incubated at near saturating concentrations (0.38 nM) was 1.63 ± 0.58 pmol/g of tissue. Chronic section of the carotid sinus nerve (14 days) resulted in a 64% reduction ( P < 0.05) in specific binding. We conclude that the majority of specific dopaminergic receptors are located on carotid sinus nerve afferent terminals.

Inhibition of neurotransmitter receptor binding by ergot derivatives.

Bromocriptine, lergotrile, lisuride, metergoline, and the Sandoz ergot derivatives 25-397, 29-712, and 29-717 have been tested for their ability to inhibit the synaptic receptor binding of spiroperidol, 5-hydroxytryptamine (5-HT), d-lysergic acid diethylamide (LSD), quinuclidinyl benzilate (QNB), WB.4101, and gamma-aminobutyric acid (GABA). Only GABA binding was not affected, and QNB binding was decreased only by lergotrile and metergoline at high concentrations. The most potent inhibitors of the other ligands were bromocriptine and lisuride for spiroperidol (1-2 nMM), metergoline for 5-HT (29 nM), lisuride for LSD (15 nM), and lergotrile for WB.4101 (17 nM). The direct receptor effects of the ergot derivatives in vitro may contribute to understanding their in vivo effects on behavior and in predicting their therapeutic potential in neurological and neuroendocrine disorders.

Sodium-dependent interaction of benzamides with dopamine receptors in rat and dog anterior pituitary glands.

Sulpiride and other benzamides' displacement of [3H]-spiroperidol binding to rat and dog anterior pituitary dopaminergic receptors was found to be selectively sodium dependent, while typical neuroleptic activity was not influenced by NaCl. These results indicate the existence in the anterior pituitary of a subpopulation of dopaminergic receptors with which benzamides interact.

Brain locations controlling the behavioral effects of chronic amphetamine intoxication.

Rats were administered D-amphetamine repeatedly for 4 days. After day 1 of treatment, the amphetamine-induced increases in ambulation, rearing, and stereotyped activity were augmented. However, after 4 days treatment, the rearing and ambulatory responses became attenuated while the stereotyped activities remained augmented. Micro-injection studies revealed that both the augmentation and attenuation of nonstereotyped ambulation were generated from the nucleus accumbens. The augmentation of stereotyped behaviors was generated from the caudate nucleus. Chronically treated animals who were administered 0.7 but not 1.0 mg/kg apomorphine showed augmented behavioral response. Chronic amphetamine treatment significantly decreased (3H) spiroperidol binding in both the nucleus accumbens and caudate nucleus. However, no effect on the DA-stimulated adenyl cyclase activity was observed in either brain region. It is concluded that repeated D-amphetamine administration selectively augments and attenuates D-amphetamine-induced behaviors and that these selective effects are mediated by different dopamine systems.

Aging process affects a single class of dopamine receptors

[ 3HSpiroperidol and [ 3H](−)-sulpiride specific binding have been used to assay for D 1 and D 2 dopaminergic recognition sites in striatal membranes of aged rats. While [ 3H]spiroperidol binding shows a decreased number of binding sites, no changes have been detected in [ 3H](−)-sulpiride binding, which is a marker for D 2 dopaminergic receptors. Data obtained with GTP and DA-dependent adenylyl cyclase activity confirm the hypothesis that aging selectively affects in rats those dopaminergic receptors coupled to the formation of cyclic AMP (D 1).

3H]Spiroperidol binding in rat striatum: two high-affinity sites of differing selectivities.

[3H]Spiroperidol binding to homogenates of rat striatum is saturable and shows either monophasic or biphasic saturation isotherms under specified conditions. In poorly washed membrane fragment preparations, saturation isotherms of [3H]spiroperidol binding are monophasic, revealing an apparently homogeneous set of sites with KD 0.6 +/- 0.3 nM and density 440 +/- 80 fmol/mg protein. However, equilibrium displacement studies of [3H]spiroperidol binding at this site indicate an alpha-adrenergic component in addition to the previously described dopaminergic component. In thoroughly washed membrane fragment preparations, saturation isotherms are clearly biphasic, showing an additional high-affinity site with an approximate KD of 24 +/- 10 pM and an approximate density of 110 +/- 20 fmol/mg protein at a protein concentration of 2.0 mg/ml. Selectivity at this site appears classically dopaminergic, suggesting that the lower affinity site is the primary source of the alpha-adrenergic component of spiroperidol binding.

Decrease in lymphocyte [ 3H]spiroperidol binding sites in parkinsonism

[ 3H]spiroperidol binding has been measured in lymphocytes from patients with Parkinson's disease and age matched healthy volunteers. A dramatic decrease (73%) in the number of binding sites ( Bmax) without any variation of the affinity ( KD) has been observed in Parkinsonian patients. This decrease in Bmax is linearly correlated with the degree of disability of the Parkinsonian patients (r = 0.891, p <0.001). This decrease appeared to be relatively selective since no variation was observed with patients suffering of other neurological disorders (vascular hemiplegia, Alzeihmer's disease, olivopontocerebellar degeneration, Huntington's chorea).

Increased renal neuroleptic binding in spontaneously hypertensive rats

The present experiments demonstrate high affinity stereospecific binding of the dopaminergic ligand [ 3H]spiroperidol to rat membranes. The binding was saturable, regionally distrubuted, and showed the same specificity as the caudate [ 3H]spiroperidol site for displacement by dopaminergic antagonists. Since there is an association between hypertension and abnormalities of renal blood flow, which appears to be regulated in part by dopamine, [ 3H]spiroperidol binding was measured in kidneys of spontaneously hypertensive rats. In two independent experiments renal cortical [ 3H]spiroperidol stereospecific binding was increased in spontaneously hypertensive rats.

Inhibition of [3H]spiroperidol binding by in vitro addition of ethanol.

The in vitro addition of ethanol at concentrations greater than 0.2% produced an inhibition of [3H]spiroperidol binding. This inhibition was competitive, lowering the affinity without altering the maximum number of sites. Both the rates of association and dissociation were altered such that the ligand spent less time attached to the receptor. Since the binding was competitive, little inhibition was seen at ligand concentrations greater than 300 pM. These results are important for in vitro studies when drugs are dissolved in ethanol, and also for in vivo studies on the effects of ethanol.

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Dithiothreitol (DTT), a disulfide reducing agent, diminished the specific binding of [ 3H] dopamine to partially purified calf striatal membranes (P 2) but did not have an effect on [ 3H] spiroperidol binding. The thiol reagents, p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM) and iodoacetamide (IA), were also tested for inhibitory effects on agonist and antagonist binding to the dopamine receptor. PCMB inhibited both [ 3H] dopamine and [ 3H] spiroperidol binding by changing the affinity (K d) and the number of binding sites (B max) for both of these ligands. This effect of PCMB was reversed by the addition of DTT. NEM inhibited binding to the dopamine agonist site but not to the antagonist site, while IA was ineffective on either site. These results indicate that a DTT-reducible disulfide bond may be an essential component for agonist binding to the dopamine receptor. Furthermore, the experiments with PCMB, NEM and IA suggest that the exposure of thiol groups in the dopamine receptor may play an important role in agonist and antagonist binding.

Sodium-dependent interaction of benzamides with dopamine receptors

Sulpiride and other benzamide (BM)-displacing activity on [ 3H]-spiroperidol ([ 3H]SPIR) binding by rat striatal dopaminergic receptors was found to be uniquely sodium-dependent, while classical neuroleptic (NL) activity was not influenced by NaCl. These results suggest the existence of at least two populations of striatal dopaminergic receptors, sodium-dependent and sodium-independent, through which BM and NL respectively interact.

Lack of effect of repeated electroconvulsive shock on [3H]spiroperidol and [2H]5-hydroxytryptamine binding and cholinergic parameters in rat brain.

Repeated electroconvulsive shock (ECS) administered on alternate days for 10 days produced no changes in rat striatal [3H]spiroperidol binding measured 24 h after the last shock compared to anaesthetised controls. Similarly, there was no change in while brain specific [3H]5-HT binding. Sodium-dependent high affinity [3H]choline uptake (HAUC) and ChAT were also unaltered in striatal and hippocampal samples following repeated ECS. Acute administration of Pentylenetetrazol did produce an increase in hippocampal HAUC immediately postictally. However, ECS (Xl) did not change HAUC measured 1 h postictally. An effect of halothane on HAUC was noted in these experiments indicating the importance of an evaluation of anaesthetic effects in ECS studies.

Properties of dopamine agonist and antagonist binding sites in mammalian retina

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.

Decreased spiroperidol and LSD binding in rat brain after continuous amphetamine

Rats were implanted with a silicone tubing pellet continuously releasing amphetamine base for several days. After five days of this treatment specific binding of spiroperidol (dopamine receptors) and LSD (serotonin receptors) was decreased in the corpus striatum and frontal cortex. In the striatum the number of dopamine receptors was decreased while the affinity was unchanged. These results indicate that dopamine and serotonin receptors can be regulated by the release of their own neurotransmitter.