Spiroperidol Binding Sites Research Articles

3H]spiroperidol binding in crude membrane suspensions of gill tissue from the marine mussel, Mytilus californianus

1. Saturable, high-affinity binding sites for [ 3H]spiroperidol can be demonstrated in crude suspensions of mussel gill tissue. 2. This binding shows stereospecificity toward the d- and l-isomers of butaclamol. 3. Competition studies show that both dopamine and serotonin can displace [ 3H]spiroperidol from binding sites at nanomolar concentrations. 4. Evidence is presented that suggests that the [ 3H]spiroperidol-binding sites can be divided into two distinct groups: those with high affinity for dopamine and those with high affinity for serotonin.

Effect of chronic phencyclidine or d-amphetamine treatment on [ 3H]spiroperidol binding in rat caudate-putamen and nucleus accumbens

Chronic treatment (20 days) of rats with d-amphetamine decreased the density, but had no effect on the apparent affinity of [ 3H]spiroperidol binding sites in caudate-putamen and nucleus accumbens. In contrast, chronic phenycyclidine treatment had no effect either on the number or on the affinity of [ 3H]spiroperidol binding sites in these two brain regions.

Dopamine receptors in the striatum and limbic system of various strains of mice: relation to differences in responses to apomorphine.

Dopamine receptors, defined as [3H]spiroperidol binding sites, had similar population parameters in the limbic forebrain of C57BL/6, Albino Swiss and DBA/2 mice, but the parameters of the striatal populations were different: not only the densities differed among themselves, but the KD value of the striatal dopamine receptors of DBA/2 mice was significantly higher than that in the two remaining strains. Behavioral responses of Albino Swiss mice to apomorphine: biphasic effect of apomorphine on locomotor activity and stereotypy characterized by high motility, frequent rearing and sharp, not very frequent bites, were similar to those described earlier for C57BL/6 mice, and differed from those reported for DBA/2 mice. The results suggest that the difference in responding to apomorphine in various strains of mice may be related to differences in their striatal dopamine receptors.

Effect of right middle cerebral artery occlusion on striatal dopaminergic function.

Following right middle cerebral artery occlusion in the rat, striatal dopaminergic system alterations were studied. Dopamine turnover was assessed by measuring 3,4-dihydroxyphenylacetic acid concentrations and dopamine receptor function, by measuring (3H)-Spiroperidol binding. There was a transient decrease in 3,4-dihydroxyphenylacetic acid and permanent damage to dopamine receptors, as indicated by a time-dependent progressive reduction in the number of (3H)-Spiroperidol binding sites. The receptor deficit also manifested as turning behaviour towards the lesioned side 4 weeks after the lesion following subcutaneous apomorphine. Long-term changes of dopaminergic receptor activity in this experimental model of cerebral infarction may be secondary to cortical degeneration following middle cerebral artery occlusion.

Sodium ion modulates D2 receptor characteristics of dopamine agonist and antagonist binding sites in striatum and retina.

Sodium ion (Na(+)) influences binding of both dopamine agonists and antagonists to D(2) receptors in striatum and retina. Also, Na(+) markedly potentiates the loss of high-affinity agonist binding due to the GTP analogue p[NH]ppG. 2-Amino-6, 7-dihydroxy-1,2,3,4-tetrahydro[5,8-(3)H]naphthalene ([(3)H]ADTN) binds exclusively to an agonist conformation of D(2) receptor in both striatum and retina, distinct from the antagonist conformation labeled by [(3)H]spiroperidol or [(3)H]domperidone in striatum or by [(3)H]spiroperidol in retina. Na(+) is not required for interaction of [(3)H]ADTN or antagonist radioligand sites with the selective D(2) agonist LY-141865, the D(2) antagonist domperidone, or nonselective dopamine agonists or antagonists; however, Na(+) is necessary for high affinity interaction of those radioligand sites with the D(2) antagonists molindone and metoclopramide. With Na(+) present, striatal sites for [(3)H]ADTN, [(3)H]spiroperidol, and [(3)H]domperidone have similar affinities for antagonists but only [(3)H]ADTN sites have high affinity for agonists. Na(+) further decreases the low affinity of dopamine agonists for [(3)H]spiroperidol binding sites. Also, Na(+) enhances [(3)H]spiroperidol and decreases [(3)H]ADTN binding. Na(+) alone causes bound [(3)H]ADTN to dissociate from at least 30% of striatal and 50% of retinal sites, and with Na(+) present [(3)H]ADTN rapidly dissociates from the remaining sites upon addition of p[NH]ppG. It is proposed that D(2) receptors in striatum and retina exist in distinct but interconvertible conformational states, with different properties depending on the presence or absence of Na(+) and of guanine nucleotide.

Affinity of 10-(4-methylpiperazino)dibenz[b,f]oxepins for clozapine and spiroperidol binding sites in rat brain

10-(4-Methylpiperazino)dibenz[b,f]oxepins were prepared and evaluated as potential antipsychotic agents using specific clozapine [8-chloro-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]diazepine] binding sites in rat forebrain that are noncholinergic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. [3H]Clozapine binding in the presence of atropine represents nonmuscarinic binding, while binding in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity for dopamine binding sites was determined by displacement of [3H]spiroperidol from binding sites in rat caudate nuclei. Thus, clozapine, its 2-chloro isomer, its dechloro analogue, and their 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepine analogues have about the same relative affinity for the nonmuscarinic clozapine binding sites. At the spiroperidol (dopaminergic) sites, both the nature of the tricyclic system and the presence of a chlorine atom on the tricyclic system have a substantial effect on the binding affinity. Within each series, shift or a chlorine atom from the position distal to the piperazino group to the proximal position increases the binding affinity by a factor of about nine, but removal of the chlorine atom substantially decreases the binding affinity. Nevertheless, 10-(4-methylpiperazino)dibenz[b,f]oxepin has a threefold greater affinity for the dopaminergic binding sites than does clozapine itself.

Modification of morphine-induced change in striatal ( 3H)-spiroperidol binding and stereotype behavior by cyclo(Leu-Gly)

Recent reports from our laboratories have indicated that the peptide cyclo(Leu-Gly), an analog of MIF (Pro-Leu-Gly-NH 2), administered prior to chronic exposure to morphine, prevents the development of both analgesic tolerance and some signs of physical dependence. The same peptide treatment also prevented the development of morphine-induced increases in certain behavioral responses to the dopamine agonist apomorphine. The present study investigated behavioral (stereotypy) and neurochemical receptor changes (specific ( 3H)-spiroperidol binding) occuring in the rat striatal dopamine (DA) system following chronic morphine treatment with and without prior cyclo(Leu-Gly)administration. While chronic morphine treatment (s.c. 5 pellet implant for 3 days, each pellet contained 65 mg morphine free base) did not alter the total number of high-affinity striatal ( 3H)-spiroperidol binding sites (28 fmol/mg tissue), it did increase the affinity of the receptor for the ligand (K D decreased from 40 to 24 pM). Cyclo(Leu-Gly) (8 mg/kg) prevented the morphine induced increase in dopamine receptor affinity. In parallel, cyclo(Leu-Gly) prevented the increase in apomorphine-induced stereotypy which was observed in chronic morphine treated rats. The peptide alone did not alter any of the binding characteristics. These data suggest that the ability of the peptide to block the development of physical dependence induced by morphine may involve the ability of the peptide to interfere with morphine-induced changes in dopaminergic systems.

Inhibitory effects of ascorbic acid on the binding of [3H]dopamine antagonists to neostriatal membrane preparations: relationship to lipid peroxidation.

Ascorbic acid, sodium ascorbate, and isoascorbic acid (the stereoisomer of ascorbic acid) inhibited the stereospecific binding of [3H]spiroperidol to neostriatal membrane preparations. Greater inhibitory effects were obtained at intermediate concentrations of the three ascorbic acid analogs (i.e., 0.06 and 0.6 mM) than at higher (6 mM) or lower (0.006 mM) concentrations. In parallel experiments, the three ascorbic acid analogs induced lipid peroxidation, which was also greater at the two intermediate than at higher or lower concentrations. Several known inhibitors of lipid peroxidation, including propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, alpha-naphthol, and cobalt chloride, as well as the iron chelating agents EDTA and DETAPAC (diethylenetriaminepentaacetic acid) were able to counteract the effects of the ascorbic acid analogs on both lipid peroxidation and on [3H]spiroperidol binding. These data strongly suggest that an iron-catalyzed lipid peroxidation is responsible for the observed inhibitory effects on binding. In other experiments, neostriatal membrane preparations that were preincubated with ascorbic acid (0.6 mM) and subsequently washed still had greatly diminished capacity to bind [3H]spiroperidol, indicating that ascorbic acid need not be physically present during the binding assay in order to affect binding. This experimental procedure also appears to be a way in which [3H]spiroperidol binding sites can be inactivated and washed free of the inactivating agent.

Differential affinities of molindone, metoclopramide and domperidone for classes of [ 3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors

Rat striatum contains two populations of dopaminergic [ 3H]spiroperidol binding sites. The two populations are similar in their affinities for chloropromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D 2 antagonists molindone, metochlopramide and domperidone. Hence, this population may represent D 2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D 2 receptor sites.

Effects of chronic lithium treatment on dopamine receptors in the rat corpus striatum. II. No effect on denervation or neuroleptic-induced supersensitivity

The influence of chronic dietary lithium administration was evaluated on dopamine receptor supersensitivity in the rat corpus striatum. Supersensitivity was induced with either unilateral destruction of dopamine-containing fibers in the nigrostriatal pathway or with 3 weeks of treatment with haloperidol (HAL). Both treatments elevated [ 3H]spiroperidol binding sites, but in neither case was this increase in ligand binding affected by chronic dietary Li (brain levels 0.8 to 1.2 mEq/l tissue). Our rats receiving 21 daily injections of HAL did show a behavioral supersensitivity to the dopamine agonist, apomorphine, and this effect was attenuated by concurrent treatment with dietary Li (accompanying paper). However, in contrast to previous data 20, this behavioral attenuation could not be linked to the prevention of increased [ 3H]spiroperidol binding in the corpus striatum. Furthermore, co-administration of dietary Li to subjects injected with HAL for 3 weeks did not reverse the increased density of [ 3H]spiroperidol binding sites which developed in the corpus striatum. Neither HAL nor Li treatment altered the affinity of the radioligand for its binding site. In the same animals, neostriatal dopamine-sensitive adenylate cyclase was not affected by either long-term dietary Li or chronic neuroleptic treatment, supporting the view that membrane antagonist and agonist sites differentially adapt to chronic alterations of synaptic input. Taken together, the results are incompatible with the hypothesis that the anti-manic action of Li is related to its ability to prevent dopamine receptor supersensitivity.

Synthesis of clozapine analogues and their affinity for clozapine and spiroperidol binding sites in rat brain.

Analogues of clozapine, some prepared by a novel, shorter synthesis than those described previously, were evaluated as potential antipsychotic agents using clozapine binding sites in rat forebrain that are nonmuscarinic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. The binding of clozapine to muscarinic sites is inhibited in the presence of atropine. Displacement of [3H]clozapine by an analogue of clozapine in the presence of atropine represents nonmuscarinic binding, while displacement in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity of the analogues for dopamine binding sites was determined by their ability to displace [3H]spiroperidol from binding sites in rat caudate nuclei. To the extent which binding affinity for nonmuscarinic clozapine sites in rat forebrain reflects the antipsychotic potential of a particular drug, dibenzo-5H-cycloheptene analogues of clozapine are as effective as clozapine itself. Strong binding to nonmuscarinic clozapine sites is not dependent on the presence of a chlorine atom on th tricyclic system. One or both of the nitrogen atoms in the dibenzo-5H-[1,4]diazepine ring of clozapine appear to be necessary for the strong inhibition of clozapine binding to spiroperidol sites in rat caudate nuclei. Anticholinergic activity is substantially higher for clozapine and its dibenz[1,4]oxazepine analogue than for its benzo-5H-cycloheptene analogue.

Rat dopaminergic function in the retina during aging

Parameters of dopaminergic transmission were studied in the retina of mature (3–4 months) and aged (23–24 months) rats. In the retina of senescent rats we found significantly higher dihydroxyphenylacetic acid (DOPAC) levels and a higher number of ( 3H)-spiroperidol binding sites. We detected also an increase of ( 3H)-methionine-enkephalin binding sites. The changes in the density of ( 3H)-spiroperidol and ( 3H)-Metenkephalin binding sites in the retina are opposite to those observed in the brain of aged rats.

3H]Dihydroergocryptine binding to bovine striatal membranes defined by a low d-butaclamol concentration : Antagonism by substituted benzamides

An apparent dopamine-D2 receptor binding system was characterized using [ 3H]dihydroergocryptine (DHE) as the ligand. Specific binding to bovine striatal membranes was defined as the difference between binding in the absence and in the presence of 4 nM d-butaclamol. These high affinity binding sites ( K d = 0.24 nM) were saturable ( B max = 75 fmoles/mg protein), apparently homogeneous, non-interacting, and stereospecific. Specific binding was greatest in areas rich in dopaminergic innervation. In competition experiments, apomorphine ( ic 50 = 100 nM) was fifty times more potent than dopamine which, in turn, was nine times more potent than l-norepinephrine and serotonin. Antipsychotic drugs including the substituted benzamides—metoclopramide, sulpiride and sultopride—were active in competing with [ 3H]DHE for these binding sites ( ic 50 = 100 nM ), and their affinities agreed well with their anti-dopaminergic potencies in vivo. Potencies of antipsychotic drugs in competing for DHE binding sites were increased compared to their potencies in competing for [ 3H]spiroperidol binding sites using 1 μM d-butaclamol as the masking ligand. This relative increase was about 10-fold greater for the substituted benzamides than for other antipsychotic drugs. The relative inactivity of substituted benzamides in competing for binding sites labeled by spiroperidol was accounted for by their greater selectivity for dopamine receptors. In binding studies using a non-selective radio-ligand, selectivity could be enhanced by defining specific binding sites with a low concentration of masking ligand.

The [ 3H]5-HT and [ 3H]spiroperidol binding sites in the rat frontal cortex are thermally inactivated at different rates

The rate of thermal inactivation of [ 3H]5-HT, [ 3H]spiroperidol and [ 3H]LSD binding sites in a membrane preparation from the rat frontal cortex has been studied. The binding sites were inactivated as different rates and the results are interpreted as supporting the claim that [ 3H]5-HT and [ 3H]spiroperidol bind to different sites within the frontal cortex of the rat.

Central dopaminergic neurons during development of genetic and doca-salt hypertension in the rat

The in vivo binding of [ 3H]spiroperidol was measured in discrete areas of the brain in 7-, 9- and 16-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) controls. An increase in the [ 3H]spiroperidol binding in the striatum, tuberculum olfactorium and frontal cortex but not in the cerebellum was detected at all ages in SHR. The increase was more pronounced in 7- than in 9- or 16-week-old SHR. In vitro data indicated an increase in B max but no variation in K d in the striatum of 7-week-old SHR. Moreover no difference was detectable in the dopaminergic cell bodies (A9, A10). This increase was specific to [ 3H]spiroperidol binding sites since no difference was observed in the in vivo binding of [ 3H]QNB and [ 3H]LSD in the same brain regions. No variation in dopamine level or dopamine utilization, as estimated by measuring the disappearance of the amine induced by a-methyl- p-tyrosine, was observed. The DOPA accumulation after injection of the DOPA decarboxylase inhibitor NSD 1015 was greater in the tuberculum olfactorium from 7-week-old SHR. An increase in [ 3H]spiroperidol binding sites was also observed in the striatum and tuberculum olfactorium after 7 weeks of DOCA-salt treatment. These results suggest that dopaminergic neurons might be implicated in the onset of hypertension in the rat.

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).

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.

Evidence for loss of brain [ 3H]spiroperidol and [ 3H]ADTN binding sites in rabbit brain with aging

[3H]Spiroperidol and [3H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [3H]spiroperidol binding sites and a 140% higher number of [3H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [3H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [3H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [3H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.

Identification of stereospecific [ 3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [ 3H]spiroperidol. The specific binding is saturable with two components (K D1 = 1.9 nM, K D2 = 36.2 nM). Determination of the K D by kinetic studies measuring rate constants for association and dissociation provided K D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [ 3H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (−)butaclamol. The relative potencies of different antidopaminergic agents in competing for [ 3H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.

3H]-Spiroperidol labels dopamine receptors in membranes from rabbit mesenteric artery.

The potent dopamine receptor antagonist [3H]-spiroperidol was used to label binding sites in a membrane fraction derived from rabbit mesenteric artery which had characteristics expected for dopamine receptors. The binding was of high affinity with an equilibrium dissociation constant (KD) of 13.1 nM; it was saturable with 110 fmol of [3H]-spiroperidol bound/mg protein at maximal occupancy of the sites. Binding at 37 degrees C was rapid and readily reversible with rate constants of 0.0154nM-1 min-1 and 0.114min-1 for forward and reverse reaction, respectively. Dopamine receptor antagonists were about 100--200 times more potent than alpha-adrenolytic drugs in competing for the [3H]-spiroperidol binding sites and dopamine was much more potent than (-)-noradrenaline, (-)-isoprenaline, clonidine or serotonin. It is concluded that in a membrane fraction of the rabbit mesenteric artery there exist binding sites for [eH]-spiroperidol indistinguishable from dopamine receptors. Thus the present results support the view that in vascular smooth muscle there exist specific dopamine receptors.