Characterization of [3H]pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both [3H]quinuclidinyl benzilate [( 3H]QNB) and [3H]pirenzepine [( 3H]PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound [3H]QNB binding sites. The Kd values for [3H]QNB and [3H]PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, [3H]PZ appeared to label fewer solubilized binding sites than did [3H]QNB. Maximum binding values for [3H]PZ and [3H]QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of [3H]QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for [3H]PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of [3H]QNB and [3H]PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for [3H]QNB and [3H]PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.     

Comparison of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain

The properties of [3H]quinuclidinylbenzilate ( [3H]QNB) binding and [3H]pirenzepine ( [3H]PZ) binding to various regions of rat brain were compared. [3H]PZ appeared to bind with high affinity to a single site, with a Kd value of approximately 15 nM in the cerebral cortex. The rank order of potencies of muscarinic drugs to inhibit binding of either [3H]QNB or [3H]PZ was QNB greater than atropine = scopolamine greater than pirenzepine greater than oxotremorine greater than bethanechol. Muscarinic antagonists (except PZ) inhibited both [3H]PZ and [3H]QNB binding with Hill coefficients of approximately 1. PZ inhibited [3H]QNB binding in cortex with a Hill coefficient of 0.7, but inhibited [3H]PZ binding with a Hill coefficient of 1.0. Hill coefficients for agonists were less than 1. The density of [3H]PZ binding sites was approximately half the density of [3H]QNB binding sites in cortex, striatum and hippocampus. In pons-medulla and cerebellum, the densities of [3H]PZ binding sites were 20 and 0%, respectively, relative to the densities of [3H]QNB binding sites. When unlabeled PZ was used to compete for [3H]QNB binding, the relative number of high-affinity PZ binding sites in cortex, pons and cerebellum agreed with the relative number of [3H]PZ binding sites in those regions. The binding of [3H]PZ and [3H]QNB was nonadditive in cortex. GTP inhibited high-affinity oxotremorine binding, but not PZ binding. Together, these data suggest that [3H]PZ binds to a subset of [3H]QNB binding sites. Whether this subset reflects the existence of subtypes of muscarinic receptors or is a consequence of coupling to another membrane protein remains to be seen.     

The interaction of amitriptyline, doxepin, imipramine and their N-methyl quaternary ammonium derivatives with subtypes of muscarinic receptors in brain and heart

The interaction of amitriptyline, doxepin, imipramine and their N-methyl quaternary derivatives with muscarinic receptors was investigated in the brain and heart. The potency of the tricyclic derivatives for inhibiting the binding of 11[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b] [1,4] benzodiazepine-6-one to M2 muscarinic receptors in cerebral cortex was similar to that measured in competitive binding experiments with the nonselective muscarinic antagonist [3H]N-methylscopolamine in the corpus striatum and heart. Moreover, the tricyclic derivatives antagonized muscarinic receptor-mediated inhibition of adenylate cyclase activity with similar potency in the corpus striatum and heart, and there was good agreement between the affinities of the tricyclic derivatives when measured by radioligand binding and by antagonism of the adenylate cyclase response. Our results show that amitriptyline, doxepin and imipramine lack selectivity for subtypes of the muscarinic receptor.     

Differential ontogeny of putative M1 and M2 muscarinic receptor binding sites in the murine cerebral cortex and heart

Studies with [3H]pirenzepine [( 3H]PZ) suggest that this nonclassical muscarinic antagonist selectively identifies putative M1 muscarinic receptors. We now compare the ontogeny of these putative M1 sites, identified by high-affinity [3H]PZ binding, with sites identified by the classical antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) in murine cerebral cortical and cardiac homogenates. Dissociation constants (Kd) for [3H]PZ (2.1-6 nM in the cortex and 2.0-21 nM in the heart) and for (-)-[3H]QNB (10-28 pM in the cortex and 10-39 pM in the heart) are similar in adult and neonatal tissues, whereas receptor density (maximum binding, femtomoles per milligram of protein) varies significantly. Cerebral cortical [3H]PZ binding rises from 14% at birth, to 88% of adult levels by day 14, peaks at 128% at day 28 and falls to the mean adult level of 606 fmol/mg of protein. Cerebral cortical (-)-[3H]QNB binding parallels [3H]PZ binding. Conversely, parallel studies show cardiac (-)-[3H]QNB density is 3- to 17-fold greater than the comparable density of high-affinity [3H]PZ binding sites throughout ontogeny. We conclude that: 1) the high ratio of [3H]PZ binding to (-)-[3H]QNB binding identifies the murine cerebral cortex as a tissue which contains predominantly putative M1 muscarinic binding sites; 2) the relatively low ratio of [3H]PZ binding to (-)-[3H]QNB binding throughout ontogeny identifies the murine heart as a tissue which contains primarily the putative M2 muscarinic binding site; and 3) M1 and M2 receptor binding sites show distinct developmental curves in the cerebral cortex and heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of subtypes of the muscarinic receptor to adenylate cyclase in the corpus striatum and heart

The binding properties of a series of muscarinic antagonists were compared with their ability to antagonize muscarinic receptor mediated inhibition of adenylate cyclase activity in homogenates of the corpus striatum and heart of rats. When measured by the competitive inhibition of the binding of the muscarinic antagonist N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists in the corpus stratum and cerebral cortex were consistent with a model incorporating a minimum of three populations of muscarinic receptors, a high affinity site for pirenzepine (M1), a high affinity site for AF-DX 116 [11] [2-[ (diethylamino)methyl]-1-piperidinyl] acetyl] -5, 11-dihydro-6H-pyrido [2,3-b] 1,4] benzodiazepine-6-one (M2) and a third population (non-Ml, non-M2 sites) displaying low affinity for the latter antagonists. The results of similar experiments on the heart showed that this tissue contained a uniform population of M2 muscarinic receptors. The binding properties of the M2 receptor in cerebral cortex and corpus stratum were also investigated directly in antagonist [3H] AF-DX 116 competition experiments and, although the high affinity AF-DX 116 site in brain (M2) exhibited selectivity for the cardioselective antagonists AF-DX 116 and gallamine, some differences were noted between M2 sites in brain and heart. The muscarinic adenylate cyclase response in the corpus striatum was relatively insensitive to the M2 selective antagonists AF-DX 116 and gallamine as well as the M1 selective antagonist pirenzepine, suggesting that non-M1, non-M2 sites inhibit adenylate cyclase activity in the corpus striatum. In contrast, the effects of muscarinic antagonists on the muscarinic adenylate cyclase response in the heart were consistent with the postulate that M2 receptors inhibit adenylate cyclase activity in this tissue.     

A cholinergic antagonist identifies a subclass of muscarinic receptors in isolated rat pancreatic acini

Atropine, pirenzepine (PZ) and the novel antimuscarinic drug [11- [[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) were used to subclassify the pancreatic muscarinic receptor by correlating their effects on carbachol-mediated amylase release with their actions on the binding of [3H]N-methylscopolamine in rat pancreatic acini. Maximal stimulation of amylase release occurred at 3 microM carbachol. Atropine, PZ and AF-DX 116 inhibited carbachol-mediated amylase release with the following pA2 values: atropine = 9.1, PZ = 6.5 and AF-DX 116 = 5.7. There was parallel inhibition of [3H]N-methylscopolamine binding, with the following inhibition constants: atropine = 2.38 nM, PZ = 426 nM and AF-DX 116 = 3660 nM. Using the same animals, these compounds inhibited [3H]N-methylscopolamine binding in homogenates from both cerebral cortex and heart. The order of potency was the same in the cerebral cortex as in the pancreas: atropine = 0.67 nM, PZ = 85 nM and AF-DX 116 = 440 nM. However, in the cortex, the binding data with PZ also exhibited a high-affinity site with a KH value of 11 nM. In the heart, the order of potency was shifted to atropine greater than AF-DX 116 greater than PZ, with inhibition constants of 1.55, 12 and 110 nM, respectively. Thus, the muscarinic receptors in the pancreas and the heart exhibited the characteristics of the putative M2 receptor subtype, having lower affinities for PZ than the muscarinic receptors in the cerebral cortex. However, the heart had a significantly higher affinity for AF-DX 116.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative antidopaminergic properties of thioridazine, mesoridazine and sulforidazine on the corpus striatum

The functional antidopaminergic potencies of the atypical antipsychotic drug thioridazine (THD), as well as its active metabolites mesoridazine (MES) and sulforidazine (SUL), were assessed by testing their blockade of the inhibitory effects of endogenous dopamine (DA) or apomorphine on the electrically evoked release of radiolabeled DA and acetylcholine (ACh) from perfused rabbit striatal slices. These functional comparisons (reflecting presynaptic and postsynaptic DA receptors, respectively) were correlated with potency estimations of these drugs in competing for D2 DA receptors (i.e., [3H]spiperone binding sites) in rabbit striatal homogenates. Similar orders of potency (SUL greater than MES much greater than THD) were found for blockade of pre- and postsynaptic DA receptors modulating DA and ACh release, respectively, as well as in competing for [3H]spiperone binding sites in the striatum. MES, SUL and haloperidol were 2 to 3 times more potent at DA release modulatory receptors than postsynaptic DA receptors. In contrast, THD was 8 times more potent at antagonizing the apomorphine-induced inhibition of DA release than against apomorphine's effect on ACh release. THD was virtually inactive in antagonizing the inhibition of ACh release induced when nomifensine was used to increase endogenous synaptic DA, despite significantly antagonizing these effects on DA release in the same slices. Together, these data indicate that: 1) MES and SUL are responsible for a significant part of the antidopaminergic effects attributed to THD; 2) THD should produce less cholinergic activation than other neuroleptics; and 3) that nonreceptor-mediated effects at high THD concentrations may mask effects due to receptor blockade.     

Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [3H]quinuclidinyl benzilate or [3H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [3H]quinuclidinyl benzilate in a biphasic manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of high affinity [3H]pirenzepine and (-)-[3H] quinuclidinyl benzilate binding to muscarinic cholinergic receptors in rabbit peripheral lung

We have characterized the binding of the selective muscarinic antagonist [3H]pirenzepine ([3H])PZ) and the classical muscarinic antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) to muscarinic cholinergic sites in rabbit peripheral lung membranes. For both radioligands, high affinity binding with pharmacologic specificity was demonstrated. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 4.5 nM and the Kd for (-)-[3H]QNB binding was 6.2 pM. Comparison of the total binding capacity values determined by saturation experiments with [3H] PZ and (-)-[3H]QNB demonstrates that approximately 78% of the total muscarinic binding sites in rabbit peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (-)-[3H]QNB binding by the muscarinic agonist carbachol in peripheral lung membranes. If the pulmonary muscarinic receptor with high affinity for PZ proves to have an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.     

Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist [3H]pirenzepine [( 3H]PZ) and the classical muscarinic antagonist [3H](-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for [3H](-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of [3H](-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.     

Kappa receptor regulation of dopamine release from striatum and cortex of rats and guinea pigs

The effects of opioid agonists with selectivity for kappa, mu and delta types of opioid receptors on the K+-stimulated release of [3H]dopamine (DA) from striatum and cortex of rat and guinea pig loaded previously with the monoamine have been studied. The kappa agonist U50488H did not affect base-line release of [3H]DA measured in 5 mM K+, but produced a dose-dependent inhibition of the release of [3H]DA stimulated by 20 mM K+ from slices of striatum in rat and guinea pig, with an IC50 of about 0.5 nM in each case. In contrast, the mu-selective agonist, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and the delta-selective agonist, [D-Pen2-D-Pen5]enkephalin, did not inhibit stimulated release from the slice preparations at concentrations up to 1 microM. The inhibitory effects of U50488H were antagonized by naloxone, and the potent and selective kappa antagonist, nor-binaltorphimine (nor-BNI). Similar results were obtained when release of [3H]DA from rat and guinea pig cortex slices was examined. In guinea pig cortex, the maximum inhibition of DA release induced by U50488H was 80% of control-stimulated fractional release. In rat cortex and in striatum of both species the maximum release was about 40% of control fractional release. Thus, in the guinea pig, the mesocortical dopaminergic pathway appears more sensitive to the inhibitory effects of U50488H than the nigrostriatal system. The effects of the opioids on the K+ (12.5 mM)-stimulated release of [3H]DA from guinea pig striatal synaptosomes also were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacologic characterization and functional role of muscarinic autoreceptors in the rabbit striatum

The objectives of the present studies were 1) to pharmacologically characterize the muscarinic autoreceptors in the striatum and 2) to examine their role in the regulation of physiologic acetylcholine (ACh) release. Schild plots were generated for atropine and pirenzepine against oxotremorine-induced inhibition of [3H]ACh release. Atropine, a nonselective antagonist, yielded a pA2 of 8.92. The pA2 for pirenzepine, a purported M1-selective antagonist, was 7.14. Both Schild plots had slopes not significantly different from one. Four agonists [oxotremorine, carbachol (CARB), McN-A-343 and pilocarpine] were tested for their effectiveness in inhibiting [3H]ACh release. McN-A-343 and pilocarpine have been reported to be selective for M1 receptors. Oxotremorine and carbachol were effective and potent inhibitors of [3H] ACh release, whereas McN-A-343 and pilocarpine were weak. Although the existence of muscarinic receptor subtypes remains an open question, these data are consistent with the "low" pirenzepine affinity (M2) subtype. Chronic treatments (14 days) with several agents were carried out (in vivo) to assess the role of muscarinic autoreceptors in the regulation of physiologic ACh release. Scatchard analyses of binding studies with [3H]quinuclidinyl benzilate were also performed to assess changes in the muscarinic receptor population in the striatum. Chronic treatment with scopolamine caused a 100% increase in the Bmax for [3H]quinuclidinyl benzilate binding but had no effect on the sensitivity of [3H]ACh release to inhibition by CARB. Fourteen-day treatment with physostigmine (3 mg/kg) produced a decrease in the sensitivity of [3H]ACh release to CARB plus a 42% decrease in Bmax and a 48% decrease in Kd for [3H]quinuclidinyl benzilate binding. Chronic haloperidol treatment caused an increase in the sensitivity of [3H]ACh release to CARB accompanied by a 46% increase in Bmax for 3H quinuclidinyl benzilate binding. These data suggest that muscarinic autoreceptors in the striatum do not regulate physiologic ACh release in the presence of intact acetylcholinesterase and that the interaction of dopaminergic and cholinergic neurons in the striatum may not be simple trans-synaptic inhibition.     

Inhibitors of arachidonic acid metabolism: effects on rat striatal dopamine release and uptake.

The purpose of this study was to investigate the possibility that arachidonic acid metabolites mediate D-2 dopamine (DA) receptor inhibition of striatal DA release. The phospholipase A2 inhibitor p-bromophenacyl bromide (BPB; 10 microM) increased electrically evoked overflow of endogenous DA from rat striatal slices and appeared to partially block the modulatory effects of the D-2 DA receptor agonist N-0437 on this release. However, BPB also increased spontaneous DA outflow in a dose-dependent manner. U-73122 (10 microM), another phospholipase A2 inhibitor, decreased evoked overflow of DA, did not affect the action of N-0437 but also increased spontaneous outflow of DA. In contrast, arachidonic acid (30 microM) produced no effects. In slices prelabeled with [3H]DA, exposure to BPB, U-73122 and nordihydroguaiaretic acid (a lipoxygenase inhibitor) significantly increased spontaneous outflow of tritium whereas the cyclooxygenase inhibitors aspirin and indomethacin did not. In low micromolar concentrations, BPB, U-73122 and nordihydroguaiaretic acid, but not aspirin and indomethacin, inhibited uptake of [3H]DA into striatal synaptosomes and binding of [3H]mazindol to the DA transporter. Only U-73122 affected D-2 DA receptor binding. Taken together, these results suggest that it is unlikely that arachidonic acid metabolites mediate the actions of release-modulating D-2 DA autoreceptors in the striatum. However, the results also suggest that certain inhibitors of arachidonic acid metabolism are relatively potent DA uptake blockers/releasers and that this action is unrelated to their inhibition of enzymes in the arachidonic acid cascade. Caution should be used when using BPB and nordihydroguaiaretic acid to study mechanisms involved in DA release, because these compounds may increase DA release and thereby appear to antagonize the effects of activation of presynaptic receptors.     

LACK OF EVIDENCE FOR AXONAL TRANSPORT OF D1 AND D2 RECEPTORS IN THE NIGRO-STRIATAL PATHWAY OF THE RAT

The possibility that D1 and D2 dopamine receptors are axonally transported in the nigro-striatal pathway has been investigated in the rat by placing a coronal knife cut (sparing the striato-nigral pathway) through the medial forebrain bundle (MFB) and autoradiographically examining the density of D2 (as labeled by [3H]spiperone in the presence of ketanserin) and D1 (as labeled by [3H]SCH 23390) receptors. The efficacy of MFB transection has been assessed by measuring in parallel the binding of [3]ketanserin, a ligand that has been reported to be axonally transported in this bundle. At 12 h post-transection, there was a minor accumulation of [3H]spiperone binding on both sides of the transection. However, (+)butaclamol (1 microM) failed to displace the ligand build-up at the knife cut, thus demonstrating the nonspecific nature of [3H]spiperone accumulation. Similar results were observed at 24, 48, and 72 h after severing the MFB. MFB transection also failed to cause changes in specific [3H]SCH 23390 binding at the knife cut at 12-72 h post-surgery. In contrast, a dramatic accumulation of [3H]ketanserin binding sites was observed rostral and caudal to the cut at 12 h post-transection, attesting to the efficacy of the lesion. These results confirm the existence of both anterograde and retrograde transport of [3H]ketanserin binding sites and suggest that D1 and D2 receptors are not axonally transported in fibers of the nigro-striatal pathway.     

Effect of chronic administration of U-50,488H on tolerance to its pharmacological actions and on multiple opioid receptors in rat brain regions and spinal cord

The effects of chronic administration of U-50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamide ), a selective kappa opioid agonist, on the development of tolerance to its analgesic and hypothermic effects and on mu, delta and kappa opioid receptors in brain regions and spinal cord of male Sprague-Dawley rats were determined. Rats were injected i.p. twice daily with 25 mg/kg of U-50,488H for 4 days. The development of tolerance to the analgesic and hypothermic effects of U-50,488H was almost complete after 4 days of treatment. [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr (DSTLE) and [3H]ethylketocyclazocine (EK) were used as ligands for mu, delta and kappa opioid receptors, respectively. The binding of [3H]DAMGO to membranes prepared from various brain regions (pons + medulla, midbrain, hypothalamus, corpus striatum and cortex) and spinal cord was unaffected by chronic administration of U-50,488H. The binding of [3H]DSTLE in U-50,488H-treated rats was decreased in spinal cord and increased in corpus striatum. The binding of [3H]EK to membranes prepared from pons + medulla, midbrain, cortex and spinal cord was decreased whereas it was increased in the corpus striatum. The changes in the binding of [3H]DSTLE and [3H]EK after chronic treatment with U-50,488H were due to changes in the maximum binding values and not in the Kd values. The results indicate that, in the rat, chronic administration of U-50,488H results in the development of tolerance to its analgesic and hypothermic effects and down-regulation of kappa and delta opioid receptors in the spinal cord and an up-regulation in the corpus striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylphenidate-induced increases in vesicular dopamine sequestration and dopamine release in the striatum: the role of muscarinic and dopamine D2 receptors

Methylphenidate (MPD) administration alters the subcellular distribution of vesicular monoamine transporter-2 (VMAT-2)-containing vesicles in rat striatum. This report reveals previously undescribed pharmacological features of MPD by elucidating its receptor-mediated effects on VMAT-2-containing vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) and on striatal dopamine (DA) release. MPD administration increased DA transport into, and decreased the VMAT-2 immunoreactivity of, the membrane-associated vesicle subcellular fraction. These effects were mimicked by the D2 receptor agonist quinpirole and blocked by the D2 receptor antagonist eticlopride. Both MPD and quinpirole increased vesicular DA content. However, MPD increased, whereas quinpirole decreased, K(+)-stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K(+)-stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K(+)-stimulated DA release, whereas the N-methyl-d-aspartate receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was without effect. This suggests that D2 receptors mediate both the MPD-induced redistribution of vesicles away from synaptosomal membranes and the MPD-induced up-regulation of vesicles remaining at the membrane. This results in a redistribution of DA within the striatum from the cytoplasm into vesicles, leading to increased DA release. However, D2 receptor activation alone is not sufficient to mediate the MPD-induced increases in striatal DA release because muscarinic receptor activation is also required. These novel findings provide insight into the mechanism of action of MPD, regulation of DA sequestration/release, and treatment of disorders affecting DA disposition, including attention-deficit hyperactivity disorder, substance abuse, and Parkinson's disease.     

Comparative anticholinergic properties of thioridazine, mesoridazine and sulforidazine

The anticholinergic properties of thioridazine (THD) and its metabolites mesoridazine (MES) and sulforidazine (SUL) were compared to the antimuscarinics atropine and quinuclidinylbenzilate (QNB). THD, MES and SUL were virtually inactive in antagonizing the carbachol-induced inhibition of evoked ACh release from perfused rabbit striatal slices. This lack of effect was seen even when dopamine influences were abolished by treatment with reserpine and alpha-methyl-p-tyrosine. The lack of functional anticholinergic potency contrasted with the affinity of THD for muscarinic receptors measured as competition for [3H]QNB binding sites in striatal homogenates (Ki values: atropine, 2.7 nM; THD 14 nM; SUL, 66 nM; and MES, 90 nM). Both atropine and QNB blocked carbachol-induced inhibition of ACh release in a dose-dependent manner (IC50 values vs. 3 microM carbachol: 0.5 nM for QNB; 1.25 nM for atropine). THD, only 5 times less potent than atropine in competing for [3H]QNB binding sites, was inactive in antagonizing carbachol-induced ACh release. At very high concentrations (3-30 microM), THD, MES and SUL did enhance dopamine efflux and inhibit ACh release. In summary, the lack of effect of THD on release modulatory muscarinic receptors suggest that THD is selective for the M1 subtype. Because the M2 subtype is a small fraction of the total population in the striatum, it is not surprising that they would escape recognition in the QNB binding assays. These data suggest that inhibition of ACh release may contribute to the actions of THD only at very high doses, or when drug accumulation is abnormal.     

Muscarinic receptors mediating inhibition of gamma-aminobutyric acid release in rat corpus striatum and their pharmacological characterization

The effects of acetylcholine (ACh) and of cholinergic agonists on the release of tritiated gamma-aminobutyric acid ([3H]GABA) were studied in superfused synaptosomes prepared from rat corpus striatum and prelabeled with the radioactive amino acid. ACh, oxotremorine or (-)-nicotine, all tested at 100 microM had no effect on the spontaneous outflow of [3H]GABA. The depolarization-evoked overflow obtained by exposing the synaptosomes to 9 mM KCl was decreased in a concentration-dependent manner by ACh, oxotremorine, oxotremorine-M or carbachol. The maximal inhibition caused by ACh was 50%. The EC50 (agonist concentration causing half-maximal effect) amounted to 1 microM. Oxotremorine and oxotremorine-M were almost equipotent to ACh, whereas the concentration-response curve of carbachol was slightly (although not significantly) shifted to the right with respect to that of ACh. (-)-Nicotine (100 microM) did not affect the K(+)-evoked [3H]GABA overflow. ACh also inhibited the K(+)-evoked release of endogenous GABA. The inhibitory effect of 10 microM ACh on the release of [3H]GABA evoked by 9 mM KCl was insensitive to the nicotinic antagonist mecamylamine (10 microM) but it was potently blocked by the muscarinic antagonist atropine (IC50 = 5 nM) and weakly antagonized by pirenzepine, dicyclomine and AF-DX 116. The pharmacological profile of this receptor was very similar to that of the muscarinic autoreceptors regulating [3H]ACh release. The extent of [3H]GABA release inhibition caused by ACh did not differ between dorsal and ventral striatum. The inhibitory effect of ACh was much less pronounced in hippocampus and cortex than in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of dopamine release in the guinea-pig retina by G(i)- but not by G(s)- or G(q)-protein-coupled receptors

The modulation of dopamine release from the guinea-pig retina was studied using maximally effective concentrations of 10 agonists acting on G(i)-, G(s)- or G(q)-protein-coupled receptors (PCRs). Retinal discs were preincubated with [(3)H]noradrenaline and superfused; tritium overflow was evoked electrically. The following compounds acting on G(i)-PCRs reduced the tritium overflow, which represents quasi-physiological dopamine release under the experimental conditions of our study: the dopamine and alpha(2)-adrenoceptor agonist B-HT 920 by 95%, the muscarinic agonist oxotremorine by 96%, melatonin by 94%, the cannabinoid agonist WIN 55,212-2 by 71% and histamine by 66%. Tritium overflow was not affected by serotonin or by agonists acting on G(s)-PCRs (ACTH1-24 and the beta-adrenoceptor agonist procaterol) and G(q)-PCRs (angiotensin II and bradykinin). The effects of B-HT 920, oxotremorine and melatonin were studied in more detail using appropriate antagonists. The inhibitory effect of a submaximally active concentration of B-HT 920 was counteracted by the dopamine D(2/3) antagonist haloperidol but not affected by the alpha(2)-adrenoceptor antagonist phentolamine. The muscarinic antagonist atropine shifted to the right the concentration-response curve of oxotremorine (pA(2) 8.7) and the melatonin MT(2) antagonist 4-P-PDOT produced a rightward shift of the concentration-response curve of melatonin (pA(2) 10.6). Melatonin was also studied in superfused brain slices (from the guinea-pig) preincubated with [(3)H]noradrenaline. The electrically evoked tritium overflow in cerebrocortical, hippocampal and hypothalamic slices (representing quasi-physiological noradrenaline release) and in striatal slices (representing quasi-physiological dopamine release) was not affected by melatonin at a concentration that causes the maximum effect in retinal discs. In conclusion, dopamine release in the guinea-pig retina is inhibited via G(i)-PCRs including dopamine (D(2/3)), muscarinic and melatonin (MT(2)) receptors but not affected via any of the G(s)- or G(q)-PCRs under study. Unlike in the retina, melatonin fails to inhibit monoamine release in four brain regions of the guinea-pig.     

Identification of nonserotonergic [3H]ketanserin binding sites associated with nerve terminals in rat brain and with platelets; relation with release of biogenic amine metabolites induced by ketanserin- and tetrabenazine-like drugs

In mammalian striatal tissue and cat platelets, [3H]ketanserin labels besides serotonin-S2 receptors nonserotonergic saturable binding sites. The sites have been distinguished and characterized in [3H]ketanserin binding assays by selective inhibition with tetrabenazine (Ki = 4 nM), a monoamine depleting agent. In rats, the nonserotonergic ketanserin sites were enriched in the striatum (KD = 12.4 +/- 0.3 nM, maximal number of binding sites = 53.2 +/- 11.8 fmol/mg of tissue at pH 7.7, 37 degrees C) and nucleus accumbens. The sites were decreased by 65 to 78% after 6-hydroxydopamine lesions, suggesting an association with dopaminergic nerve terminals. In in vitro superfusion experiments using [3H]dopamine, [3H]norepinephrine and [3H]serotonin loaded rat brain tissue and [3H]serotonin loaded human platelets, 5 min superfusion with 10(-6) M ketanserin, tetrabenazine and reserpine caused instantaneously a marked increase in tritium efflux. The effect was attenuated by the monoamine oxidase inhibitor, pargyline, in brain slices but not in platelets. High-performance liquid chromatography analysis of endogenous catecholamines, serotonin and metabolites in superfusates from striatal slices revealed that stimulation with these drugs provoked mainly release of 3,4-dihydroxybenzeneacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. Potencies of a series of ketanserin derivatives, benzoquinolizine derivatives and a variety of drugs affecting neurotransmission were assessed in the in vitro release test using [3H]dopamine loaded striatal slices, and in [3H]ketanserin binding assays to nonserotonergic sites in the striatum and to serotonin-S2 receptors in brain tissue. Activities of drugs in the release test correlated strongly with their binding affinities for nonserotonergic ketanserin sites (rs = 0.83, n = 30, P less than .001). High potency in the latter two tests was confined to few close structural congeners of ketanserin and tetrabenazine. Distinct structural activity relationships for interaction with nonserotonergic ketanserin sites and serotonin-S2 receptors were found. It was concluded that nonserotonergic ketanserin sites mediate release of oxidated metabolites of biogenic amines from nerve endings and of serotonin from platelets. Hence release of biogenic amine metabolites or of cytoplasmic amines is probably not a mere diffusion process but involves specific membranous molecules. Unlike tetrabenazine, ketanserin caused no obvious depletion of central catecholamine and indoleamine stores. Implications of these findings for the mechanism of action of the drugs are discussed.