Both GABAA and GABAB receptors participate in suppression of [CA2+]i pulsing in toad melanotrophs.

The receptor mechanisms involved in the inhibitory effect of gamma-aminobutyric acid (GABA) in suppressing spontaneous [Ca2+]i pulsing in melanotrophs of Xenopus laevis were investigated. The selective GABAB receptor agonist, baclofen reversibly arrested [Ca2+]i pulsing. This inhibition was unaffected by the selective GABAA receptor antagonist, bicuculline methiodide, but was blocked by the selective GABAB receptor antagonist, CGP 35348 (3-aminopropyl diethyoxymethyl phosphinic acid). The selective GABAA receptor agonist, muscimol, also arrested [Ca2+]i pulsing after causing a transient rise in [Ca2+]i. This biphasic response to muscimol was unaffected by CGP 35348, but was blocked by bicuculline. The inhibitory effect of GABA was unaffected by either CGP 35348 or bicuculline when given alone, but was blocked by both antagonists given together. In cells pretreated with pertussis toxin, the response to baclofen was completely lost, whereas responses to GABA and muscimol persisted; the response to GABA was blocked by bicuculline alone. Thus, both GABAA and GABAB receptors are involved in the inhibitory effect of GABA in suppressing spontaneous [Ca2+]i pulsing in Xenopus melanotrophs.

Animal models with potential applications for screening compounds for the treatment of obsessive-compulsive disorder.

The availability of an animal model for obsessive-compulsive disorder (OCD) is necessary for the development of novel pharmacological treatments. To be useful, the model must be predictive of clinical performance, possess characteristic criteria and distinguish anti-OCD from antidepressant compounds. Due to the lack of OCD models useful for drug discovery, all compounds currently used for OCD were developed first as antidepressants. In this article, we discuss the relative merits of: stereotypic behaviours (canine acral lick, feather picking, amphetamine- and 5-HT-induced stereotypy); adjunctive and displacement behaviours (schedule-induced polydipsia, wheel running, resident-intruder grooming); anxiolytic tests (separation and shock-induced ultrasonic vocalisation and marble burying); and depression tests (inescapable shock-induced escape and immobility in forced swim) as potential OCD models. We conclude that adjunctive and displacement behaviours, and in particular schedule-induced polydipsia, may prove to be the best models for compulsive behaviour in animals that can be used for the discovery of novel anti-OCD agents.

Iloperidone binding to human and rat dopamine and 5-HT receptors.

Iloperidone (HP 873; 1-[4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy] -3- methoxyphenyl]ethanone) is a compound currently in clinical trials for the treatment of schizophrenia. Iloperidone displays affinity for dopamine D2 receptors and for 5-HT2A receptors and has a variety of in vivo activities suggestive of an atypical antipsychotic. Here we present an examination of the affinity of iloperidone to a variety of human and rat homologs of dopamine and 5-HT receptor subtypes. We employed receptor binding assays using membranes from cells stably expressing human dopamine D1, D2S, D2L, D3, D4 and D5 and 5-HT2A and 5-HT2C receptors and rat 5-HT6 and 5-HT7 receptors. Iloperidone displayed higher affinity for the dopamine D3 receptor (Ki = 7.1 nM) than for the dopamine D4 receptor (Ki = 25 nM). Iloperidone displayed high affinity for the 5-HT6 and 5-HT7 receptors (Ki = 42.7 and 21.6 nM, respectively), and was found to have higher affinity for the 5-HT2A (Ki = 5.6 nM) than for the 5-HT2C receptor (Ki = 42.8 nM). The potential implications of this receptor binding profile are discussed in comparison with data for other antipsychotic compounds.

Structure-activity relationships of a series of novel (piperazinylbutyl)thiazolidinone antipsychotic agents related to 3-[4-[4-(6-fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl]-2,5,5- trimethyl-4-thiazolidinone maleate.

HP-236 (3-[4-[4-(6-Fluorobenzo[b]thien-3-yl)-1-piperazinyl]butyl]-2,5,5- trimethyl-4-thiazolidinone maleate; P-9236) (54) displayed a pharmacological profile indicative of potential atypical antipsychotic activity. A series of piperazinyl butyl thiazolidinones structurally related to this compound were prepared and evaluated in vitro for dopamine D2 and serotonin 5HT2 and 5HT1A receptor affinity. The compounds were examined in vivo in animal models of potential antipsychotic activity and screened in models predictive of extrapyramidal side effect (EPS) liability. The synthesis of these compounds, details of their structure-activity relationships, and discovery of a new lead, compound 50, as well as further development of the profiles of compounds 50 and 54 are described.

Serotonergic activity of HP 184: does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl) -1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 microM HP 184 enhanced spontaneous release of [3H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30-40% after a 14-21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain:plasma ratio of HP 184 was approximately 2:1, with brain concentrations of 1.6 micrograms/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.

Frequency-dependent inhibition of neurotransmitter release by besipirdine and HP 184.

We have described the interaction of besipirdine (HP 749, N-(n-propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride) with voltage-dependent Na+ channels (Tang et al., 1995, Br. J. Pharmacol. 116,2468). Here we describe studies with besipirdine and a related compound, HP 184 (N-(n-propyl)-3-fluoro-4-pyridinyl)-1H-3-methylindol-1-amine hydrochloride), showing that this interaction is voltage-dependent and leads to frequency-dependent inhibition of electrically stimulated neurotransmitter release. Thus, the inhibition of veratridine-induced increases in intracellular Ca2+ was enhanced by depolarization with KCl (IC50 shifted from 23.8 +/- 1.4 microM in 5 mM KCl to 7.3 +/- 1.2 microM in 15 mM KCl for besipirdine and from 58.2 +/- 1.3 microM for HP 184). Moreover, the enhancement of electrically stimulated [3H]norepinephrine release by besipirdine was diminished at higher frequencies of stimulation. As has been previously suggested for such compounds, we predict that besipirdine would act as a filter in the brain allowing signalling at low frequencies but blocking transmission at high frequencies.

Besipirdine (HP 749) reduces schedule-induced polydipsia in rats.

The aim of the present paper is to report on the adrenergic and serotonergic effects of besipirdine (HP 749) in vivo and to discuss its potential use in the treatment of obsessive compulsive disorder. Besipirdine inhibited biogenic amine uptake in vitro. It prevented tetrabenazine-induced ptosis in mice and potentiated the 5-hydroxytryptophan-induced serotonin syndrome in rats. Furthermore, it decreased schedule-induced polydipsic behavior in rats. Schedule-induced polydipsia may be a model for obsessive compulsive disorder. Previous results from our group have shown that certain selective serotonin reuptake inhibitors decrease schedule-induced polydipsia after 14-21 days of treatment. Besipirdine reduced schedule-induced polydipsic behavior immediately and this reduction lasted throughout the duration of the experiment (29 days).

Effects of besipirdine at the voltage-dependent sodium channel.

1. Besipirdine (HP 749) is a compound undergoing clinical trials for efficacy in treating Alzheimer's disease. Among other pharmacological effects, besipirdine inhibits voltage-dependent sodium and potassium channels. This paper presents a pharmacological study of the interaction of besipirdine with voltage-dependent sodium channels. 2. Besipirdine inhibited [3H]-batrachotoxin binding (IC50 = 5.5 +/- 0.2 microM) in a rat brain vesicular preparation and concentration-dependently inhibited veratridine (25 microM)-stimulated increases in intracellular free sodium ([Na+]i) and calcium ([Ca2+]i) in primary cultured cortical neurones of rat. 3. Besipirdine (30-100 microM) concentration-dependently inhibited (up to 100%) veratridine-stimulated release of [3H]-noradrenaline (NA) from rat cortical slices. 4. When examined in greater detail, besipirdine was found to inhibit [3H]-batrachotoxin binding in vesicular membranes competitively. However, when examined in rat brain synaptosomes, we found that the antagonism by besipirdine was not competitive; that is, the maximal stimulation of [Ca2+]i induced by veratridine decreased with increasing concentrations of besipirdine. 5. These results show that besipirdine is an inhibitor of voltage-sensitive sodium channels and appears to bind to a site close to the batrachotoxin/veratridine binding site.

Changes in paroxetine binding in the cerebral cortex of polydipsic rats.

Schedule-induced polydipsia was induced when food-deprived rats were subjected to a fixed-time (60 s) feeding schedule for 150 min daily for 3 weeks (training period). Subsequent chronic administration of the serotonin reuptake inhibitor fluoxetine reduces schedule-induced polydipsia over 2-4 weeks. We asked whether changes in the serotonin reuptake carrier occur following the development of schedule-induced polydipsia and its reduction by fluoxetine. Using [3H]paroxetine binding, we found a 40% increase in Kd and a 50% decrease in Bmax in polydipsic rats; both were reversed by fluoxetine. Food deprivation alone did not affect these parameters. These observations suggest that changes in the serotonin reuptake carrier correlate with the development and reversal of schedule-induced polydipsia.

HP 749 enhances calcium-independent release of [3H]norepinephrine from rat cortical slices and synaptosomes.

Previous studies have shown that, at concentrations of 1 microM and 10 microM, HP 749 increased electrically-stimulated release of [3H]norepinephrine (NE) from rat cortical slices. These effects were Ca(2+)-dependent, indicating an effect on release from vesicular stores. At 100 microM, HP 749 had two effects. In addition to enhancing the Ca(2+)-dependent electrically-evoked release, it also induced a rise in the basal efflux (spontaneous release) of [3H]NE, which was observed in both cortical slices and synaptosomes. The spontaneous release effect was (1) not blocked by the reuptake inhibitor nomifensine, (2) not affected by removal of external calcium, (3) not blocked by vesicular depletion with reserpine, and (4) not inhibited by the sodium channel blocker tetrodotoxin (TTX). As would be expected, the spontaneous [3H]NE release induced by the cytoplasmic releaser tyramine and the sodium channel activator veratridine were blocked by nomifensine and TTX, respectively. Notably, however, the Ca(2+)-independent veratridine-induced release was completely blocked by 100 microM HP 749. The mechanism of spontaneous release of [3H]NE caused by 100 microM HP 749 is unresolved at present; however, the data are consistent with this release originating from a cytoplasmic source.

Melanotrophs of Xenopus laevis do respond directly to neuropeptide-Y as evidenced by reductions in secretion and cytosolic calcium pulsing in isolated cells.

Neuropeptide-Y (NPY) is present, along with dopamine and gamma-aminobutyric acid, in the neurons innervating the intermediate lobe of the pituitary gland of Xenopus laevis, and all three neurotransmitters have been shown to inhibit melanotroph secretion from isolated neurointermediate lobes. However, unlike dopamine and gamma-aminobutyric acid, NPY has been reported to be without inhibitory effect on secretion from dispersions of intermediate lobe cells. Moreover, binding studies have been taken as indicating that Xenopus melanotrophs lack NPY receptors, although such receptors appear to be present on folliculostellate cells. For these reasons, NPY has been considered to act indirectly on Xenopus melanotrophs; the putative intermediary is supposed to be the folliculo-stellate cell. However, the present experiments show that NPY does strongly inhibit melanotroph secretion from cells dispersed from Xenopus intermediate lobes. In addition, they demonstrate that NPY acts directly on individual Xenopus melanotrophs (immunohistochemically identified and under conditions that preclude any interaction between cells) to inhibit the intermittent rises in cytosolic free Ca (cytosolic Ca pulsing). From these observations, we conclude that NPY does act directly on melanotrophs of Xenopus.

Effectiveness of GABAB antagonists in inhibiting baclofen-induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat.

1. The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs. 2. Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura-2. 3. (-)-Baclofen lowered [Ca2+]i in a concentration-dependent manner with an EC50 of 0.96 microM. The reduction in [Ca2+]i produced by (-)-baclofen at a maximally effective concentration (100 microM) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nM), or by perifusion with a nominally Ca-free solution. 4. The GABAB receptor antagonists, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2-hydroxy saclofen, phaclofen and 4-amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (-)-baclofen (3 microM). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 microM, compared to 120 to 400 microM for the others. CGP 35348 acted competitively. 5. CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nM) or the specific GABAA receptor agonist, muscimol (10 microM). 6. The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.

Why are several inhibitory transmitters present in the innervation of pituitary melanotrophs? Actions and interactions of dopamine, GABA and neuropeptide Y on secretion from neurointermediate lobes of Xenopus laevis.

The aim of the present experiments was to determine whether some rationale for the presence of the several inhibitory neurotransmitters in the innervation to the toad melanotroph might be found in differences in their individual effects or in possible cooperative interactions affecting secretion. Measurements of peptide release from isolated, perifused neurointermediate lobes of the toad Xenopus laevis showed that each of the three identified inhibitory transmitters, dopamine, GABA and NPY, was able to inhibit secretion profoundly and no less effectively than omission of Ca. Moreover, the inhibitory effects were rather similar in onset, duration and recovery. Furthermore, there was no evidence of any cooperative interactions when the transmitters were given in various combinations. And finally, the inhibitory response to each of the transmitters was abolished by pretreatment with pertussis toxin. While not excluding differential postsynaptic effects on other parameters of melanotroph function, the similarities observed have encouraged alternative speculations on the significance of the apparent redundancy of inhibitory transmitters.

Studies on pituitary melanotrophs reveal the novel GABAB antagonist CGP 35-348 to be the first such compound effective on endocrine cells.

One obstacle to understanding the action and physiological significance of the responsiveness of various endocrine cells to gamma-aminobutyric acid (GABA) has been that previously available substances, all active as GABAB antagonists in the nervous system, are ineffective on endocrine cells. The introduction of a potent new member of this class, CGP 35-348, of very different chemical structure, encouraged us to examine its effect on endocrine cells. For this purpose, we studied melanotroph secretion from pituitary neurointermediate lobes. We found that CGP 35-348, in contrast to previously available members of this class, suppressed completely, in rat and toad, secretory responses to baclofen, the classic GABAB agonist. Analysis, in toad, showed CGP 35-348 did not affect responses to GABAA agonists (muscimol; isoguvacine), dopamine, or neuropeptide Y. When tested against GABA, the physiological ligand present in the innervation of melanotrophs (along with dopamine and neuropeptide Y), CGP 35-348 completely suppressed the secretory response, which, in toad, is purely inhibitory and unaffected by bicuculline, the specific GABAA antagonist. In addition, CGP 35-348 unmasked a stimulant effect that bicuculline blocked. In CGP 35-348, we thus have a new tool with which to analyse responses to GABA and their physiological involvement in endocrine cells.

Alpha-adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating.

In sympathetic neurons, catecholamines interact with prejunctional alpha-adrenergic receptors to reduce delivery of transmitter to postjunctional target organs. This autoinhibitory feedback is a general phenomenon seen in diverse neurons containing a variety of transmitters. The underlying mechanisms of alpha-adrenergic inhibition are not clear, although decreases in cyclic AMP and cAMP-mediated phosphorylation have been implicated. We have studied depolarization-induced catecholamine release and calcium-channel currents in frog sympathetic neurons. Here we show that alpha-adrenergic inhibition of transmitter release can be explained by inhibition of Ca2+-channel currents and not by modulation of intracellular proteins. Noradrenaline strongly reduces the activity of N-type Ca2+ channels, the dominant calcium entry pathway triggering sympathetic transmitter release, whereas L-type Ca2+ channels are not significantly inhibited. The down-modulation of N-type channels involves changes in rapid gating kinetics but not in unitary flux. This is the first detailed description of inhibition of a high-voltage activated neuronal Ca2+ channel at the single-channel level. The coupling between alpha-adrenergic receptors and N-type channels involves a G protein, but not a readily diffusible cytoplasmic messenger or protein kinase C, and may be well suited for rapid and spatially localized feedback-control of transmitter release.

'Slow' K+-stimulated Ca2+ influx is mediated by Na+-Ca2+ exchange: a pharmacological study.

K+-stimulated 45Ca2+ influx was measured in rat brain presynaptic nerve terminals that were predepolarized in a K+-rich solution for 15 s prior to addition of 45Ca2+. This 'slow' Ca2+ influx was compared to influx stimulated by Na+ removal, presumably mediated by Na+-Ca2+ exchange. The K+-stimulated Ca2+ influx in predepolarized synaptosomes, and the Na+-removal-dependent Ca2+ influx were both saturating functions of the external Ca2+ concentration; and both were half-saturated at 0.3 mM Ca2+. Both were reduced about 50% by 20 microM Hg2+, 20 microM Cu2+ or 0.45 mM Mn2+. Neither the K+-stimulated nor the Na+-removal-dependent Ca2+ influx was inhibited by 1 microM Cd2+, La3+ or Pb2+, treatments that almost completely inhibited K+-stimulated Ca2+ influx in synaptosomes that were not predepolarized. The relative permeabilities of K+-stimulated Ca2+, Sr2+ or Ba2+ influx in predepolarized synaptosomes (10:3:1) and the corresponding selectivity ratio for Na+-removal-dependent divalent cation uptake (10:2:1) were similar. These results strongly suggest that the K+-stimulated 'slow' Ca2+ influx in predepolarized synaptosomes and the Na+-removal-dependent Ca2+ influx are mediated by a common mechanism, the Na+-Ca2+ exchanger.

Measurement of the cytosolic sodium ion concentration in rat brain synaptosomes by a fluorescence method.

A method for the measurement of the cytosolic Na+ concentration in intact synaptosomes is described. This method makes use of a pH sensitive dye (BCECF) that can be loaded into the cytosol and a relatively specific ionophore (monensin) that can exchange Na+ for H+ across the synaptosomal membrane. By setting conditions such that there is no electrochemical potential difference for H+ across the membrane (no membrane potential and pHi = pHo), addition of ionophore would induce a H+ flux only if there is a concentration difference for Na+. Thus, when there is no fluorescence change (no cytosolic pH change) extracellular [Na+] equals intrasynaptosomal [Na+]. The intrasynaptosomal [Na+] concentration was determined to be 7 +/- 3 mM (n = 5; mean +/- S.E.). The results obtained with this fluorescence method are compared with estimates obtained by atomic absorption spectrometry. Limitations and applications of the method are discussed.

Nerve growth factor modulates the drug sensitivity of neurotransmitter release from PC-12 cells.

The release of catecholamines from adrenal chromaffin cells is known to be blocked by dihydropyridines, such as nitrendipine, and enhanced by others, such as BAY K8644. On the other hand, release from sympathetic neurons is predominantly insensitive to these agents. Release of [3H]norepinephrine from undifferentiated PC-12 pheochromocytoma cells resembles that from chromaffin cells in that it is extremely sensitive to dihydropyridines. Following differentiation, however, release of catecholamine becomes predominantly insensitive to both nitrendipine and BAY K8644. Under both growth conditions, release remains completely blocked by 3 mM Co2+ or by removal of Ca2+ from the release media. Dose-response curves to K+ show that following differentiation, cells become more sensitive, releasing transmitter at lower K+ concentrations. In contrast, depolarization-induced uptake of 45Ca2+ remains sensitive to dihydropyridines and shows similar sensitivity to K+ stimulation in both growth conditions. These results can be explained by invoking a model involving dihydropyridine-sensitive and -insensitive types of voltage-sensitive calcium channels.

Maitotoxin-induced release of gamma-[3H]aminobutyric acid from cultures of striatal neurons.

The potent marine toxin, maitotoxin, induced the release of gamma-[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose-dependent manner. Maitotoxin-induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin-induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D-600 at concentrations of 10(-6) M did not block maitotoxin-induced or 70 mM K+-induced release. High concentrations of D-600 (10(-4) M) partially blocked both maitotoxin- and 70 mM K+-induced release. The dihydropyridine calcium agonist BAY K8644 (10(-6) M) did not enhance maitotoxin-induced or 70 mM K+-induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage-sensitive calcium channels.