Evidence that a deviation in the kynurenine pathway aggravates atherosclerotic disease in humans.

BACKGROUND: The metabolism of tryptophan (Trp) along the kynurenine pathway has been shown to carry strong immunoregulatory properties. Several experimental studies indicate that this pathway is a major regulator of vascular inflammation and influences atherogenesis. Knowledge of the role of this pathway in human atherosclerosis remains incomplete. OBJECTIVES: In this study, we performed a multiplatform analysis of tissue samples, in vitro and in vivo functional assays to elucidate the potential role of the kynurenine pathway in human atherosclerosis. METHODS AND RESULTS: Comparison of transcriptomic data from carotid plaques and control arteries revealed an upregulation of enzymes within the quinolinic branch of the kynurenine pathway in the disease state, whilst the branch leading to the formation of kynurenic acid (KynA) was downregulated. Further analyses indicated that local inflammatory responses are closely tied to the deviation of the kynurenine pathway in the vascular wall. Analysis of cerebrovascular symptomatic and asymptomatic carotid stenosis data showed that the downregulation of KynA branch enzymes and reduced KynA production were associated with an increased probability of patients to undergo surgery due to an unstable disease. In vitro, we showed that KynA-mediated signalling through aryl hydrocarbon receptor (AhR) is a major regulator of human macrophage activation. Using a mouse model of peritoneal inflammation, we showed that KynA inhibits leukocyte recruitment. CONCLUSIONS: We have found that a deviation in the kynurenine pathway is associated with an increased probability of developing symptomatic unstable atherosclerotic disease. Our study suggests that KynA-mediated signalling through AhR is an important mechanism involved in the regulation of vascular inflammation.

A systems biology approach to understand gut microbiota and host metabolism in morbid obesity: design of the BARIA Longitudinal Cohort Study.

INTRODUCTION: Prevalence of obesity and associated diseases, including type 2 diabetes mellitus, dyslipidaemia and non-alcoholic fatty liver disease (NAFLD), are increasing. Underlying mechanisms, especially in humans, are unclear. Bariatric surgery provides the unique opportunity to obtain biopsies and portal vein blood-samples. METHODS: The BARIA Study aims to assess how microbiota and their metabolites affect transcription in key tissues and clinical outcome in obese subjects and how baseline anthropometric and metabolic characteristics determine weight loss and glucose homeostasis after bariatric surgery. We phenotype patients undergoing bariatric surgery (predominantly laparoscopic Roux-en-Y gastric bypass), before weight loss, with biometrics, dietary and psychological questionnaires, mixed meal test (MMT) and collect fecal-samples and intra-operative biopsies from liver, adipose tissues and jejunum. We aim to include 1500 patients. A subset (approximately 25%) will undergo intra-operative portal vein blood-sampling. Fecal-samples are analyzed with shotgun metagenomics and targeted metabolomics, fasted and postprandial plasma-samples are subjected to metabolomics, and RNA is extracted from the tissues for RNAseq-analyses. Data will be integrated using state-of-the-art neuronal networks and metabolic modeling. Patient follow-up will be ten years. RESULTS: Preoperative MMT of 170 patients were analysed and clear differences were observed in glucose homeostasis between individuals. Repeated MMT in 10 patients showed satisfactory intra-individual reproducibility, with differences in plasma glucose, insulin and triglycerides within 20% of the mean difference. CONCLUSION: The BARIA study can add more understanding in how gut-microbiota affect metabolism, especially with regard to obesity, glucose metabolism and NAFLD. Identification of key factors may provide diagnostic and therapeutic leads to control the obesity-associated disease epidemic.

(3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752) -a Novel Cortical-Preferring Catecholamine Transmission- and Cognition-Promoting Agent.

Here we describe for the first time the distinctive pharmacological profile for (3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752), a new phenyl-pyrrolidine derivative with regioselective central nervous system transmission-enhancing properties. IRL752 (3.7-150 µmol/kg, s.c.) was characterized through extensive in vivo studies using behavioral, tissue neurochemical, and gene expression as well as microdialysis methods. Behaviorally, the compound normalized tetrabenazine-induced hypoactivity, whereas it was unable to stimulate basal locomotion in normal animals or either accentuate or reverse hyperactivity induced by amphetamine or MK-801. IRL752 induced but minor changes in monoaminergic tissue neurochemistry across noradrenaline (NA)- and dopamine (DA)-dominated brain regions. The expression of neuronal activity-, plasticity-, and cognition-related immediate early genes (IEGs), however, increased by 1.5-fold to 2-fold. Furthermore, IRL752 dose-dependently enhanced cortical catecholamine dialysate output to 600%-750% above baseline, whereas striatal DA remained unaltered, and NA rose to ∼250%; cortical and hippocampal dialysate acetylcholine (ACh) increased to ∼250% and 190% above corresponding baseline, respectively. In line with this cortically preferential transmission-promoting action, the drug was also procognitive in the novel object recognition and reversal learning tests. In vitro neurotarget affinity and functional data coupled to drug exposure support the hypothesis that 5-hydroxytryptamine 7 receptor and α2(C)-adrenoceptor antagonism are key contributors to the in vivo efficacy and original profile of IRL752. The cortical-preferring facilitatory impact on catecholamine (and ACh) neurotransmission, along with effects on IEG expression and cognition-enhancing features, are in line with the potential clinical usefulness of IRL752 in conditions wherein these aspects may be dysregulated, such as in axial motor and cognitive deficits in Parkinson disease. SIGNIFICANCE STATEMENT: This report describes the distinctive preclinical profile of (3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752). Its in vivo neurochemical, behavioral, microdialysis, and gene expression properties are consistent with a cortically regioselective facilitatory impact on catecholaminergic and cholinergic neurotransmission accompanied by cognitive impairment-reversing features. The pharmacological characteristics of IRL752 are in line with the clinical usefulness of IRL752 in conditions wherein these aspects may be dysregulated, such as in axial motor and cognitive deficits in Parkinson disease.

The selective 5-hydroxytryptamine 1A antagonist, AZD7371 [3(R)-(N,N-dicyclobutylamino)-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide (R,R)-tartrate monohydrate] (robalzotan tartrate monohydrate), inhibits visceral pain-related visceromotor, but not autonomic cardiovascular, responses to colorectal distension in rats.

5-Hydroxytryptamine 1A (5-HT(1A)) receptors have been suggested as a target for the treatment of irritable bowel syndrome (IBS). A recent clinical trial investigating the efficacy of the selective 5-HT(1A) antagonist AZD7371 [3(R)-(N,N-dicyclobutylamino)-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide (R,R)-tartrate monohydrate] showed no symptomatic improvement in IBS patients. We characterized the mechanisms mediating potential analgesic effects of AZD7371 in a model of colorectal distension (CRD)-induced visceral pain in rats to understand its mechanism of action and the lack of clinical efficacy. Visceromotor and cardiovascular responses (telemetry) were assessed in conscious rats during noxious CRD (80 mm Hg). Effects of AZD7371 (3-300 nmol/kg i.v.; 1-30 micromol/kg p.o.) and a reference 5-HT(1A) antagonist, WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide maleate salt; 3-300 nmol/kg i.v.), were assessed. Effects of intracerebroventricular AZD7371 were also evaluated. Intravenous AZD7371 or WAY-100635 and oral AZD7371 dose-dependently inhibited visceromotor responses to CRD (ED(50), 203, 231, and 14 micromol/kg, respectively). In telemetrized rats, oral AZD7371 inhibited visceromotor responses to CRD without affecting the concomitant hypertensive and tachycardic responses. Intracerebroventricular AZD7371 did not affect visceromotor responses, whereas it inhibited micturition. None of the doses tested induced visible gross side effects. AZD7371, likely acting at a spinal site, inhibited the visceromotor but not the cardiovascular responses to visceral pain in the CRD model in rats. Although agents effective on multiple pain-related readouts in the CRD model (e.g., pregabalin or clonidine) alleviate IBS symptoms, AZD7371, which is effective on only one pain-related pseudoaffective readout, does not. Data from preclinical CRD models of visceral pain need to be interpreted cautiously as it relates to their clinical translational value.

The orphan receptor GPR55 is a novel cannabinoid receptor.

BACKGROUND: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes. EXPERIMENTAL APPROACH: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways. KEY RESULTS: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1. CONCLUSIONS: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Constitutive activity of glucagon receptor mutants.

Increased constitutive activity has been observed in the PTH receptor in association with naturally occurring mutations of two residues that are conserved between members of the glucagon/vasoactive intestinal peptide/calcitonin 7TM receptor family. Here, the corresponding residues of the glucagon receptor, His178 and Thr352, were probed by mutagenesis. An elevated level of basal cAMP production was observed after the exchange of His178 into Arg, but not for the exchange into Lys, Ala, or Glu. However, for all of these His178 substitutions, an increased binding affinity for glucagon was observed [dissociation constant (Kd) ranging from 1.1-6.4 nM, wild type: Kd = 12.0 nM]. A further increase in cAMP production was observed for the [H178R] construct upon stimulation with glucagon, albeit the EC50 surprisingly was increased approximately 10-fold relative to the wild-type receptor. Substitution of Thr352, located at the intracellular end of transmembrane segment VI, with Ala led to a slightly elevated basal cAMP level, while the introduction of Pro or Ser at this position affected rather the binding affinity of glucagon or the EC50 for stimulation of cAMP production. The large extracellular segment, which is essential for glucagon binding, was not required for constitutive activation of the glucagon receptor as the introduction of the [H178R] mutation into an N-terminally truncated construct exhibited an elevated basal level of cAMP production. The analog des-His1-[Glu9]glucagon amide, which in vivo is a glucagon antagonist, was an agonist on both the wild-type and the [H178R] receptor and did not display any activity as an inverse agonist. It is concluded that the various phenotypes displayed by the constitutively active glucagon receptor mutants reflect the existence of multiple agonist-preferring receptor conformers, which include functionally active as well as inactive states. This view agrees with a recent multi-state extension of the ternary complex model for 7TM receptor activation.

The growth hormone (GH)-binding protein cloned from human IM-9 lymphocytes modulates the down-regulation of GH receptors by 22- and 20-kilodalton human GH in IM-9 lymphocytes and the biological effects of the hormone in Nb2 lymphoma cells.

The cDNA coding for the 246-amino acid long N-terminal extracellular portion of the human (h) GH receptor, corresponding to the circulating GH-binding protein (hGHBP), was cloned by polymerase chain reaction from human IM-9 lymphocytes. The cDNA sequence was identical to that reported for human liver and placenta and demonstrated alternative splicing of exon 3. The protein with the exon 3-encoded domain was expressed and secreted in glycosylated form from baby hamster kidney (BHK) cells, purified to homogeneity, and sequenced; the amino acid sequence was identical to that predicted from liver cDNA. The cloned hGHBP competed in a dose-dependent fashion for binding of 125I-labeled 22-kilodalton (kDa) hGH, and at higher concentrations for binding of 125I-labeled 20-kDa hGH, to IM-9 lymphocytes. hGHBP decreased the association rate of [125I]hGH to the cells without decreasing the dissociation rate. hGHBP blocked the down-regulation of GH receptor in IM-9 cells by both 22- and 20-kDa hGH. hGHBP also blocked the binding of [125I]hGH to PRL receptors on Nb2 lymphoma cells and the effect of the hormone on thymidine incorporation. Binding of both 22- and 20-kDa hGH to the binding protein was demonstrated directly by immunoprecipitation with monoclonal antibody 263. The present work thus establishes the identity of the IM-9 human GHBP from those of liver and placenta, and demonstrates its ability to bind both 22- and 20-kDa hGH with good affinity and to block their biological actions mediated though both somatogenic and lactogenic receptors. The modulation of receptor down-regulation by the BP may be a relevant facet of its physiological role.

Catecholamine-containing biodegradable microsphere implants as a novel approach in the treatment of CNS neurodegenerative disease. A review of experimental studies in DA-lesioned rats.

Biodegradable controlled-release microsphere systems made with the biocompatible biodegradable polyester excipient poly(DL-lactide-co-glycolide) constitute an exciting new technology for drug delivery to the central nervous system (CNS). Implantable controlled-release microspheres containing dopamine (DA) or norepinephrine (NE) provide a novel means to compare DA- or NE -induced restitution of function in unilateral 6-hydroxydopamine lesioned rats. A suspension of 3 microL of DA- or NE-containing microspheres or empty microspheres was implanted in 2 sites of the DA denervated striatum of rats previously unilaterally lesioned with 6-hydroxydopamine. Contralateral-rotational behavior induced by apomorphine was used as an index of lesion success and, following implantation of the microspheres, also as an index of functional recovery. Interestingly, both DA- and NE-microsphere-implanted rats displayed a 30-50% reduction in the number of apomorphine-induced rotations up to 8 wk postimplantation. Rats implanted with empty microspheres did not demonstrate significant changes in contralateral rotational behavior. Behavioral studies following implantation of a mixture of DA and NE microspheres revealed an 80% decrease in the number of apomorphine induced rotations up to 4 wk. On conclusion of the studies, immunocytochemical examination revealed growth of DA and tyrosine hydroxylase immunoreactive fibers in the striatum of DA and NE microsphere-implanted rats. Functional behavior appeared to correlate with the degree of fiber growth. Preliminary electron microscopic studies showed signs of axonal sprouting in the vicinity of the implanted microspheres. No growth was noted in rats implanted with empty microspheres. This report reviews the abilities of both microencapsulated NE and DA to assure functional recovery and to promote DA fiber (re)growth in parkinsonian rats. This novel means to deliver these substances to the central nervous system could be of therapeutic usefulness in Parkinson's disease.

Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2.

Starting from total pancreatic mRNAs, the classical guinea pig pancreatic lipase was cloned using rapid amplification of 3' and 5' cDNA ends. Internal oligonucleotide primers were designed from a partial cDNA clone including the region coding for the lid domain. Using this strategy, we did not amplify the cDNA corresponding to the pancreatic lipase related protein 2 in which the lid domain is deleted. Amino acid sequences of the classical guinea pig pancreatic lipase and the related protein 2 were compared based on the primary and tertiary structures of the classical human pancreatic lipase. Their distinct physiological roles are discussed in the light of functional amino acid differences.

One-step purification and characterization of human pancreatic lipase expressed in insect cells.

A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post-infection using both anti-HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation-exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N-terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re-activation by colipase.

(-)-Pindolol stereospecifically inhibits rat brain serotonin (5-HT) synthesis.

(-)-Pindolol (4 mg/kg, i.p.) stereospecifically and selectively inhibited rat brain 5-HT synthesis. The DA and NA synthesis remained unaffected by pindolol (either enantiomer) treatment, and the (-)-pindolol-induced decrease in 5-HT synthesis was not prevented by reserpine pretreatment. The results indicate that, in addition to its beta-adrenergic properties, (-)-pindolol may act as an agonist at synthesis-controlling 5-HT receptors (autoreceptors ?) in the rat CNS.

Lack of 5-HT1A autoreceptor desensitization following chronic citalopram treatment, as determined by in vivo microdialysis.

Electrophysiological studies suggest that 5-HT autoreceptor desensitization may be responsible for the delayed clinical efficacy of some antidepressant drugs, such as selective 5-HT reuptake inhibitors (SSRI) and certain MAO inhibitors (MAOI). In the present study we have used in vivo microdialysis to test this hypothesis. Rats were treated for 2 weeks with the antidepressant SSRI citalopram (5 mg/kg, s.c., b.i.d.). After 24 hr withdrawal, dialysis probes were implanted in the dorsal hippocampus (DH) and the frontal cortex (FCx). The rats then received as acute challenge, a 5-HT1A autoreceptor-active dose of the reference 5-HT1A agonist 8-OH-DPAT (0.025 mg/kg s.c.). The 8-OH-DPAT-induced changes in dialysate 5-HT from the DH and the FCx were monitored and taken as an index of autoreceptor sensitivity. Chronic citalopram and control animals responded similarly to 8-OH-DPAT with a drop of 5-HT of about 50-65%; no significant difference between the chronic citalopram and control groups were obtained, either in the DH or in the FCx. These data suggest that cell body 5-HT1A autoreceptors do not desensitize in response to repeated administration with antidepressant SSRI drugs such as citalopram.

The role of 5-HT1A autoreceptors and alpha 1-adrenoceptors in the inhibition of 5-HT release--II NAN-190 and SDZ 216-525.

Novel 5-HT1A receptor antagonists, WAY 100135 and WAY 100635, were used to test the involvement of 5-HT1A receptors in the decrease of hippocampal extracellular 5-HT induced by the 5-HT1A/alpha 1 ligands, NAN-190 and SDZ 216-525. Using microdialysis in anaesthetized rats, it was found that WAY 100135 (3 mg/kg s.c.) and WAY 100635 (0.3 mg/kg s.c.) antagonised the decrease of 5-HT induced by the 5-HT1A receptor agonist 8-OH-DPAT (0.025 mg/kg s.c.) but did not alter 5-HT when administered alone. Both NAN-190 (0.03 and 0.3 mg/kg s.c.) and SDZ 216-525 (1 mg/kg s.c.) decreased 5-HT. The effect of 0.03 mg/kg s.c. NAN-190 was antagonised by WAY 100135 (3 mg/kg s.c.) and WAY 100635 (0.3 mg/kg s.c.). The effect of SDZ 216-525 (1 mg/kg s.c.) was also blocked by WAY 100635 (0.3 mg/kg s.c.). However, the 5-HT response to a high dose of NAN-190 (0.3 mg/kg s.c.) was not antagonised by WAY 100635 (0.3 or 3 mg/kg s.c.). Our experiments using WAY 100635 and WAY 100135 provide clear evidence that NAN-190 and SDZ 216-525 act as agonists at the 5-HT1A autoreceptor, supporting our earlier studies using the non-selective 5-HT1A antagonist, pindolol. However, our data reveal that, at least in the case of NAN-190, non-5-HT1A receptor mechanisms mediate the decrease of 5-HT induced by higher doses. A lack of specificity of NAN-190 (and possibly SDZ 216-525) at high doses may explain the failure of previous studies to detect a 5-HT1A receptor agonist action.

Central monoaminergic effects of two aporphine analogues to the putative serotonin-receptor agonist, 8-hydroxy-2-di-n-propylaminotetralin.

Two aporphine analogues to the potent and selective serotonin (5-HT) agonist, 8-hydroxy-2-di-n-propylaminotetralin (8-OH-DPAT) were studied with regard to their actions upon central monoaminergic systems. The biochemical and behavioural findings indicate that 8-hydroxy-N-methyl-aporphine (8-OH aporphine) and its N-n-propyl analogue (8-OH PNA) possess weak dopamine/noradrenaline antagonist- and agonist-like properties, respectively. In addition, both aporphines seemed to only weakly influence serotoninergic transmission. The latter findings are compared to those of known serotonin agonists (e.g. 8-OH-DPAT), and are discussed with particular reference to the structural requirements for activation of central 5-HT receptors.

Differential inhibition of serotonin release by 5-HT and NA reuptake blockers after systemic administration.

The inhibition of serotonin (5-HT) release produced by antidepressants varying in relative selectivity for blocking uptake of 5-HT and noradrenaline (NA) was compared. Release was measured by microdialysis in anesthetized rats with nerve terminal 5-HT uptake inhibited by local infusion of citalopram (1 microM) through a dialysis probe in hippocampus. With 5-HT uptake first blocked in hippocampus, systemic injection of uptake inhibitors produced decreases in dialysate 5-HT, presumably due to autoreceptor stimulation in the raphe. The largest decreases (about 60-70%) in 5-HT were produced by the selective 5-HT uptake inhibitors sertraline, paroxetine and citalopram. Nonselective blockers caused less suppression of release. Thus, the maximum decrease in 5-HT was 35% after clomipramine, a less selective 5-HT uptake inhibitor, and < or = 30% after the nonselective 5-HT/NA uptake blockers imipramine and amitriptyline, 5-HT was not decreased after maprotiline, a selective NA uptake blocker. Pretreatment with (+)WAY100135 to block 5-HT1A autoreceptors, abolished the inhibition of 5-HT release produced by systemic sertraline, clomipramine and imipramine. One explanation for the difference between selective and nonselective inhibitors with respect to central 5-HT release, is the excitatory effect of (alpha 1) adrenergic receptor stimulation on 5-HT neuronal discharge. However, pretreatment with alpha-methyl-p-tyrosine to deplete NA, did not influence the inhibition of 5-HT release produced by imipramine.

WAY100635-induced augmentation of the 5-HT-elevating action of citalopram: relative importance of the dose of the 5-HT1A (auto)receptor blocker versus that of the 5-HT reuptake inhibitor.

The elevation of extracellular 5-HT after systemic administration of 5-HT reuptake inhibiting drugs is strongly potentiated by agents capable of blocking 5-HT1A autoreceptors in the midbrain raphe. The present in vivo microdialysis study was aimed at assessing the relative importance of 5-HT reuptake inhibition versus 5-HT1A autoreceptor blockade in this interaction. Citalopram (0.5 or 5.0 mg/kg s.c.) dose-dependently increased dialysate 5-HT in the rat ventral hippocampus, maximally doubling the initial baseline values within 60 min after injection. The selective 5-HT1A receptor blocker, WAY100635 (0.01-0.3 mg/kg s.c.), further augmented, in a dose-dependent manner, the high-dose citalopram response (to approximately 4-5 x the pre-citalopram baseline). For comparison, the effect of low-dose (0.5 mg/kg s.c.) citalopram was mildly, but not significantly, potentiated by WAY100635 (0.3 mg/kg). WAY100635 given alone does not alter 5-HT under these conditions. The data confirm previous findings that 5-HT1A autoreceptor blockade enhances the citalopram-induced increase of extracellular 5-HT in the forebrain. To the extent the extracellular levels of 5-HT is a valid index, through 5-HT reuptake blockade appears to be the primary prerequisite for this interaction to occur. New drugs and/or treatment regimes based on the SSRI/5-HT1A autoreceptor blocker combination concept should, therefore, emphasize the former property.

Autoreceptor antagonists enhance the effect of the reuptake inhibitor citalopram on extracellular 5-HT: this effect persists after repeated citalopram treatment.

The effect of repeated administration of the reuptake inhibitor citalopram (10 mg/kg s.c., b.i.d. for 14 days) or saline on extracellular 5-hydroxytryptamine (5-HT) and autoreceptor sensitivity was assessed using microdialysis in the frontal cortex (FCx) and dorsal hippocampus (DH) of unanesthetized rats. Acute citalopram (5 mg/kg s.c.) challenge produced significant increases in DH and FCx 5-HT. The nonselective 5-HT1A/1B receptor antagonist (-)+penbutolol (8 mg/kg s.c.), administered 2 hr after citalopram challenge, significantly enhanced 5-HT in FCx and DH of both the chronic citalopram and saline pretreatment groups. Administration of the selective 5-HT1A receptor antagonist WAY 100635 (0.3 mg/kg s.c.) after citalopram challenge significantly enhanced 5-HT in FCx but not DH of both pretreatment groups. This suggests that there may be differences between DH and FCx in regulation of 5-HT release. Nevertheless, these results provide evidence that 5-HT autoreceptors are still active in restraining 5-HT release. Nevertheless, these results provide evidence that 5-HT autoreceptors are still active in restraining 5-HT release even after repeated administration of an antidepressant drug.

The role of 5-HT1A autoreceptors and alpha1-adrenoceptors in the modulation of 5-HT release--III. Clozapine and the novel putative antipsychotic S 16924.

Clozapine and the novel putative, antipsychotic S 16924 ((1-(benzodioxane-5-yl)-3-[3-(4-fluorophenacyl)pyrrolidine]-1-o xapropane HCl) share significant affinity for alpha1-adrenoceptors and 5-HT1A autoreceptors in vitro and display an 'atypical' behavioural profile in in vivo models used for detecting potential neuroleptic effects. In the present study, in vivo microdialysis was used to examine the effect of clozapine and S 16924 on 5-HT overflow in the rat ventral hippocampus, and to assess the relative role of putative alpha1-adrenoceptor antagonist and 5-HT1A autoreceptor agonist properties of the drugs in this regard. S 16924 (0.1-3 mg/kg, s.c.) reduced dialysate 5-HT in a dose- and time-dependent fashion by maximally approximately 70% from baseline 40-60 min after injection. Clozapine (0.1-10 mg/kg, s.c.) reduced 5-HT overflow in the same manner, with a maximum effect of approximately 60% from baseline, obtained after 60-80 min. The 5-HT decrease elicited by S 16924 (1.0 mg/kg, s.c.) was significantly, though only partially, antagonized by pretreatment with the selective 5-HT1A receptor antagonist WAY 100635 (0.3 mg/kg, s.c.). The selective alpha1-adrenoceptor agonist cirazoline (0.02 mg/kg, i.p.) alone did not significantly attenuate the effect of S 16924 (1.0 mg/kg, s.c.) on 5-HT overflow. Combined treatment with both WAY 100635 and cirazoline, however, totally reversed the 5-HT-suppressing effect of S 16924 (1.0 mg/kg, s.c.). By comparison, when given separately, neither WAY 100635 (0.3 mg/kg, s.c.) nor cirazoline (0.02 mg/kg, i.p.) antagonized the clozapine (0.3 mg/kg, s.c.)-induced decrease of 5-HT in ventral hippocampus dialysates. In the presence of both WAY 100635 and cirazoline, the response to this dose of clozapine was however significantly, though modestly, attenuated. In contrast, the WAY 100635/cirazoline combination failed to antagonise the 5-HT decrease resulting from a higher dose (3.0 mg/kg, s.c.) of clozapine. We conclude that both alpha1-adrenoceptor antagonist and 5-HT1A receptor agonist properties of clozapine and S 16924 contribute to the 5-HT release-reducing action of these drugs. Whereas these factors apparently explain the effect of S 16924 fully, additional mechanism(s) appear to be involved in the case of clozapine. With regard to the interplay between alpha1-adrenoceptor and 5-HT1A (auto)receptor mechanisms in the control of 5-HT release in the rat forebrain, the present data suggest that an excitation mediated by the former is outweighed by the simultaneous activation of the latter-inhibitory-receptors.

Studies on the role of 5-HT1A autoreceptors and alpha 1-adrenoceptors in the inhibition of 5-HT release--I. BMY7378 and prazosin.

UNLABELLED: The present study utilized in vivo microdialysis to investigate the importance of 5-HT1A autoreceptors and alpha 1-adrenoceptors in the decreased 5-HT release obtained following administration of the mixed 5-HT1A autoreceptor partial agonist/alpha 1-adrenoceptor antagonist BMY7378, the selective 5-HT1A receptor agonist 8-OH-DPAT and the alpha 1-adrenoceptor antagonist prazosin. BMY7378 (0.25 mg/kg, s.c.), 8-OH-DPAT (0.025 mg/kg, s.c.) and prazosin (0.1-1.0 mg/kg, s.c.) all suppressed ventral hippocampal 5-HT efflux. The BMY7378- and 8-OH-DPAT-induced inhibition of 5-HT release were reversed by a 40 min pre-treatment with either (+/-)pindolol (8 mg/kg, s.c.) or WAY-100635 (0.3 mg/kg, s.c.), to block 5-HT1A autoreceptors. Neitehr of these antagonists altered the prazosin-induced (0.3 mg/kg, s.c.) 5-HT disease. THE RESULTS: (i) confirm that both an alpha 1-adrenoceptor antagonist (prazosin) and 5-HT1A autoreceptor stimulants (BMY7378 and 8-OH-DPAT) may reduce cerebral 5-HT release; (ii) support that the BMY7378-induced decrease in 5-HT release results from 5-HT1A autoreceptor agonism, rather than alpha 1-adrenoceptor blockade; and (iii) argue against "physiological" antagonism (i.e. via blockade of beta-adrenoceptors, 5-HT1B receptors or some other mechanism) as an explanation for the reversal by pindolol of 5-HT1A autoreceptor agonist-induced suppression of 5-HT release. These data support the usefulness of pindolol, as well as the more specific compound WAY-100635, to block 5-HT1A autoreceptors.

Pivaloyl esters of N,N-dialkylated dopamine congeners. Central dopamine-receptor stimulating activity.

In order to test for dopamine-receptor stimulating activity a new, sensitive biochemical screening method was designed. For behavioral studies and for determination of the duration of action on the compounds, motor activity measurements were used. O,O'-Dipivaloyl-N,N-dipropyldopamine (4) was the only derivative of a series of dipivaloyl-N,N-dialkyldopamines studied that showed any significant activity. However, the monopivaloyl ester 2-(3-pivaloyloxyphenyl)-N,N-dipropylethylamine (8) seemed to be more potent. The same relationship was found for the corresponding phenols, N,N-dipropyldopamine (3) and 2-(3-hydroxyphenyl)-N,N-dipropylethylamine (7), although both were more active than their pivaloyl esters.