Regulation of presynaptic neurotransmission by macroautophagy.

mTOR is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and autophagic degradation of cellular components. When triggered by mTOR inactivation, macroautophagy degrades long-lived proteins and organelles via sequestration into autophagic vacuoles. mTOR further regulates synaptic plasticity, and neurodegeneration occurs when macroautophagy is deficient. It is nevertheless unknown whether macroautophagy modulates presynaptic function. We find that the mTOR inhibitor rapamycin induces formation of autophagic vacuoles in prejunctional dopaminergic axons with associated decreased axonal profile volumes, synaptic vesicle numbers, and evoked dopamine release. Evoked dopamine secretion was enhanced and recovery was accelerated in transgenic mice in which macroautophagy deficiency was restricted to dopaminergic neurons; rapamycin failed to decrease evoked dopamine release in the striatum of these mice. Macroautophagy that follows mTOR inhibition in presynaptic terminals, therefore, rapidly alters presynaptic structure and neurotransmission.

The pharmacokinetics and pharmacological effect of (S)-5-OH-DPAT following controlled delivery with transdermal iontophoresis.

The pharmacokinetic (PK) and pharmacodynamic (PD) properties of the active (S)-enantiomer of the potent dopamine (DA) agonist 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT) were investigated in a novel anesthetized animal model. First, the relationship between current density, in vivo transport, and plasma profile was characterized. Second, the effect of the anesthetic mixture, transdermal iontophoresis, and blood sampling on the striatal DA release (PD end point) was investigated. Third, the PK-PD relationship following transdermal iontophoresis was investigated during a controlled reversible pharmacological response. Given that striatal DA levels are unaltered during experimental procedures, this rat model can be used to investigate the PK-PD relationship. The in vivo flux was linearly correlated with the current density, indicating that drug delivery can be titrated by the current density. Following transdermal iontophoresis and intravenous infusion, a strong reversible effect was observed. Compartmental modeling showed that the relationship between plasma concentration and biomarker response is best characterized by an effect compartment, rather than an indirect response model. In addition, covariate analysis suggested that the delivery rate can affect the PD efficiency. Finally, PK-PD analysis revealed that steady delivery rates are translated into continuous dopaminergic stimulation. This can be of benefit for reducing side effects in the symptomatic treatment of Parkinson's disease with 5-OH-DPAT.

The in vitro and in vivo evaluation of new synthesized prodrugs of 5-OH-DPAT for iontophoretic delivery.

The feasibility of transdermal iontophoretic transport of 4 novel ester prodrugs of 5-OH-DPAT (glycine-, proline-, valine- and beta-alanine-5-OH-DPAT) was investigated in vitro and in vivo. Based on the chemical stability of the prodrugs, the best candidates were selected for in vitro transport studies across human skin. The pharmacokinetics and pharmacodynamic effects of the prodrug with highest transport efficiency, were investigated in a rat model. The in vitro transport, plasma profile and pharmacological response were analyzed with compartmental modeling. Valine- and beta-alanine-5-OH-DPAT were acceptably stable in the donor phase and showed a 4-fold and 14-fold increase in solubility compared to 5-OH-DPAT. Compared to 5-OH-DPAT, valine- and beta-alanine-5-OH-DPAT were transported less and more efficiently across human skin, respectively. Despite a higher in vitro transport, lower plasma concentration was observed following 1.5h current application (250 microAcm(2)) of beta-alanine-S-5-OH-DPAT in comparison to S-5-OH-DPAT. However the prodrug showed higher plasma concentrations post-iontophoresis, explained by a delayed release due to hydrolysis and skin depot formation. This resulted in a pharmacological effect with the same maximum as 5-OH-DPAT, but the effect lasted for a longer time. The current findings suggest that beta-alanine-5-OH-DPAT is a promising prodrug, with a good balance between stability, transport efficiency and enzymatic conversion.

(+)-UH 232, a partial agonist of the D3 dopamine receptors, attenuates cognitive effects of angiotensin IV and des-Phe(6)-angiotensin IV in rats.

We have recently found that postsynaptic D3 dopamine (DA) receptors appear not to participate in the memory enhancing effects of the angiotensin AT4 receptor agonists angiotensin IV (Ang IV) and des-Phe(6)-Ang IV. In this study we evaluated role of the presynaptic DA D3 receptors in these effects. For that purpose effect of (+)-UH 232, a selective D3 DA receptors partial agonist preferring presynaptic sites, on the pro-cognitive action of intracerebroventricularly (icv) injected Ang IV and des-Phe(6)-Ang IV was examined. Male Wistar rats weighing 180-200 g were used. Both peptides given at the dose of 1 nmol facilitated recall of a passive avoidance (PA) behaviour, improved object recognition (OR), and increased apomorphine-induced stereotype behaviour. In the auxiliary tests performed to control for the unspecific influence of motor (open field, OF) and emotional ('plus' maze, PM) effects of our treatments on the results of the memory tests they had either no (OF) or negligible (PM) effects. Intraperitoneal pre-treatment of the animals with an ineffective on its own dose (1 mg/kg) of (+)-UH 232 abolished or markedly diminished effects of both peptides on PA and OR but did not influence enhancement of stereotypy caused by the peptides.

Mechanistic studies of the transdermal iontophoretic delivery of 5-OH-DPAT in vitro.

A characterization and optimization of the in vitro transdermal iontophoretic transport of 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT) is presented. The utility of acetaminophen as a marker of electroosmotic flow was studied as well. The following parameters of iontophoretic transport of 5-OH-DPAT were examined: drug donor concentration, electroosmotic contribution, influence of co-ions, current density, and composition of the acceptor phase. The steady-state flux (Flux(ss)) of acetaminophen was linearly correlated with the donor concentration and co-iontophoresis of acetaminophen did not influence the iontophoretic flux of 5-OH-DPAT, indicating that acetaminophen is an excellent marker of electroosmotic flow. Lowering the Na(+) concentration from 78 to 10 mM in the donor phase, resulted in a 2.5-fold enhancement of the Flux(ss). The Flux(ss) showed a nonlinear relation with the drug donor concentration and an excellent linear correlation with the current density. Reducing the pH of the acceptor phase from 7.4 to 6.2 resulted in a dramatic decrease of the Flux(ss) of 5-OH-DPAT, explained by a reduced electroosmotic flow and an increased counter-ion flow. Optimization of the conditions resulted in a maximum Flux(ss) of 5-OH-DPAT of 1.0 micromol x cm(-2) h(-1) demonstrating the potential of the iontophoretic delivery of this dopamine agonist for the symptomatic treatment of Parkinson's disease.

Circadian and ultradian rhythms in the crayfish caudal photoreceptor.

The study of circadian clocks in crustaceans has led to the hypothesis of a distributed circadian system of pacemakers. In this review, we investigate the role of the crayfish caudal photoreceptor (CPR) as a candidate to form part of this pacemaking circadian system. Two circadian rhythms are documented for CPR electrical activity. These rhythms correspond to the spontaneous and light-induced discharge of action potentials. The intrinsic characterization of the rhythms is made through the analysis of the firing rate of the corresponding action potentials. The discharges were extracellularly recorded in the isolated 6th abdominal ganglion (AG) in an organ culture kept at constant temperature for up to 5 days. For preparations kept in the dark, spontaneous activity varies in a circadian manner, with a period of 24.7 h and the acrophase at subjective nighttime (2140). For light-induced activity, pulses of constant intensity applied regularly throughout the 24-h cycle show that the firing rate at peak and latency vary rhythmically. The period for this rhythm is 24.24 h and the acrophase is at subjective dawn (0326). Additionally, an ultradian rhythm of a approximately 12-h period was observed for both rhythms. When tested with light pulses of different intensities, the CPR responsiveness at night is almost one log unit greater than in daytime. The effect of temperature on both activities is also described. The phase-shift caused by temperature for these circadian rhythms depends on the application time. These results show that the 6th AG is capable of generating a circadian rhythm of electrical activity in the CPR, which in turn is likely to be part of the crayfish circadian system. A possible interaction of different pacemakers forming the distributed circadian system is also discussed. The role of serotonin as a possible modulator of the CPR electrical activity is documented. In addition, the level of the 5-HT(1A) receptors displays a diurnal rhythm in the 6th AG, with the acrophase at twilight (1849). We suggest that the 5-HT(1A) receptor does participate in this modulation. Finally, the hypothesis of the expression of two circadian oscillators in a single identified neuron is presented.

Involvement of brain serotonin 5-HT1A receptors in genetic predisposition to aggressive behavior.

Experiments were performed on Norwegian rats selected over more than 59 generations for high and low levels of high-affective defensive aggressivity and on highly aggressive (offensive) Tg8 mice with irreversible monoamine oxidase A knockout. There were significant differences in the functional state and expression of 5-HT(1A) receptors between highly aggressive and non-aggressive animals. Functional activity assessed in terms of hypothermia evoked by a 5-HT(1A) agonist was significantly greater in non-aggressive rats and mice than in aggressive animals. The high level of functional activity in non-aggressive rats coincided with a greater level of expression of 5-HT(1A) receptors in the midbrain. The level of 5-HT(1A) receptor mRNA in aggressive mice was unchanged in the midbrain and hypothalamus and was increased in the frontal cortex and amygdaloid complex. These results led to the conclusion that 5-HT(1A) receptors play a significant role in the mechanisms of genetic predisposition to aggressive behavior.

Assays for enhanced activity of low efficacy partial agonists at the D(2) dopamine receptor.

BACKGROUND AND PURPOSE: Low efficacy partial agonists at the D2 dopamine receptor may be useful for treating schizophrenia. In this report we describe a method for assessing the efficacy of these compounds based on stimulation of [35S]GTPgammaS binding. EXPERIMENTAL APPROACH: Agonist efficacy was assessed from [(35)S]GTPgammaS binding to membranes of CHO cells expressing D2 dopamine receptors in buffers with and without Na+. Effects of Na+ on receptor/G protein coupling were assessed using agonist/[3H]spiperone competition binding assays. KEY RESULTS: When [35S]GTPgammaS binding assays were performed in buffers containing Na+, some agonists (aripiprazole, AJ-76, UH-232) exhibited very low efficacy whereas other agonists exhibited measurable efficacy. When Na+ was substituted by N-methyl D-glucamine, the efficacy of all agonists increased (relative to that of dopamine) but particularly for aripiprazole, aplindore, AJ-76, (-)-3-PPP and UH-232. In ligand binding assays, substitution of Na+ by N-methyl D-glucamine increased receptor/G protein coupling for some agonists -. aplindore, dopamine and (-)-3-PPP - but for aripiprazole, AJ-76 and UH-232 there was little effect on receptor/G protein coupling. CONCLUSIONS AND IMPLICATIONS: Substitution of Na+ by NMDG increases sensitivity in [(35)S]GTPgammaS binding assays so that very low efficacy agonists were detected clearly. For some agonists the effect seems to be mediated via enhanced receptor/G protein coupling whereas for others the effect is mediated at another point in the G protein activation cycle. AJ-76, aripiprazole and UH-232 seem particularly sensitive to this change in assay conditions. This work provides a new method to discover these very low efficacy agonists.

The 5-HT(1A) receptor active compounds (R)-8-OH-DPAT and (S)-UH-301 modulate auditory evoked EEG responses in rats.

Schizophrenics commonly demonstrate abnormalities in central filtering capability following repetitive sensory stimuli. Such sensory inhibition deficits can be mirrored in rodents following administration of psycho-stimulatory drugs. In the present study, male Sprague-Dawley rats were implanted with brain surface electrodes to record auditory evoked EEG potentials in a paired-stimulus paradigm, using 87 dB clicks delivered 0.5 s apart. Amphetamine (1.83 mg/kg, i.p.) produced the expected loss of sensory inhibition, as defined by an increase in the ratio between test (T) and conditioning (C) amplitudes at N40, a mid-latency peak of the evoked potentials. Also, the 5-HT(1A) agonist (R)-8-OH-DPAT caused a significant increase in the TC ratio at the highest dose studied (0.5 mg/kg s.c.), while the 5-HT(1A) antagonist (S)-UH-301 did not significantly affect the TC ratio at any dose studied (0.1-5 mg/kg s.c.). When administered with amphetamine, a lower dose of 8-OH-DPAT (0.1 mg/kg) and the highest dose of UH-301 tested (5 mg/kg, s.c.) were able to reverse the amphetamine-induced increase in TC ratio. The findings suggest that 5-HT(1A) signaling is involved in sensory inhibition and support the evaluation of 5-HT(1A) receptor active compounds in conditions with central filtering deficits, such as schizophrenia.

Pharmacokinetics and pharmacodynamics analysis of transdermal iontophoresis of 5-OH-DPAT in rats: in vitro-in vivo correlation.

Pharmacokinetics and dopaminergic effect of dopamine agonist 5-OH-DPAT in vivo were determined following transdermal iontophoresis in rats based on drug concentration in plasma (C(p)) and dopamine levels in striatum (C(DA)). Correlation of the in vitro transport with the pharmacokinetic-pharmacodynamic (PK-PD) profiles was characterized in the transport in dermatomed rat skin (DRS) and rat stratum corneum (RSC). The integrated in vivo PK-PD and in vitro transport models successfully described time course of C(p), C(DA), and in vitro flux in DRS and RSC. Population value of steady-state flux (J(ss)) in vivo (31 nmol/cm(2) . h with 95% confidence interval (CI) = 20-41) is closer to J(ss) in vitro in DRS (61 nmol/cm(2) . h, CI = 54-67) than in vitro J(ss) in RSC (98 nmol/cm(2) . h, CI = 79-117). On the other hand, skin release rate constant (K(R)) in vivo was similar to the K(R) in RSC (4.8/h, CI = 2.4-7.1 vs. 2.6/h, CI = 2.5-2.6). Kinetic lag time (t(L)) in vivo was negligible, which is close to in vitro t(L) in RSC (0.0 h, CI = 0.0-0.1). Based on nonlinear mixed-effect modeling, profiles of C(p) and C(DA) were successfully predicted using in vitro values of J(ss) in DRS with K(R) and t(L) in RSC. A considerable dopaminergic effect was achieved, indicating the feasibility to reach therapeutically effective concentrations of 5-OH-DPAT upon transdermal iontophoresis.

Reduction in 5-HT1A receptor density, 5-HT1A mRNA expression, and functional correlates for 5-HT1A receptors in genetically defined aggressive rats.

The present experiments tested the hypothesis that one of the critical mechanisms underlying genetically defined aggressiveness involves brain serotonin 5-HT1A receptors. 5-HT1A receptor density, the receptor mRNA expression in brain structures, and functional correlates for 5-HT1A receptors identified as 8-OH-DPAT-induced hypothermia and lower lip retraction (LLR) were studied in Norway rats bred for 59 generations for the lack of aggressiveness and for high affective aggressiveness with respect to man. Considerable differences between the highly aggressive and the nonaggressive rats were shown in all three traits. A significant decrease in B(max) of specific receptor binding of [3H]8-OH-DPAT in the frontal cortex, hypothalamus, and amygdala and a reduction in 5-HT1A receptor mRNA expression in the midbrain of aggressive rats were found. 5-HT1A receptor agonist 8-OH-DPAT (0.5 mg/kg, i.p.) produced a distinct hypothermic reaction in nonaggressive rats and did not affect significantly the body temperature in aggressive rats. Similar differences were revealed in 8-OH-DPAT-induced LLR: LLR was expressed much more in nonaggressive than in aggressive animals. Additionally, 8-OH-DPAT (0.5 mg/kg i.p.) treatment significantly attenuated the aggressive response to man. The results demonstrated an association of aggressiveness with reduced 5-HT1A receptor expression and function, thereby providing support for the view favoring the idea that brain HT1A receptor contributes to the genetically defined individual differences in aggressiveness.

Transdermal iontophoresis of the dopamine agonist 5-OH-DPAT in human skin in vitro.

The feasibility of transdermal iontophoretic delivery of a potent dopamine agonist 5-OH-DPAT was studied in vitro in side by side diffusion cells across human stratum corneum (HSC) and dermatomed human skin (DHS) according to the following protocol: 6 h of passive diffusion, 9 h of iontophoresis and 5 h of passive diffusion. The influences of the following parameters on the flux were studied: donor solution pH, NaCl concentration, drug donor concentration, current density and skin type. A current density of 0.5 mA cm(-2) was used, except for one series of experiments to study the current density effect. Probably due to the influence of the skin perm-selectivity and the competition with H(+), increase in pH from 3 to 5 resulted in a significant increase in flux. Further increase in pH to 6 did not further increase the flux. The iontophoretic transport was found to increase linearly with concentration and current density, providing a convenient way to manage dose titration for Parkinson's disease therapy. Increase in concentration of NaCl dramatically reduced the flux of 5-OH-DPAT as a result of ion competition to the transport. When DHS was used, the iontophoretic transport was less. Also, with DHS the response in flux profile, by switching the current on and off, was shallower than that with HSC. With the optimum condition, a delivery of 104 microg of 5-OH-DPAT per cm(2) patch per hour is feasible, indicating that the therapeutic level could be achieved with a smaller patch size than required in case of rotigotine. Thus, based on this in vitro study, transdermal iontophoretic delivery of 5-OH-DPAT is very promising.

Binding characteristics of high-affinity dopamine D2/D3 receptor agonists, 11C-PPHT and 11C-ZYY-339 in rodents and imaging in non-human primates by PET.

We have evaluated the in vitro autoradiographic binding characteristics and in vivo brain distribution of two high-affinity dopamine D2/D3 receptor agonists, (+/-)-2-(N-phenethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-PPHT) and (+/-)-2-(N-cyclohexylethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-ZYY-339) in rodents and in monkeys using positron emission tomography (PET). In vitro autoradiograms in rat brain slices with (11)C-PPHT and 11C-ZYY-339 revealed binding to dopaminergic regions in the striata, which was substantially (>90%) displaced by 10 microM sulpiride. Striatal binding was also removed in the presence of 5-guanylylimidophosphate (Gpp(NH)p), indicative of binding of these radiotracers to the high-affinity (HA) state. The results of in vivo studies in rats exhibited binding of the two radiotracers to the striata (striata/cerebellum approached 2 in 30 min). The regional selectivity to the striata was reduced by preadministration of haloperidol. PET studies in male rhesus monkeys using an ECAT EXACT HR+ scanner indicated localization of 11C-PPHT and 11C-ZYY-339 in the striata and thalamus. Striata to cerebellum and thalamus to cerebellum ratios were low (1.5 and 1.3, respectively, at 30 min postinjection) for both 11C-PPHT and 11C-ZYY-339, apparently due to the slower nonspecific clearance from cerebellum. These findings with 11C-PPHT and 11C-ZYY-339 indicate the possibility of in vivo imaging of high-affinity state of dopamine D2/D3 receptors in both the striata and the thalamus.

Serotonin reuptake inhibitors do not prevent 5,7-dihydroxytryptamine-induced depletion of serotonin in rat brain.

Although the selective toxicity of 5,7-dihydroxytryptamine (5,7-DHT) is thought to depend on the drug's transport into serotonin (5HT) neurons via the 5HT transporter, few studies have critically examined this postulation. We therefore evaluated if 5,7-DHT-induced reductions in 5HT concentrations and synthesis rate in rat brain are blocked by pretreatment with 5HT-selective reuptake inhibitors. Rats pretreated with desipramine (DMI) (to prevent norepinephrine depletion) received intracerebroventricular injections of 5,7-DHT (5, 50, 100, 200 microg/rat) 30 min after fluoxetine (20 mg/kg ip). Forty-eight hours later, they received m-hydroxybenzylhydrazine 30 min before sacrifice. The concentrations of 5HT and 5-hydroxytryptophan (5HTP, an index of 5HT synthesis) were measured in hypothalamus, cortex and brainstem. Each 5,7-DHT dose produced significant reductions in 5HT and 5HTP concentrations in all regions examined (5 microg reduced 5HT but not 5HTP), effects that were not blocked by fluoxetine. Two other 5HT reuptake blockers (chlorimipramine, alaproclate) also failed to block the 5HT and 5HTP depleting actions of 5,7-DHT. Desipramine blocked 5,7-DHT-induced norepinephrine (NE) depletion. Pretreatment with the 5HT receptor antagonist metergoline, or the 5HT(1A) agonist 8-hydroxy-(di-n-propylamino)tetralin (to slow 5HT neuronal firing rate) also failed to antagonize the 5HT depleting action of 5,7-DHT. Together, the data strongly suggest that the mechanism by which 5,7-DHT depletes the brain of serotonin does not involve 5HT-transporter-mediated concentration of neurotoxin in 5HT neurons, may not involve 5HT receptor interaction, and does not depend on the firing rate of the 5HT neuron.

Nad-299 antagonises 5-HT-stimulated and spiperone-inhibited [35S]GTPgammaS binding in cloned 5-HT1A receptors.

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxytryptamine1A A (5-HT1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-3H]-3,4-dihydro-2H-1-benzopyan-5-carboxamide ([3H]NAD-299) exhibited high affinity (Kd = 0.16 nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-3H]di-n-propylamino)tetralin ([3H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [35S]GTPgammaS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [35S]GTPgammaS binding 2.5-fold while spiperone and methiothepin inhibited [35S]GTPgammaS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [35S]GTPgammaS binding to basal levels. The KiL/KiH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (+/-)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [35S] GTPgammaS (r = 0.97).

Stereoselective synthesis of tetrasubstituted (Z)-alkenes from aryl alkyl ketones utilizing the Horner-Wadsworth-Emmons reaction.

Tetrasubstituted (Z)-alkenes were readily prepared through the Horner-Wadsworth-Emmons reactions of methyl 2-[bis(2,2,2-trifluoroethyl)phosphono]propionate with aryl alkyl ketones by employing Sn(OSO(2)CF(3))(2) and N-ethylpiperidine.

Fear behavior and regional brain monoamines distribution after R(+)-8-OHDPAT and R(+)-UH-301 injections into the dorsal raphe nucleus in rats.

The effects of 8-OHDPAT and UH-301 injection into the dorsal raphe nucleus (DRN) on fear behavior of the light-dark transitions test and regional brain monoamines (NA, DA, 5-HT) and their metabolites (MHPG, DOPAC, 5-HIAA) in the hypothalamus (HPT), midbrain central gray matter (MID), amygdala (AMY), hippocampus (HIP) and pons (PO) were examined. An injection of 8-OHDPAT (300 ng) as well UH-301 (300 ng) into the DRN evoked an increase in the number of head dipping from dark to the illuminated compartment of chamber, an increase of time of motionless in the dark compartment and decrease of time of locomotion activity in the illuminated compartment. HPLC analysis showed reduction of 5-HIAA/5-HT ratio in the HPT, HIP and PO, increase of MHPG/NA ratio in the HIP and PO, and increase of DA content in the HPT, AMY and HIP after 8-OHDPAT injection. But injection of UH-301 reduced 5-HT in the MID and increased in the AMY, reduced 5-HIAA content in the HIP and increased in the MID and decreased MHPG/NA ratio in the PO. These results indicate that both 5-HT1A receptor agonists, acting on the 5HT1A autoreceptors caused the anxiolytic effects, reduced fear behavior on the rat connected with infringement of dynamic balance between the serotonergic and catecholaminergics systems.

An integrative pharmacokinetic and pharmacodynamic study of the 5-HT1A receptor antagonist (S)-UH-301 in the rat.

(S)-UH-301 ((-)-(S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin hydrochloride) is a well known 5-HT(1A) receptor antagonist. The present study describes the pharmacokinetic properties of (S)-UH-301 after subcutaneous administration in rats, using a newly developed HPLC-UV bioanalytical method. The relationships between (S)-UH-301 concentrations and some pharmacodynamic effects were also studied. The AUC of (S)-UH-301 in brain, but not in plasma, increased in proportion to dose (1-100 mumol/kg). However, at doses above 32 mumol/kg, peak concentrations of the drug did not increase in proportion to dose, and there was a doubling of its apparent half-life. There was a good correspondence between the time courses for the antagonism of 8-OH-DPAT-induced motor behaviours and hypothermia and the tissue concentrations of (S)-UH-301. Doses of (S)-UH-301 above 10 mumol/kg decreased 5-HT and dopamine synthesis. Therefore, a selective 5-HT(1A) antagonistic dose range of (S)-UH-301 should be 0.1-10 mumol/kg s.c., corresponding to concentrations below approximately 10 nmol/g in brain and approximately 1 nmol/ml in plasma.

Real-time analysis of dopamine: antagonist interactions at recombinant human D2long receptor upon modulation of its activation state.

1. Antipsychotic drugs may mediate their therapeutic effects not only by preventing the binding of dopamine but also by decreasing the propensity of the dopamine receptor to assume an active R* state. Ligand-mediated activation and blockade of the recombinant human D(2long) receptor was investigated in CHO-K1 cells upon modulation of its R* state. 2. Both the Ala(371)Lys (A371K) and Thr(372)Arg (T372R) D2long receptor mutants could be activated in a ligand-dependent manner via a chimeric G(alphaq/o) protein, and more efficaciously so than with the promiscuous G(alpha15) protein. 3. Dopamine and partial agonists (E(max): lisuride >> (+)-UH 232 approximately bromerguride) displayed dissimilar Ca(2+) kinetic properties at wild-type and mutant receptors. A371K and T372R D2long receptor mutants demonstrated an attenuated and enhanced maximal response to these partial agonists, respectively. 4. Dopamine antagonists were unable to block the transient high-magnitude Ca(2+) phase at the wild-type D2long receptor upon simultaneous exposure to antagonist and dopamine, while full blockade of the low-magnitude Ca(2+) phase did occur at a later time (onset-time: haloperidol < bromerguride < (+)-butaclamol). A similar, though more efficacious, antagonist profile was also found at the A371K mutant receptor. Conversely, the blockade of the low-magnitude Ca(2+) phase was attenuated (haloperidol) or almost absent [(+)-butaclamol and bromerguride] at the T372R mutant receptor. 5. In conclusion, mutagenesis of the Ala(371) and Thr(372) positions affects in an opposite way the ligand-dependent activation and blockade of the D2long receptor. The observed attenuation of dopamine-mediated Ca(2+) signal generation with different decay-times may underlie distinct properties of the dopaminergic ligands.