Involvement of 5-HT6 receptors in nigro-striatal function in rodents

4-Amino-N-(2,4 bis-methylamino-pyrimidin-4-yl) benzene sulphonamide (Ro 04-6790) is a potent, selective and competitive antagonist for the 5-HT6 receptor which can be detected in the cerebro-spinal fluid (CSF) of rats following intraperitoneal administration. Since 5-HT6 receptor mRNA and 5-HT6 receptor-like immunoreactivity have been shown to be present in the striatum, the purpose of the present study was to evaluate the effect of 5-HT6 receptor antagonism on haloperidol- and SCH 23390-induced catalepsy in mice and on the turning behaviour of rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle. Ro 04-6790 (3, 10 and 30 mg kg(-1) i.p.) did not induce catalepsy and had no effect on catalepsy induced by either haloperidol or SCH 23390. Ro 04-6790 (3, 10 and 30 mg kg(-1) i.p.) did not itself induce rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle nor did it affect the rotational behaviour induced by either L-Dopa or amphetamine. 5-HT6 receptor antagonism inhibited the rotational behaviour of 6-OHDA lesioned rats induced by treatment with the muscarinic antagonists scopolamine and atropine. The data support earlier conclusions from experiments with antisense oligonucleotides that the 5-HT6 receptor is involved in the control of acetylcholine neurotransmission in the rat brain.     

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection

The title compounds (6-9) were prepared and evaluated for serotonin 5-HT4 agonistic activity in in vitro tests. Introducing a propyl or allyl group at the 3-position of benzamide caused only a slight enhancement of agonistic activity. Construction of the benzo[b]furan skeleton and 2,3-dihydrobenzo[b]furan skeleton caused a significant enhancement of the activity. 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2-methylbenzo[b]furan-7-carboxamide (7b) hemifumarate was as potent as cisapride. 4-Amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-methylbenzo[b]furan-7-carboxamide (8a) hemifumarate, 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-ethylbenzo[b]furan-7-carboxamide (8c) hemifumarate, and 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dimethylbenzo[b]furan-7-carboxamide (8d) hemifumarate were more potent than cisapride. Furthermore, 8a hemifumarate was free from dopamine D1, D2, serotonin 5-HT1, 5-HT2 and muscarine M1, M2 receptor binding activity in the in in vitro tests. On the other hand, construction of the indole skeleton caused a remarkable decrease in activity.     

Design and synthesis of a series of 6-substituted-2-pyridinylmethylamine derivatives as novel, high-affinity, selective agonists at 5-HT1A receptors

A search for novel, selective agonists with high intrinsic activity at the 5-HT1A subtype of serotonin (5-HT) receptors was undertaken. Mechanistic and thermodynamic considerations led to the design of 6-substituted-2-pyridinylmethylamine as a potential 5-HT1A pharmacophore. Various adducts derived from the 6-substituted-2-pyridinylmethylamine moiety were tested for their affinity at 5-HT1A, alpha1-adrenergic, and D2-dopaminergic receptors. Compounds with high affinity for 5-HT1A receptors (pKi >/= 8) were examined for agonist properties by measuring their ability to inhibit forskolin-stimulated cAMP production in HA7 cells (i.e., HeLa cells permanently transfected with the h5-HT1A receptor gene and expressing the h5-HT1A receptor protein). Several compounds of the type aryl?4-[(6-substituted-pyridin-2-ylmethylamino)methyl]piperidin -1-yl? methanone had nanomolar affinity for 5-HT1A binding sites and were more than 500-fold selective with respect to alpha1 and D2 sites. Importantly, their 5-HT1A agonist properties were demonstrated in HA7 cells where they behaved as potent inhibitors of cAMP accumulation. In particular, (3, 4-dichlorophenyl)?4-[(6-oxazol-5-ylpyridin-2-ylmethylamin o)methyl]pip eridin-1-yl?methanone (70) and (3, 4-dichlorophenyl)?4-[(6-azetidinopyridin-2-ylmethylamino)met hyl]piper idin-1-yl?methanone (36) appeared to be more potent than, and at least as efficacious as, the prototypical 5-HT1A agonist (+/-)-8-OH-DPAT. SAR studies revealed that the pyridine nitrogen atom and the nature and the position of the substituents on the pyridine ring were critically involved in the ability of the compounds to recognize and activate 5-HT1A receptors. Structural modifications of the nonpharmacophoric part of the molecule showed, however, that the entire structure was required for affinity at 5-HT1A binding sites.     

Functional characterisation of the human cloned 5-HT7 receptor (long form); antagonist profile of SB-258719

1. The functional profile of the long form of the human cloned 5-HT7 receptor (designated h5-HT7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT7(a) receptor. 2. The receptor binding profile, determined by competition with [3H]-5-CT, was consistent with that previously reported for the h5-HT7(a) receptor. The selective 5-HT7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]ben zene sulfonamide) displayed high affinity (pKi 7.5) for the receptor. 3. In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT > 5-HT > 8-OH-DPAT) was the same in functional and binding studies. 4. Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA2 of 7.2+/-0.2 and slope not significantly different from 1, consistent with competitive antagonism. 5. The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT7 receptor-mediated and to reflect inverse agonism. 6. It is concluded that in this expression system, the h5-HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.     

F 11440, a potent, selective, high efficacy 5-HT1A receptor agonist with marked anxiolytic and antidepressant potential

F 11440 (4-methyl-2-[4-(4-(pyrimidin-2-yl)-piperazino)-butyl]-2H, 4H-1,2,4-triazin-3,5-dione) was the outcome of a research effort guided by the hypothesis that the magnitude of the intrinsic activity of agonists at 5-HT1A receptors determines the magnitude of their antidepressant and anxiolytic-like effects. The affinity of F 11440 for 5-HT1A binding sites (pKi, 8.33) was higher than that of buspirone (pKi, 7.50), and somewhat lower than that of flesinoxan (pKi, 8.91). In vivo, F 11440 was 4- to 20-fold more potent than flesinoxan, and 30- to 60-fold more potent than buspirone, in exerting 5-HT1A agonist activity at pre- and postsynaptic receptors in rats (measured by, for example, its ability to decrease hippocampal extracellular serotonin (5-HT) levels and to increase plasma corticosterone levels, respectively). F 11440 did not have detectable antidopaminergic activity (unlike buspirone, which inhibited all of the directly observable behavioral effects of methylphenidate in rats), showed no evidence of antihistaminergic activity (unlike flesinoxan, which protected against the effects of a histamine aerosol in guinea pigs), and had a 70-fold separation between its 5-HT1A agonist and alpha-1 adrenergic antagonist properties (measured as the ability to inhibit the methoxamineinduced increase in blood pressure in rats), unlike flesinoxan, which showed a <3-fold separation. In HeLa cells expressing human 5-HT1A receptors, F 11440 decreased the forskolin-induced increase in AMP, and, based on its maximal effect, was found to have an intrinsic activity of 1.0 relative to that of 5-HT, which was significantly higher than that of buspirone (0.49), ipsapirone (0.46) and flesinoxan (0.93). Consistent with the aforementioned hypothesis, F 11440 produced anxiolytic- and antidepressant-like effects in animal models (i.e., increased punished responding in a pigeon conflict procedure and decreased immobility in a rat forced swimming test, respectively) that were more substantial than those of buspirone, ipsapirone and flesinoxan. Thus, F 11440, shown here to be a potent, selective, high efficacy 5-HT1A receptor agonist, appears to have the potential to exert marked anxiolytic and antidepressant activity in humans.     

SC-53606, a potent and selective antagonist of 5-hydroxytryptamine4 receptors in isolated rat esophageal tunica muscularis mucosae

(1-S,8-S)-N-[(hexahydro-1H-pyrrolizin-1-yl)methyl]-6-chloroimi+ ++- dazo[1,2-a]pyridine-8-carboxamide hydrochloride (SC-53606) acts as an antagonist of 5-hydroxytryptamine4 (5-HT4) receptor-mediated relaxation of carbachol-induced contractions in rat esophageal tunica muscular mucosae, but does not possess 5-HT4 agonist activity. SC-53606 demonstrated a pA2 value against 5-HT in this tissue of 7.91 +/- 0.08 (Ki = 12.3 +/- 1.17 nM). Similar pA2 values of 7.68 +/- 0.06, 7.67 +/- 0.06 and 7.63 +/- 0.05 were determined for the synthetic 5-HT4 receptor agonists SC-53116, 5-methoxytryptamine and renzapride, respectively. In addition, slopes of Schild plots for antagonism of these four agonists by SC-53606 were 1.07 +/- 0.02, 0.98 +/- 0.03, 1.04 +/- 0.02 and 0.96 +/- 0.06, respectively, and did not deviate from unity. The pA2 values for 5-HT4 antagonism against 5-HT were determined to be 6.80 +/- 0.09 for tropisetron and 7.36 +/- 0.08 for 2-methoxy-4-amino-S- chlorobenzoic acid-2-(diethylamino)ethyl ester SDZ 205-557), indicating that SC-53606 is more a potent 5-HT4 antagonist than either of the reference antagonists. Radioligand binding studies also demonstrated that SC-53606 is a selective antagonist with more affinity for 5-HT4 than for other 5-HT receptors. Displacement of radioligand binding from 5-HT1 and 5-HT2 receptors by SC-53606 was less than 50% at a 10 microM concentration. Similarly, SC-53606 displayed little binding affinity at alpha 1, alpha 2 and beta adrenergic, dopamine1, dopamine2 and muscarinic cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative receptor mapping of serotoninergic 5-HT3 and 5-HT4 binding sites

The clinical use of currently available drugs acting at the 5-HT4 receptor has been hampered by their lack of selectivity over 5-HT3 binding sites. For this reason, there is considerable interest in the medicinal chemistry of these serotonin receptor subtypes, and significant effort has been made towards the discovery of potent and selective ligands. Computer-aided conformational analysis was used to characterize serotoninergic 5-HT3 and 5-HT4 receptor recognition. On the basis of the generally accepted model of the 5-HT3 antagonist pharmacophore, we have performed a receptor mapping of this receptor binding site, following the active analog approach (AAA) defined by Marshall. The receptor excluded volume was calculated as the union of the van der Waals density maps of nine active ligands (pKi > or = 8.9), superimposed in pharmacophoric conformations. Six inactive analogs (pKi < 7.0) were subsequently used to define the essential volume, which in its turn can be used to define the regions of steric intolerance of the 5-HT3 receptor. Five active ligands (pKi > or = 9.3) at 5-HT4 receptors were used to construct an antagonist pharmacophore for this receptor, and to determine its excluded volume by superimposition of pharmacophoric conformations. The volume defined by the superimposition of five inactive 5-HT4 receptor analogs that possess the pharmacophoric elements (pKi < or = 6.6) did not exceed the excluded volume calculated for this receptor. In this case, the inactivity may be due to the lack of positive interaction of the amino moiety with a hypothetical hydrophobic pocket, which would interact with the voluminous substituents of the basic nitrogen of active ligands. The difference between the excluded volumes of both receptors has confirmed that the main difference is indeed in the basic moiety. Thus, the 5-HT3 receptor can only accommodate small substituents in the position of the nitrogen atom, whereas the 5-HT4 receptor requires more voluminous groups. Also, the basic nitrogen is located at ca. 8.0 A from the aromatic moiety in the 5-HT4 antagonist pharmacophore, whereas this distance is ca. 7.5 A in the 5-HT3 antagonist model. The comparative mapping of both serotoninergic receptors has allowed us to confirm the three-component pharmacophore accepted for the 5-HT3 receptor, as well as to propose a steric model for the 5-HT4 receptor binding site. This study offers structural insights to aid the design of new selective ligands, and the resulting models have received some support from the synthesis of two new active and selective ligands: 24 (Ki(5-HT3) = 3.7 nM; Ki(5-HT4) > 1000 nM) and 25 (Ki(5-HT4) = 13.7 nM; Ki(5-HT3) > 10,000 nM).     

RS-102221: a novel high affinity and selective, 5-HT2C receptor antagonist

The 5-HT2C receptor is one of three closely related receptor subtypes in the 5-HT2 receptor family. 5-HT2A and 5-HT2B selective antagonists have been described. However, no 5-HT2C selective antagonists have yet been disclosed. As part of an effort to further explore the function of 5-HT2C receptors, we have developed a selective 5-HT2C receptor antagonist, RS-102221 (a benzenesulfonamide of 8-[5-(5-amino-2,4-dimethoxyphenyl) 5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione). This compound exhibited nanomolar affinity for human (pKi = 8.4) and rat (pKi = 8.5) 5-HT2C receptors. The compound also demonstrated nearly 100-fold selectivity for the 5-HT2C receptor as compared to the 5-HT2A and 5-HT2B receptors. RS-102221 acted as an antagonist in a cell-based microphysiometry functional assay (pA2 = 8.1) and had no detectable intrinsic efficacy. Consistent with its action as a 5-HT2C receptor antagonist, daily dosing with RS-102221 (2 mg/kg intraperitoneal) increased food-intake and weight-gain in rats. Surprisingly, RS-102221 failed to reverse the hypolocomotion induced by the 5-HT2 receptor agonist 1-(3-chlorophenyl)piperazine (m-CPP). It is concluded that RS-102221 is the first selective, high affinity 5-HT2C receptor antagonist to be described.     

Behavioural response to pharmacologic manipulation of serotonin receptors in the genetically dystonic hamster

The genetically dystonic (dtsz) hamster is an autosomal recessive mutant that shares several features with paroxysmal dystonia, i.e., a subcategory of inherited idiopathic dystonia in humans. Because the serotonin (5-HT) system has been suggested to be involved in dystonia, we examined the functional responsiveness of the 5-HT system in dystonic hamsters by administering various 5-HT agonists and antagonists selective for different receptor subtypes and observing the effects on dystonic attacks as well as the behavioural responses associated with drug administration. Paradoxically, marked prodystonic effects (i.e., increased severity and/or decreased latency of dystonic attacks) were seen with both the selective 5-HT1A receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT) and the selective and "silent" 5-HT1A receptor antagonist, N-tert-butyl-3[4-(2-methoxyphenyl)piperazin-1-yl]-2- phenylpropionamide [(+)-WAY-100135], whereas other 5-HT1A receptor antagonists, i.e., methyl 4[4-(4-[1,1,3-trioxo-2H-1,2-benzoiosothiazol-2-yl]butyl)-1- piperazinyl]1-H-indole-2-carboxylate (SDZ 216-525) and N1-bromoacetyl-N8-3'-(4-indolyloxy)-2'-hydroxypropyl-(Z)-1,8- diamino-p-methane (pindobind-5-HT1A) did not alter dystonia to any comparable extent. Because among these 5-HT1A receptor antagonists, (+)-WAY-100135 is the only drug known to be not only silent at postsynaptic but also presynaptic (somatodendritic) 5-HT1A receptors, the marked prodystonic effect of this drug could relate to increased 5-HT release as a result of the blockade of somatodendritic 5-HT1A receptors. The only 5-HT1A receptor antagonist that exerted antidystonic effects in hamsters was pindolol, which, however, could be related to its beta-adrenoceptor blocking action. The 5-HT1A receptor partial agonist ipsapirone exerted moderate prodystonic activity. Prodystonic activity was also determined for the mixed 5-HT1A/5-HT2 receptor agonist 5-methoxy-N,N-dimethyltryptamine, although this drug was less potent in this regard than 8-OH-DPAT. The 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) exerted prodystonic effects in mutant hamsters, which, however, were also seen after the administration of the 5-HT2 receptor antagonist ritanserin. Collectively, the results of this study demonstrate that dystonia in genetically dystonic hamsters can be affected by pharmacologic manipulation of 5-HT receptors. The data may also indicate that dystonia is not a potential clinical application for selective 5-HT1A or 5-HT2 receptor antagonists.     

Serotonin efflux in the rat ventral lateral geniculate nucleus assessed by fast cyclic voltammetry is modulated by 5-HT1B and 5-HT1D autoreceptors

Fast cyclic voltammetry (FCV) was used to measure electrically stimulated monoamine efflux in the rat ventral lateral geniculate nucleus (vLGN). The electrochemical characteristics of the released species resembled 5-HT but not dopamine or noradrenaline. Amine efflux was abolished by the sodium channel blocker tetrodotoxin (0.1 microM), Ro 4-1284 (1.0 microM), the fast-acting reserpine analogue, and removal of Ca2+ from the superfusate. Amine efflux was unaffected by the monoamine oxidase inhibitor clorgyline (0.1 microM). Of paroxetine (0.1 microM), desipramine (50 nM) and vanoxerine (0.5 microM), selective blockers of 5-HT, noradrenaline and dopamine uptake respectively, only paroxetine increased monoamine efflux (to 194 +/- 25%, mean +/- SEM) and prolonged the removal half-life (to 638 +/- 105%). The non-specific 5-HT1 antagonist methiothepin (0.2 microM) increased 5-HT efflux on long (20 pulses at 20 Hz) but not short trains (20 pulses at 100 Hz). When tested on pseudo-one-pulse stimulations (5 pulses, 100 Hz), the selective 5-HT1A agonist 8-OHDPAT (1.0 microM) had no effect. CP 93129 (0.3 microM), the selective 5-HT1B agonist, decreased 5-HT efflux to 37 +/- 4% of control and was antagonised by the 5-HT1B blocker isamoltane (0.5 microM) and by the 5-HT1D/B antagonist GR 127935 (50 nM). The preferential 5-HT1D agonist sumatriptan (0.5 microM) also decreased 5-HT efflux, to 55 +/- 6% and was antagonised by GR 127935 (50 nM) but not isamoltane (0.5 microM). These results suggest that 5-HT released in the vLGN can be measured by FCV. Furthermore, released 5-HT is taken up by the 5-HT transporter and may be under the influence of 5-HT1B and 5-HT1D autoreceptors.     

Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture

The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7-12 day-old rat pups. In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in approximately 43% of the neurons (53/123); however, in a minority population ( approximately 6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential ( approximately -60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors. Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1-10 microM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50-100 microM). I5-HT displayed moderate inward rectification and had a mean reversal potential (+/-S.E.M.) of -4.3+/-6.6 mV (n=6). Application of 5-HT (dose range: 0.1-100 microM) produced a dose-response curve that was fitted by the Hill equation with EC50= approximately 3.4 microM and Hill coefficient= approximately 1.6 (n=8). The activation phase of I5-HT (10 microM 5-HT at -60 mV) was well fitted by a single exponential with mean (+/-S.E.M.) time constant of 45+/-30 ms (n=6). The desensitization phase of I5-HT was best fitted by a single exponential with mean (+/-S.E.M.) time constant of 660+/-167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (+/-S.E.M) of 2.7+/-1.5 pS (n=4) at -60 mV. In the minority ( approximately 6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 microM). Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.     

5-HT1B receptor antagonist properties of novel arylpiperazide derivatives of 1-naphthylpiperazine

A new series of arylpiperazide derivatives of 1-naphthylpiperazine of general formula 4 has been prepared and evaluated as 5-HT1B antagonists. Binding experiments at cloned human 5-HT1A, 5-HT1B, and 5-HT1D receptors show that these derivatives are potent and selective ligands for 5-HT1B/1D subtypes with increased binding selectivity versus the 5-HT1A receptor when compared to 1-naphthylpiperazine (1-NP). Studies of inhibition of the forskolin-stimulated cAMP formation mediated by the human 5-HT1B receptor demonstrate that the nature of the arylpiperazide substituent modulates the intrinsic activity of these 1-NP derivatives. Among them, 2-[[8-(4-methylpiperazin-1-yl)naphthalen-2-yl]oxy] -1-(4-o-tolylpiperazin-1-yl)ethanone (4a) was identified as a potent neutral 5-HT1B antagonist able to antagonize the inhibition of 5-HT release induced by 5-CT (5-carbamoyltryptamine) in guinea pig hypothalamus slices. Moreover, 4a was found to potently antagonize the hypothermia induced by a selective 5-HT1B/1D agonist in vivo in the guinea pig following oral administration (ED50 = 0.13 mg/kg).     

Characterization of monoclonal antibodies recognizing alpha and beta subunits of human chorionic gonadotropin hormone

The purpose of this study was to characterize the pharmacological effects of 2-[[4-(o-methoxyphenyl)piperazin-1-yl]methyl]-1,3-dioxoperhydro imidazo[1,5-a]pyridine (B-20991) by using several biochemical and behavioral assays. Results of binding studies showed that B-20991 binds with high affinity to the 5-HT1A receptor (Ki = 31.7 +/- 1.7 nM), moderate affinity to 5-HT3 receptor (Ki = 269.4 +/- 23.2 nM) and low affinity (Ki > 1000) to 5-HT2A receptor, dopamine D2 receptor, benzodiazepine receptors and alpha1-adrenoceptor. The administration of B-20991 produced a dose and time related decrease in mouse rectal temperature, increased both lower lip retraction and flat body posture behavioral scores in rat, decreased 5-hydroxytryptamine (5-HT, serotonin) neuronal activity in mouse hypothalamus, and did not alter dopamine neuronal activity nor locomotor activity. The anxiolytic activity of B-20991 was assessed by using both the social interaction and light/dark box tests. The results of these tests indicated that B-20991 caused a dose-related increase in the social interaction and light/dark box behavioral scores. Taken together, these results suggest that B-20991 is a 5-HT1A receptor agonist that exhibits anxiolytic activity.     

A twin study of antisocial and neurotic symptoms in childhood

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.     

123I-5-I-R91150, a new single-photon emission tomography ligand for 5-HT2A receptors: influence of age and gender in healthy subjects

5-HT2A receptors have been implicated in the pathophysiology of mood disorders and in the therapeutic effect of the so-called atypical antipsychotics. Recently, a new radioiodinated ligand with high affinity and selectivity for serotonin 5-HT2A receptors, 123iodinated 4-amino-N-1-[3-(4-fluorophenoxy)propyl]-4-methyl-4-piperidinyl] 5-iodo-2-methoxybenzamide (123I-5-I-R91150), has been developed and has been shown to be suitable for single-photon emission tomography (SPET) imaging. In this study the influence of age and gender on the ligand binding was investigated in normal volunteers. One hundred and fifty MBq of 123I-5-I-R91150 was administered to 26 normal volunteers (13 females and 13 males) with an age range of 23-60 years. SPET imaging was performed with a triple-headed gamma camera. For semi-quantitative analysis, ratios of ligand binding in different regions of interest to the binding in the cerebellum were calculated. Mean ratios of 1.7 were obtained. No gender difference was demonstrated. 5-HT2A binding was shown to decline with age. Over an age range of 40 years a reduction in ligand binding of 42% +/- 7% was found. These results are in agreement w in vitro and positron emission tomography findings of a decline in 5-HT2A receptor binding with age. The findings confirm the suitability of 123I-5-I-R91150 for SPET imaging of 5-HT2A receptors, and highlight the necessity for age-matched controls in clinical studies.     

Activities and therapy mediated by animals (pet-therapy): international picture and state of the art in Italy]

We tested the antinociceptive effect of intrathecal (i.t.) administration of 5-HT3 and the 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide (mCPBG), in rats submitted to a mechanical noxious stimulus and the influence of the 5-HT3 receptor selective antagonists, tropisetron and granisetron. Both 5-HT and mCPBG (0.01, 0.1, 1, 10, 20 micrograms/rat) produced a significant dose-dependent antinociception. The lowest active doses were 0.1 and 1 microgram for 5-HT and mCPBG, respectively. The effect, observed with 20 micrograms, was significantly lower with mCPBG (+33 +/- 6%) than with 5-HT (+63 +/- 7%). For 5-HT-induced antinociception, the minimal inhibitory doses were 0.001 micrograms/rat for tropisetron and 10 micrograms/rat for granisetron. In contrast, the same doses of the two antagonists (from 0.1 microgram/rat) similarly inhibited the effect of mCPBG. This study provides evidence that contrary to tropisetron, doses of granisetron able to inhibit the effect of a 5-HT3 receptor agonist failed to reduce that of 5-HT. This demonstrates a heterogeneity between 5-HT3 receptor antagonists and questions the true involvement of these receptors in spinal 5-HT-induced antinociception.     

Streptokinase entrapment in interdigitation-fusion liposomes improves thrombolysis in an experimental rabbit model

1. The 5-HT receptor involved in the effect of mucosal application of 5-HT to facilitate peristalsis was investigated in the isolated guinea pig ileum. 2. An application of 5-HT (3-100 microM) to the mucosal surface (by inclusion of 5-HT in the Krebs-Henseleit solution passing through the lumen of the ileum) caused a concentration related facilitation of peristalsis characterized by a reduction in the peristaltic threshold. 3. Peristalsis was not modified by methiothepine (0.1 microM), ritanserin (0.1 microM), ondansetron (5 microM), granisetron (1 microM) or SB 204070 (0.1 microM) administered alone to the mucosal surface. 4. The concentration-response curve to mucosally applied 5-HT was not altered by the mucosally applied 5-HT1/2 receptor antagonist methiothepine (0.1 microM), the 5-HT2 receptor antagonist ritanserin (0.1 microM) or the 5-HT4 receptor antagonist SB 204070 (0.1 microM). However, the mucosally applied 5-HT3 receptor antagonists ondansetron (5 microM) and granisetron (1 microM) shifted the response curves to mucosally applied 5-HT to the right in a parallel and surmountable manner. The pD2 values in the absence and presence of ondansetron were 5.42 +/- 0.07 and 4.12 +/- 0.10, respectively, (n = 6) and that of granisetron were 5.45 +/- 0.12 and 4.50 +/- 0.10 respectively, (n = 5). 5. Serosally applied ondansetron (5 microM) or granisetron (1 microM) had no effect on the concentration-response curve to mucosally applied 5-HT. However, the serosally applied ondansetron and granisetron antagonised the facilitatory effect of serosally applied 5-HT (10 microM) when administered in the presence of serosally applied SB 204070 (0.1 microM). 6. It is concluded that the facilitatory effect of mucosally applied 5-HT to reduce the peristaltic threshold in the guinea pig ileum is mediated via a 5-HT3 receptor located on the mucosal and not the serosal side of the ileum.