On the influence of groundwater table fluctuations on oil thickness in a well related to an LNAPL contaminated aquifer.

This paper presents a new modelling approach to describe and explain the temporal variation of oil thickness in well due to groundwater table fluctuations. This new model, which intends to be simple and easy to implement, was compared to field data obtained by continuous measurements of vertical LNAPL position in wells. Two scenarios have been studied: a pumping well where the oil layer is unconfined, and one where the oil layer is present in a confined porous media. This study shows that the time-depend fluctuation of the oil thickness observed in the wells could not be reproduced only with the differences between the residual oil saturations (Sorw and Sora) as suggested by Kemblowski and Chiang (1990). It should consider the transient mass exchange between the well and the porous media. Also, the proposed model shows that making the assumption of equilibrium conditions as suggested by Lenhard et al. (2017) for calculating the volume exchanges between the wells and its surrounding introduced errors. Considering transient transfers of oil better reflects the field observations. This observation is a key outcome for improving field data interpretation (e.g.: bail-down test data) and the remedial approach at site polluted by mineral oils.

Validation of a multilevel sampling device to determine the vertical variability of chlorinated solvent in a contaminated aquifer.

The vertical heterogeneity of contaminant concentrations in aquifers is well known, but obtaining representative samples is still a subject of debate. In this paper, the question arises from sites where numerous fully screened wells exist and there is a need to define the vertical distribution of contaminants. For this purpose, several wells were investigated with different techniques on a site contaminated with chlorinated solvents. A core-bored well shows that a tetrachloroethene (PCE) phase is sitting on and infiltrating a less permeable layer. Downstream of the cored well, the following sampling techniques were compared on fully screened wells: low flow pumping at several depths, pumping between packers and a new multilevel sampler for fully screened wells. Concerning low flow rate pumping, very low gradients were found, which may be due to the existence of vertical flow inside the well or in the gravel pack. Sampling between packers gave results comparable with the cores, separating a layer with PCE and trichloroethene from another one with cis 1,2-dichloroethene and vinyl chloride as major compounds. Detailed sampling according to pumped volume shows that even between packers, cleaning of the inter-packer volume is necessary before each sampling. Lastly, the proposed new multilevel sampler gives results similar to the packers but has the advantages of much faster sampling and a constant vertical positioning, which is fairly important for long-term monitoring in highly stratified aquifers.

F15063, a potential antipsychotic with D2/D3 antagonist, 5-HT 1A agonist and D4 partial agonist properties. I. In vitro receptor affinity and efficacy profile.

BACKGROUND AND PURPOSE: Combining 5-HT(1A) receptor activation with dopamine D(2)/D(3) receptor blockade should improve negative symptoms and cognitive deficits in schizophrenia. We describe the in vitro profile of F15063 (N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine). EXPERIMENTAL APPROACH: F15063 was characterised in tests of binding affinity and in cellular models of signal transduction at monoamine receptors. KEY RESULTS: Affinities (receptor and pK(i) values) of F15063 were: rD(2) 9.38; hD(2L) 9.44; hD(2S) 9.25; hD(3) 8.95; hD(4) 8.81; h5-HT(1A) 8.37. F15063 had little affinity (40-fold lower than D(2)) at other targets. F15063 antagonised dopamine-activated G-protein activation at hD(2), rD(2) and hD(3) receptors with potency (pK (b) values 9.19, 8.29 and 8.74 in [(35)S]GTP gamma S binding experiments) similar to haloperidol. F15063 did not exhibit any hD(2) receptor agonism, even in tests of ERK1/2 phosphorylation and G-protein activation in cells with high receptor expression. In contrast, like (+/-)8-OH-DPAT, F15063 efficaciously activated h5-HT(1A) (E(max) 70%, pEC(50) 7.57) and r5-HT(1A) receptors (52%, 7.95) in tests of [(35)S]GTP gamma S binding, cAMP accumulation (90%, 7.12) and ERK1/2 phosphorylation (93%, 7.13). F15063 acted as a partial agonist for [(35)S]GTP gamma S binding at hD(4) (29%, 8.15) and h5-HT(1D) receptors (35%, 7.68). In [(35)S]GTP gamma S autoradiography, F15063 activated G-proteins in hippocampus, cortex and septum (regions enriched in 5-HT(1A) receptors), but antagonised quinelorane-induced activation of D(2)/D(3) receptors in striatum. CONCLUSIONS AND IMPLICATIONS: F15063 antagonised dopamine D(2)/D(3) receptors, a property underlying its antipsychotic-like activity, whereas activation of 5-HT(1A) and D(4) receptors mediated its actions in models of negative symptoms and cognitive deficits of schizophrenia (see companion papers).

Induction of a high-affinity ketanserin binding site at the 5-Hydroxytryptamine(1B) receptor by modification of its carboxy-terminal intracellular portion.

Two chimeric 5-hydroxytryptamine (5-HT) receptors were constructed by exchanging the C-terminal portion of the human (h) 5-HT(1B) receptor with the equivalent domain of the h 5-HT(2A) receptor (5-HT(1B/2A)) or with this domain truncated from its last 44 amino acids (5-HT(1B/2ADelta44)). The equilibrium dissociation constant of the radioligand [(3)H]GR 125743 was similar for both chimera compared to the wild-type (wt) h 5-HT(1B) receptor upon transient expression in COS-7 cells. Ketanserin binding affinity was 21-fold increased from pK(i): 5.79 (wt h 5-HT(1B) receptor) to pK(i): 7.11 at the 5-HT(1B/2A) chimeric receptor, this latter value being close to that of the wt h 5-HT(1D) receptor (pK(i): 7.62). This enhanced ketanserin binding affinity was lost when the last 44 C-terminal amino acids of the 5-HT(2A) receptor were deleted in the chimera 5-HT(1B/2ADelta44) (pK(i): 5.80). The binding affinities of the 5-HT antagonists ritanserin, GR 125743, and SB-224289 were not modified at either chimeric 5-HT receptor. The agonists F 11356, 5-HT, zolmitriptan, and sumatriptan yielded slightly increased (2- to 6-fold) binding affinities at both chimera as compared to the wt h 5-HT(1B) receptor. The present data suggest a role for the C-terminal intracellular receptor domain in modifying ketanserin/5-HT(1B) receptor interactions.

F 11356, a novel 5-hydroxytryptamine (5-HT) derivative with potent, selective, and unique high intrinsic activity at 5-HT1B/1D receptors in models relevant to migraine.

F 11356 (4-[4-[2-(2-aminoethyl)-1H-indol-5-yloxyl]acetyl]piperazinyl-1-yl] ben zonitrile) was designed to take advantage of the superior potency and efficacy characteristics of 5-hydroxytryptamine (5-HT) compared with tryptamine at 5-HT1B/1D receptors. F 11356 has subnanomolar affinity for cloned human and nonhuman 5-HT1B and 5-HT1D receptors, and its affinity for 5-HT1A and other 5-HT receptors, including the 5-ht1F subtype, is 50-fold lower and micromolar, respectively. In C6 cells expressing human 5-HT1B or human 5-HT1D receptors, F 11356 was the most potent compound in inhibiting forskolin-induced cyclic AMP formation (pD2 = 8.9 and 9.6), and in contrast to tryptamine and derivatives, it produced maximal enhancement of [35S]guanosine-5'-O-(3-thio)triphosphate-specific binding equivalent to 5-HT. F 11356 was equipotent to 5-HT (pD2 = 7.1 versus 7.2) and more potent than tryptamine derivatives in contracting rabbit isolated saphenous vein. In isolated guinea pig trigeminal ganglion neurons, F 11356 was more potent (pD2 = 7.3 versus 6.7) and induced greater increases in outward hyperpolarizing Ca2+-dependent K+ current than sumatriptan. In anesthetized pigs, F 11356 elicited highly cranioselective, more potent (from 0.16 microgram/kg i.v.) and greater carotid vasoconstriction than tryptamine derivatives. Decreases in carotid blood flow were observed in conscious dogs from 0.63 mg/kg oral F 11356 in the absence of changes in heart rate or behavior. Oral activity was confirmed when hypothermic responses were elicited in guinea pigs (ED50 = 1.6 mg/kg), suggesting that F 11356 also accesses the brain. F 11356 thus is a selective, high-potency agonist at 5-HT1B/1D receptors, which distinguishes itself from tryptamine and derivatives in exerting high intrinsic activity at these receptors in vascular and neuronal models relevant to migraine.

5-(Sulfonyl)oxy-tryptamines and ethylamino side chain restricted derivatives. Structure-affinity relationships for h5-HT1B and h5-HT1D receptors.

A number of sulfonic acid ester derivatives of serotonin (5-hydroxytryptamine; 5-HT; 1) were prepared and their affinities are compared to that of the reference compound 5-[[(trifluoromethyl)sulfonyl]oxy]-tryptamine (8b). The structure-affinity relationship (SAFIR) is discussed in terms of in vitro binding for cloned human h5-HT1A, h5-HT1B and h5-HT1D receptors. All tryptamine derivatives exhibited the best affinities for h5-HT1D receptors but still, these were comparatively lower than that of compound 8b. 5-Tosylated tryptamine 11b (Ki = 6 nM) and the sulfamate derivatives 13b and 14b (Ki = 7 and 11 nM, respectively) were found to have the highest affinities for the h5-HT1D receptor. Other tryptamine derivatives displayed moderate binding for h5-HT1A and h5-HT1B receptors, along with Ki values ranging from 14-20 nM for the h5-HT1D sites. In addition, the syntheses of two alkylamino side chain restricted derivatives are described. 3-Amino-6-[[(trifluoromethyl)sulfonyl]oxy]-1,2,3,4-tetrahydrocarbazol e 21, as well as 4-[5-[[(trifluoromethyl)sulfonyl]oxy]-1H-indol-3-yl]piperidines 24 and 25, induced a shift in selectivity in favor of the h5-HT1B receptor. The relatively longer distance between the basic amine and a hydrogen-bond accepting oxygen in 21, 24 and 25 as compared to the non-restricted tryptamines, is likely responsible for this observation.

Multiple forms of arginase are differentially expressed from a single locus in Neurospora crassa.

The Neurospora crassa catabolic enzyme, arginase (L-arginine amidinohydrolase, EC 3.5.3.1), exists in multiple forms. Multiple forms of arginase are found in many vertebrates, but this is the only reported example in a microbial organism. The two major forms are structurally similar with subunit sizes of 36 and 41 kDa, respectively. The larger form is produced by mycelia growing in arginine-supplemented medium. Both forms are localized in the cytosol. The structural gene for arginase, aga, has been cloned and sequenced; it contains a 358-codon open reading frame with three in-frame ATGs at the amino terminus. Mutagenesis of these ATGs revealed that the first ATG initiates the 41-kDa protein and the third ATG initiates the 36-kDa protein. Mutation of the second ATG has no effect on translation. Northern analysis demonstrated that a 1.4-kilobase (kb) transcript is synthesized in minimal medium and both a 1.4- and 1.7-kb transcript are produced in arginine-supplemented medium. Primer extension identified the 5' ends of each transcript and demonstrated that the first and third ATG of the open reading frame are the initial AUGs of the 1.7- and 1. 4-kb mRNA, respectively. The results suggest that a basal promoter produces the 1.4-kb transcript and an arginine "activated" promoter is responsible for the 1.7-kb transcript. Tandem promoters are rare in eukaryotic organisms, and they often regulate developmental or tissue-specific gene expression. The possibility that arginase has a role in differentiation in N. crassa is being investigated.

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.

Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors: intrinsic activity and modulation by G-protein activation state.

Many 5-HT1B/D receptor ligands have affinity for 5-HT1A receptors. In the present study, the intrinsic activity of a series of 5-HT1B/D ligands was investigated at human 5-HT1A (h 5-HT1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [35S]GTPgammaS binding. In these two membrane preparations, the density of h 5-HT1A receptors (i.e., 246 to 320 fmol mg(-1) protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [35S]GTPgammaS binding responses induced by the 5-HT1B/D receptor ligands in the HeLa preparation at 30 microM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 microM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [35S]GTPgammaS binding responses induced by 5-HT1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 microM and higher concentrations. These features differ from those observed with 5-HT1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist's maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT1A and 5-HT1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT1A receptor activation by 5-HT1A and 5-HT1B/D agonists.

Autoradiography of serotonin 5-HT1A receptor-activated G proteins in guinea pig brain sections by agonist-stimulated [35S]GTPgammaS binding.

G protein activation mediated by serotonin 5-HT1A and 5-HT(1B/D) receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [35S]GTPgammaS binding to brain sections. [35S]GTPgammaS binding was stimulated by the mixed 5-HT1A/5-HT(1B/D) agonist L694247 in brain structures enriched in 5-HT1A binding sites, i.e., hippocampus (+140 +/- 14%), dorsal raphe (+70 +/- 8%), lateral septum (+52 +/- 12%), cingulate (+36 +/- 8%), and entorhinal cortex (+34 +/- 5%). L694247 caused little or no stimulation of [35S]GTPgammaS binding in brain regions with high densities of 5-HT(1B/D) binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [35S]GTPgammaS binding response was antagonized by WAY100635 (10 microM) and methiothepin (10 microM). In contrast, the 5-HT1B inverse agonist SB224289 (10 microM) did not affect the L694247-mediated [35S]GTPgammaS binding response, and the mixed 5-HT(1B/D) antagonist GR127935 (10 microM) yielded a partial blockade. The distribution pattern of the [35S]GTPgammaS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [35S]GTPgammaS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 microM) stimulated [35S]GTPgammaS binding in the hippocampus by 20-50%. Naratriptan, CP122638, and dihydroergotamine stimulated [35S]GTPgammaS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT1A but not 5-HT(1B/D) receptors can be measured in guinea pig brain sections.

Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT1B receptors in C6-glial cells: similarities with the human 5-HT1B receptor.

1. The guinea-pig recombinant 5-hydroxytryptamine1B (gp 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by monitoring G-protein activation in a membrane preparation with agonist-stimulated [35S]-GTPgammaS binding. The intrinsic activity of 5-HT receptor ligands was compared with that determined previously at the human recombinant 5-HT1B (h 5-HT1B) receptor under similar experimental conditions. 2. Membrane preparations of C6-glial/gp 5-HT1B cells exhibited [3H]-5-carboxamidotryptamine (5-CT) and [3H]-N-[4-methoxy-3,4-methylpiperazin-1-yl) phenyl]-3-methyl-4-(4-pyridinyl)benzamide (GR 125743) binding sites with a pKd of 9.62 to 9.85 and a Bmax between 2.1 to 6.4 fmol mg(-1) protein. The binding affinities of a series of 5-HT receptor ligands determined with [3H]-5-CT and [3H]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r2: 0.74, P<0.001) with those determined at the recombinant h 5-HT1B receptor. 3. [35S]-GTPgammaS binding to membrane preparations of C6-glial/gp 5-HT1B cells was stimulated by the 5-HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitriptan, sumatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulphonamide (CP 122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5-HT. The potency of these agonists showed a positive correlation (r2: 0.72, P=0.015) with their potency at the recombinant h 5-HT1B receptor. 1-naphthylpiperazine, (+/-)-cyanopindolol and (2'-methyl-4'-(5-methyl[1,2,4] oxadiazole-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935) elicited an even smaller response (Emax: 0.32 to 0.63). 4. The ligands 1'-methyl-5-(2'-methyl-4'-(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-carbonyl)-2,3,6,7tetrahydrospiro [furo[2,3-f]indole-3-spiro-4'-piperidine] (SB224289), methiothepin and ritanserin displayed inhibition of basal [35S]-GTPgammaS binding at concentrations relevant to their binding affinity for the gp 5-HT1B receptor. Methiothepin and SB224289 behaved as competitive antagonists at gp 5-HT1B receptors; pA2 values were 9.74 and 8.73, respectively when 5-HT was used as an agonist. These estimates accorded with the potencies measured in antagonism of zolmitriptan-mediated inhibition of forskolin-stimulated cyclic AMP formation. Ketanserin acted as a weak antagonist (pK(B): 5.87) at gp 5-HT1B receptors. 5. In conclusion, the recombinant gp 5-HT1B receptor shares important pharmacological similarities with the recombinant h 5-HT1B receptor. The finding that negative activity occurs at these receptors further suggests that SB224289, methiothepin and ritanserin are likely to be inverse agonists.

Design and synthesis of new potent, silent 5-HT1A antagonists by covalent coupling of aminopropanol derivatives with selective serotonin reuptake inhibitors.

Hybrid molecules built up by covalent coupling of aminopropanol derivatives (especially pindolol) with antidepressant drugs like fluoxetine, paroxetine or milnacipran were found to be potent and silent 5-HT1A antagonists (KB < 1 nM for 7c and 9a).

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).

How efficacious are 5-HT1B/D receptor ligands: an answer from GTP gamma S binding studies with stably transfected C6-glial cell lines.

The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1B and h5-HT1D receptor sites using a [35S]GTP gamma S binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTP gamma S binding to a membrane preparation containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTP gamma S binding assay to C6-glial/h5-HT1D membranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1 2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1D membrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1D than h5-HT1B binding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTP gamma S binding response: Hence, the h5-HT1B receptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1D receptor sites [e.g. dihydroergotamine and (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTP gamma S binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at the h5-HT1B receptor and only L694247 at the h5-HT1D receptor. GR127935 (10 microM) exerted little effect on [35S]GTP gamma S binding via h5-HT1B receptors (10% stimulation), but potently (pA2: 9.11) antagonized h5-HT1B receptor-stimulated [35S]GTP gamma S binding. Ketanserin and methiothepin inhibited [35S]GTP gamma S binding (by 13-28%) in the absence of an agonist, but were potent and competitive antagonists in the presence of an agonist via h5-HT1B (methiothepin) and h5-HT1D (methiothepin and ketanserin) receptors. The results document the utility of using [35S]GTP gamma S binding studies to assess agonist efficacy, and to characterize 5-HT1B/D receptor ligands as apparently neutral antagonists and inverse agonists at the G-protein level.

Sequence and functional analysis of cloned guinea pig and rat serotonin 5-HT1D receptors: common pharmacological features within the 5-HT1D receptor subfamily.

This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)1D receptor sites. Guinea pig, rat, and mouse 5-HT1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13-27%) in the N-terminal extracellular region of these 5-HT1D receptors. Guinea pig and rat 5-HT1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT1D receptor-mediated antagonist potency for methiothepin and the 5-HT2 receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT1D receptor subtype seems, unlike the 5-HT1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors.

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine1B (r 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3':5'-cyclic monophosphate (cycle AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT1B (h 5-HT1B) receptor under similar experimental conditions. 2. Intact C6-glial cells expressing rb HT1B receptors exhibited [3H]-5-carboxamidotryptamine (5-CT) binding sites with a Kd of 0.80 +/- 0.13 nM and a Bmax between 225 to 570 fmol mg-1 protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [3H]-5-CT or [3H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(-4 -pyridyl) benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the clones h 5-HT1B receptor site. 3. rb 5-HT1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT > 5-HT > zolmitriptan > naratriptan > rizatriptan > sumatriptan > R (+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r2 = 0.87; P < 0.002) with their potency at the cloned h 5-HT1B receptor subtype. 4. 2'-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-e-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5HT1B receptors; pKB values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments. 5. In conclusion, the recombinant saphenous vein 5-HT1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT1B receptors.

Serotonin dimers: application of the bivalent ligand approach to the design of new potent and selective 5-HT(1B/1D) agonists.

A series of serotonin dimers of formula 4 in which two serotonin moeities are linked together through their 5-hydroxyl residue has been prepared and evaluated as 5-HT(1B/1D) receptor agonists. Binding experiments at cloned human 5-HT(1B), 5-HT(1D), and 5-HT(1A) receptors show that all of these dimers are very potent ligands at 5-HT(1B/1D) receptors with increased binding selectivity vs the 5-HT(1A) receptor when compared to serotonin. Studies of inhibition of the forskolin-stimulated c-AMP formation mediated by the human 5-HT(1B) receptor (formerly the 5-HT(1Dbeta) receptor) demonstrate that all of these serotonin dimers behave as full agonists. Among them, the piperazide derivatives of bis-serotonin, 4g,j, were also identified as very potent agonists in contracting the New Zealand white rabbit saphenous vein (pD2 = 7.6 in each case compared to 5.8 for sumatriptan). Results analysis supports the hypothesis that the important increase in potency of the serotonin dimers can be attributed to the presence of two serotonin pharmacophores in the same molecule, while the enhanced selectivity for 5-HT(1B/1D) receptor subtypes may be due to the position of the spacer attachment to serotonin.

Promotion of cell growth by stimulation of cloned human 5-HT1D receptor sites in transfected C6-glial cells is highly sensitive to intrinsic activity at 5-HT1D receptors.

5-Hydroxytryptamine (serotonin, 5-HT), essentially known as a neurotransmitter and vasoactive agent, also functions as a mitogen in various cell types through several different second messenger systems. Stimulation of cloned human 5-HT1D receptor sites by sumatriptan in stably transfected rat C6-glial/5-HT1D cells promotes cell growth (Pauwels et al. (1996) Naunyn-Schmiedeberg's Arch Pharmacol 353:144-156). In the present study, the pharmacology of this growth response was investigated using a broad series of 5-HT receptor ligands. The data were compared with the responses obtained by measuring inhibition of forskolin-stimulated cAMP formation. 5-HT (EC50: 25 nM) promoted cell growth of C6-glial/5HT1D cells, and this in contrast to the absence of any measurable effect in pcDNA3-plasmid transfected and non-transfected C6-glial cells. The 5-HT effect could be mimicked by the following compounds (EC50 in nM): zolmitriptan (0.41), 2'-methyl-4'-(-methyl[1,2,4] oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amid (GR 127,935;0.86), naratriptan (0.92), metergoline (1.9), sumatriptan (2.9), (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-yl] ethylamine (MK-462; 3.0), and R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (R(+)-8-OH-DPAT; 30.7). These EC50 -values correspond to the compounds binding affinities at the human 5-HT1D receptor site and, with the exception of GR 127,935 and metergoline also to the EC50-values found by measuring over 5 min inhibition of forskolin (100 microM)-stimulated cAMP formation. Prolonged exposure of GR 127,935(3 h) and metergoline (30 min) to cells yielded EC50 values in the cAMP assay more close to those measured in the mitogenic response. The growth response to sumatriptan, 5-HT, GR 127,935 and metergoline was blocked by the apparently silent antagonist methiothepin, ritanserin and ketanserin with potencies similar to blockade of inhibition of stimulated cAMP formation. The 8-OH-DPAT effect also is likely mediated by 5-HT1D receptors; stereoselectivity was found with its enantiomers at this receptor site and the effect was blocked by ketanserin (1 microM) but not by spiperone (1 microM). Micromolar concentrations of the 5-HT1B receptor agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo[3,2-b]pyril-5-one (CP 93,129) and of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl_-2-aminopropane (DOI) induced cell growth with a potency that accorded with the affinity of these compounds for the human 5-HT1D receptor site. These effects were sensitive to ketanserin (1 microM) antagonism, but not to blockade by beta-adrenergic blockers and the 5-HT2 receptor antagonist 2-anilino-N-[2-(3-chlorophenoxy)-propyl] acetamidine hydroiodide (BW 501-C-67). The findings suggest that 5-HT1A, 5-HT1B and 5-HT2 receptors are not implicated in 5-HT-stimulated C6-glial/5-HT1D cell growth. In conclusion, human 5-HT1D receptors are involved in the growth of C6-glial/5-HT1D cells. This cellular response is highly sensitive to the intrinsic activity of compounds at 5-HT1D receptors.

Stimulation of cloned human serotonin 5-HT1D beta receptor sites in stably transfected C6 glial cells promotes cell growth.

The involvement of serotonin 5-HT1D beta receptor sites was investigated in the growth of rat C6 glial cells permanently transfected with a gene encoding a human 5-HT1D beta receptor. The 5-HT receptor identity of control and transfected C6 glial/5-HT1D beta cells was determined by reverse transcription-polymerase chain reaction using primers specific for rat 5-HT1A, rat 5-HT1B, rat 5-HT1D alpha, human 5-HT1D beta, and rat 5-HT2A receptor genes. Constitutive mRNA for 5-HT2A receptors was present in control and transfected C6 glial/5-HT1D beta cells, whereas mRNA for 5-HT1D beta receptor sites was only present in the transfected C6 glial/5-HT1D beta cell line. 5-HT inhibited forskolin-stimulated cyclic AMP formation and promoted cell growth, in contrast to the absence of any measurable effect in pcDNA3 plasmid-transfected and nontransfected C6 glial cells. The 5-HT effects could be mimicked by sumatriptan (EC50 = 44-76 nM) and were totally and partially blocked by methiothepin (IC50 = 9 nM) and GR 127,935 (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)-biphenyl-4-carbox yli c acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide; IC50 = 97 pM), respectively. No effect on cell growth was measured with the 5-HT2 receptor agonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; 10 microM], suggesting that 5-HT2A receptors are not involved in the 5-HT-stimulated C6 glial/5-HT1D beta cell growth. Dibutyryl-cyclic AMP (0.3 mM)-treated cultures did not show sumatriptan-promoted cell growth, indicating an inhibitory role for cyclic AMP in the cell growth mediated by 5-HT1D beta receptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacology of cloned human 5-HT1D receptor-mediated functional responses in stably transfected rat C6-glial cell lines: further evidence differentiating human 5-HT1D and 5-HT1B receptors.

This study was undertaken to investigate the pharmacology of human serotonin (5-HT)1D receptor sites by measuring two functional cellular responses, inhibition of forskolin-stimulated cAMP formation and promotion of cell growth, using transfected rat C6-glial cell lines and a broad series of 5-HT receptor agonists. Stable and separate transfection of a pcDNA3 or pRcRSV plasmid, each containing a cloned human 5-HT1D receptor gene, in rat C6-glial cells was confirmed with RT-PCR of 5-HT1D receptor mRNA and radioligand binding with [3H] 5-carboxamidotryptamine (5-CT) and [3H] sumatriptan. The 5-HT1D receptor density was 350 and 1050 fmol/mg protein for the C6-glial/pcDNA3/5-HT1D and C6-glial/pRcRSV/5-HT1D cell line, and forskolin (100 microM)-induced cAMP formation was inhibited by 45 and 78% in the presence of 1 microM 5-HT, respectively. A comparison of the intrinsic agonist activities for sixteen 5-HT receptor ligands with their corresponding binding affinities for the human 5-HT1D receptor site showed similar results for both cell lines with the exception of the partial agonist m-trifluoro-phenyl-piperazine (TFMPP). Three classes of compounds were observed: 1) efficacious agonists, such as 5-CT, 5-methoxytryptamine, 5-HT, sumatriptan, bufotenine, 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole (RU 24,969), tryptamine and 8-hydroxy-2(di-n-propilamino)tetralin (8-OH-DPAT), with agonist potency close to their binding affinity; 2) the partial agonists metergoline, 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrolo-(1,2-a) quinoxaline (CGS 12066B), 1-naphthylpiperazine and 2'-methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-amide (GR 127,935) with marked intrinsic agonist activity but at concentrations higher than their binding affinity; and 3) the silent antagonists ritanserin, ketanserin and methiothepin, apparently free of intrinsic agonist activity, with antagonist potency close to their binding affinity. The cAMP data were further supported by the observed promotion of cell growth by stimulation of both transfected cell lines with sumatriptan under serum-free conditions; half-maximal stimulation was obtained at 4.4 nM (C6-glial/pcDNA3/5-HT1D) fully in agreement with its EC50-value (5.7 nM) for inhibition of cAMP formation. This growth promoting effect was antagonised by 1 microM methiothepin and not observed in pcDNA3-plasmid-transfected and non-transfected C6-glial cells. A comparative study with a C6-glial/pcDNA3/5-HT1B cell line expressing a similar amount of cloned human 5-HT1B receptors (Bmax: 360 fmol/mg protein) showed almost no intrinsic agonist activity for metergoline, 1-naphtylpiperazine and GR 127,935. Together with the 5-HT1D receptor binding selectivity and antagonist activity of ketanserin and ritanserin, the findings define important pharmacological differences between cloned human 5-HT1D and 5-HT1B receptor sites.