Embryonic response to high beta-hydroxybutyrate (BHB) levels in postpartum dairy cows.

Cows at the beginning of lactation often do not meet their energy needs by feeding and therefore mobilize body fat, which produces ketone bodies, including ß-hydroxybutyrate (BHB). They are nevertheless usually inseminated around 60 d postpartum, when they are still in this characteristic period of energy deficit. The aim of this study was to observe the effects of negative energy balance on embryo quality and to identify ways to improve the fertility of dairy cows. Holstein cows (n = 18) grouped as high or low BHB based on blood measurement at day 45 postpartum were estrus-synchronized and treated with follicle-stimulating hormone to obtain multiple follicle development, induced to ovulate and inseminated with sexed semen around day 60 postpartum. Of the 290 embryos collected over 16 mo, 159 were of quality I to IV. Based on microarray analysis of gene expression, exposure to an energy deficit metabolic environment (high BHB) during early development appeared to modify signaling by the mTOR and sirtuins pathways in the embryo, implying mitochondrial dysfunction and inhibition of transcription, leading to slower cell division, thus programming the embryo to be more energy efficient. Altered methylation markers suggested that such coping mechanisms might persist into adulthood.

An immunocapture/scintillation proximity analysis of G alpha q/11 activation by native serotonin (5-HT)2A receptors in rat cortex: blockade by clozapine and mirtazapine.

Though transduction mechanisms recruited by heterologously expressed 5-HT(2A) receptors have been extensively studied, their interaction with specific subtypes of G-protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5-HT, the prototypical 5-HT(2A) agonist, DOI, and Ro60,0175 all enhanced [(35)S]GTPgammaS binding to G alpha q/11 in rat cortex with pEC(50) values of 6.22, 7.24 and 6.35, respectively. No activation of G o or G s/olf was seen at equivalent concentrations of DOI. Stimulation of G alpha q/11 by 5-HT (30 microM) and DOI (30 microM) was abolished by the selective 5-HT(2A) vs. 5-HT(2C)/5-HT(2B) antagonists, ketanserin (pK(B) values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5-HT-induced [(35)S]GTPgammaS binding to G alpha q/11 was only weakly inhibited by the preferential 5-HT(2C) receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5-HT(2B) receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5-HT(2A) receptors, blocked the recruitment of G alpha q/11 by 5-HT and DOI with pK(B) values of 8.54 and 8.14, respectively. Its actions were mimicked by the "atypical" antidepressant and 5-HT(2A) receptor antagonist, mirtazapine, which likewise blocked 5-HT and DOI-induced G alpha q/11 protein activation with pK(B) values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5-HT(2A) receptors in rat frontal cortex specifically recruit G alpha q/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5-HT(2A) receptor-mediated G alpha q/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent.

Hypocalcaemia due to nutritional calcium deficiency and hypoparathyroidism in an adult dog.

A 13-year-old intact male poodle had suffered periodic tetanic crises for two months. It was cachectic and moderately dehydrated, and during the crises blindness, a stiff gait and behavioural changes were observed. Routine haematological and biochemical profiles showed that it was severely hypocalcaemic, with a corrected plasma calcium concentration of 1.13 mmol/litre (reference range 2.25 to 3 mmol/litre). The dog was fed a home-made diet composed of chicken and basmati rice cooked with a soup bouillon cube; an analysis of its daily allowance indicated that the dog was generally malnourished and received only 0.222 g of calcium per day rather than the 0.6 g it required. In addition, the dog had a low blood concentration of parathyroid hormone of 12 ng/litre (reference range 20 to 80 ng/litre). Supplementing the dog with calcitriol for four days and correcting its diet increased its blood calcium to the lower part of the reference range and resolved the clinical signs, although its parathyroid hormone concentration was still low one year later.

Agonist and antagonist actions of (-)pindolol at recombinant, human serotonin1A (5-HT1A) receptors.

It has been proposed that the arylalkylamine, (-)pindolol, potentiates the therapeutic action of antidepressant drugs in humans by blockade of 5-HT1A autoreceptors. Its interactions at human 5-HT1A receptors have not, however, been directly characterized. Herein, we demonstrate that (-)pindolol exhibits nanomolar affinity at human 5-HT1A receptors expressed in Chinese Hamster Ovary cells (CHO-h5-HT1A; Ki = 6.4 nmol/L). In a functional test of receptor-mediated G-protein activation (stimulation of [35S]-GTP gamma S binding) (-)pindolol displays an efficacy of 20.3% relative to the endogenous agonist, 5-HT (= 100%). (-)Pindolol also antagonizes 5-HT (100 nmol/L)-stimulated [35S]-GTP gamma S binding, reducing it to 19.8% of control binding. These data indicate that (-)pindolol acts as a (weak) partial agonist at CHO-h5-HT1A receptors and that it blocks the action of 5-HT at these sites.

Clozapine is a partial agonist at cloned, human serotonin 5-HT1A receptors.

Clozapine exhibited 10-fold higher affinity than haloperidol for human 5-HT1A receptors expressed in Chinese Hamster Ovary cells (CHO-h5-HT1A) (Kis = 160 and 1910 nM respectively). Whereas haloperidol did not alter the basal binding of [35S]GTP gama S to CHO-h5HT1A membranes, clozapine stimulated it with an EC50 of 2320 nM and an efficacy of 49% (compared to 5-HT). The stimulation was antagonized by the selective 5-HT1A receptor antagonist, WAY 100635 (1 nM).

[(35)S]-GTPgammaS autoradiography reveals alpha(2) adrenoceptor-mediated G-protein activation in amygdala and lateral septum.

alpha(2)-adrenoceptor-mediated G-protein activation was examined by [(35)S]-GTPgammaS autoradiography. In alpha(2)-adrenoceptor-rich regions (amygdala, lateral septum), noradrenaline stimulated [(35)S]-GTPgammaS binding. These actions were abolished by the selective alpha(2) antagonist, atipamezole. Conversely, in caudate nucleus, which expresses few alpha(2) receptors, noradrenaline-induced stimulation was not inhibited by atipamezole, suggesting that it is not mediated by alpha(2)-adrenoceptors.

Agonist and inverse agonist efficacy at human recombinant serotonin 5-HT1A receptors as a function of receptor:G-protein stoichiometry.

Membrane preparations were made from Chinese Hamster Ovary (CHO) cells expressing 1.6 and 4.2 pmol/mg of recombinant human 5-HT1A receptors, as determined by saturation binding with the selective antagonist, [3H]-S 15535 ([3H]-4-(benzodioxan-5-yl)]-(indan-2-yl)piperazine). There was no change in the number of G-proteins activated by the full agonist, serotonin (5-HT; approximately 1.1 pmol/mg in each preparation, measured by [35S]-GTP gamma S saturation binding), therefore increasing the receptor:G-protein ratio from approximately 1.4:1 (RGlow) to approximately 4:1 (RGhigh). Agonist efficacy was measured by stimulation of [35S]-GTP gamma S binding. The serotonergic agonist, eltoprazine, behaved as a partial agonist (Emax = 52.7%) at RGlow membranes but virtually as a full agonist (Emax = 93.2%) at RGhigh membranes, relative to 5-HT (= 100%). The latter exhibited a two-fold shift to the left in its concentration-response curve in RGhigh compared to RGlow membranes (P < 0.01). WAY 100,635 (N-¿2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl¿-N-(2-pyridinyl) -cyclo-hexane-carboxamide), did not alter [35S]-GTP gamma S binding from basal levels in either membrane preparation. In contrast, spiperone displayed inverse agonist activity, decreasing [35S]-GTP gamma S binding from basal levels by 17% in RGlow membranes but by 28% in RGhigh membranes. These data indicate that an increased receptor:G-protein ratio (i) augments the potency of full agonists, (ii) increases the efficacy of partial agonists and (iii) increases the negative efficacy of inverse agonists at recombinant human 5-HT1A receptors. Furthermore, these data suggest that spiperone induces, or stabilises, a G-protein-coupled, but inactive conformation of the receptor.

Inhibition of the constitutive activity of human 5-HT1A receptors by the inverse agonist, spiperone but not the neutral antagonist, WAY 100,635.

At recombinant human 5-hydroxytryptamine (5-HT)5-HT1A receptors expressed in Chinese hamster ovary cells (CHO-5-HT1A), 5-carboxamidotryptamine (5-CT), acted as a full agonist (relative to 5-HT = 100%) for stimulation of receptor-mediated [35S]-GTP gamma S (guanylyl 5'-[gamma-thio]-tryphosphate) binding. In contrast, spiperone inhibited basal [35S]-GTP gamma S binding by 30.2% (IC50 = 55.5 nM) in CHO-5-HT1A membranes but not in control untransfected membranes. The antagonist, N-[2-[4-(2-methoxyphenyl) -1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclohexane-carboxamide (WAY 100,635), blocked both 5-CT-induced stimulation and spiperone-induced inhibition of [35S]-GTP gamma S binding without itself modifying [35S]-GTP gamma S binding. It is concluded that, in this heterologous expression system, 5-HT1A receptors display 'constitutive' activation of G-proteins and that spiperone displays inverse agonist activity whereas WAY 100,635 acts as a 'neutral' antagonist at this site.

Histopathological and immunological investigations of synthetic fibres and structures used in three prosthetic anterior cruciate ligaments: in vivo study in the rat.

Three types of prosthetic anterior cruciate ligaments were investigated by enzymatic and histological analysis of the tissue surrounding each implant and immunologically by a cytofluorometric analysis of T-cell populations in the peripheral blood of rats. Two of the prostheses had a braided construction, one made from polyester and the other from high performance polyethylene fibres. The third type also contained high performance polyethylene fibres, but had been manufactured in a knitted construction (Raschel high performance polyethylene). Five specimens from each prosthesis were implanted intraperitoneally in rats by a trocar for different periods of time up to 4 wk. A control group of rats underwent the surgery, but not the implant. No modification in peripheral T-cell populations was induced by the presence of any implant. Whilst the levels of acid phosphatase and esterase activity appeared to have increased slightly following implantation of any of the prostheses, such increases were not highly significant. Histologically, all three materials induced an intense acute inflammatory reaction at 3 d which gave way to a typical chronic response after 4 wk. The only major difference between the prostheses was that after 4 wk the polyester fibres exhibited less inflammation, and the surrounding tissue was more mature, more vascularized and more densely infiltrated with collagen than with the two high performance polyethylene implants. In conclusion, all three devices provided satisfactory biocompatibility in terms of cellular and healing response.

Novel injectable neutral solutions of chitosan form biodegradable gels in situ.

A novel approach to provide, thermally sensitive neutral solutions based on chitosan/polyol salt combinations is described. These formulations possess a physiological pH and can be held liquid below room temperature for encapsulating living cells and therapeutic proteins; they form monolithic gels at body temperature. When injected in vivo the liquid formulations turn into gel implants in situ. This system was used successfully to deliver biologically active growth factors in vivo as well as an encapsulating matrix for living chondrocytes for tissue engineering applications. This study reports for the first time the use of polymer/polyol salt aqueous solutions as gelling systems, suggesting the discovery of a prototype for a new family of thermosetting gels highly compatible with biological compounds.

Simple detection of point mutations associated with HIV-1 drug resistance.

A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS--PCR) --- a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant--wild-type population. Gene sequencing was carried out simultaneously for comparison. MS--PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS--PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS--PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS--PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest.

Dopamine D2 receptor-mediated G-protein activation in rat striatum: functional autoradiography and influence of unilateral 6-hydroxydopamine lesions of the substantia nigra.

Unilateral 6-hydroxydopamine (6-OHDA) lesions of substantia nigra pars compacta (SNPC) neurons in rats induce behavioural hypersensitivity to dopaminergic agonists. However, the role of specific dopamine receptors is unclear, and potential alterations in their transduction mechanisms remain to be evaluated. The present study addressed these issues employing the dopaminergic agonist, quinelorane, which efficaciously stimulated G-protein activation (as assessed by [35S]GTPgammaS binding) at cloned hD2 (and hD3) receptors. At rat striatal membranes, dopamine stimulated [35S]GTPgammaS binding by 1.9-fold over basal, but its actions were only partially reversed by the selective D2/D3 receptor antagonist, raclopride, indicating the involvement of other receptor subtypes. In contrast, quinelorane-induced stimulation (48% of the effect of dopamine) was abolished by raclopride, and by the D2 receptor antagonist, L741,626. Further, novel antagonists selective for D3 and D4 receptors, S33084 and S18126, respectively, blocked the actions of quinelorane at concentrations corresponding to their affinities for D2 receptors. Quinelorane potently induced contralateral rotation in unilaterally 6-OHDA-lesioned rats, an effect abolished by raclopride and L741,626, but not by D3 and D4 receptor-selective doses of S33084 and S18126, respectively. In functional ([35S]GTPgammaS) autoradiography experiments, quinelorane stimulated G-protein activation in caudate putamen and, to a lesser extent, in nucleus accumbens and cingulate cortex of naive rats. In unilaterally SNPC-lesioned rats, quinelorane-induced G-protein activation in the caudate putamen on the non-lesioned side was similar to that seen in naive animals (approximately 50% stimulation), but significantly greater on the lesioned side (approximately 80%). This increase was both pharmacologically and regionally specific since it was reversed by raclopride, and was not observed in nucleus accumbens or cingulate cortex. In conclusion, the present data indicate that, in rat striatum, the actions of quinelorane are mediated primarily by D2 receptors, and suggest that behavioural hypersensitivity to this agonist, induced by unilateral SNPC lesions, is associated with an increase in D2, but not D3 or D4, receptor-mediated G-protein activation.

S 15535 and WAY 100,635 antagonise 5-HT-stimulated [35S]GTP gamma S binding at cloned human 5-HT1A receptors.

In Chinese hamster ovary (CHO) cells expressing cloned human 5-HT1A receptors, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) exhibited high affinity (Ki = 0.79 nM), similar to that of 5-HT (0.61 nM), (+/-)-8-hydroxy-3-(di-n-propylamino)tetralin ((+/-)-8-OH-DPAT; 0.58 nM) and N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N- (2-pyridinyl)cyclo-hexanecarboxamide (WAY 100.635; 0.56 nM). In these cells, 5-HT stimulated [35S]GTP gamma S binding 3-fold (EC50 = 15 nM) whereas (+/-)-8-OH-DPAT exhibited 73% efficacy relative to 5-HT (EC50 = 6.0 nM). WAY 100.635 completely blocked 5-HT- and (+/-)-8-OH-DPAT-stimulated [35S]GTP gamma S binding. Likewise, S 15535 antagonised 5-HT-stimulated [35S]GTP gamma S binding, reducing it to 30.1% of control values. S 15535 (100 nM) also shifted the 5-HT and (+/-)-8-OH-DPAT stimulation curves to the right, to EC50 values of 870 and 313 nM, respectively. However, unlike WAY 100.635, which by itself did not stimulate [35S]GTP gamma S binding, S 15535 alone increased it by 34.7% relative to 5-HT (EC50 = 5.8 nM). In conclusion, S 15535 antagonises the stimulation of 5-HT1A receptors by 5-HT, whilst itself exerting weak partial agonist activity at these sites.

The 5HT(1A) receptor ligand, S15535, antagonises G-protein activation: a [35S]GTPgammaS and [3H]S15535 autoradiography study.

4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.]. The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

Down-regulation of rat beta-adrenoceptors by clenbuterol or desipramine does not require chronic treatment: [3H] CGP-12177 binding reveals rapid (24 hour) modulation.

Desipramine (DMI, 15 mg/kg, s.c.) decreased [3H]CGP-12177-labelled cortical beta-adrenoceptor density (Bmax) by 30% upon chronic (14 day) treatment. However, even a single dose (in mg/kg) of DMI (15) or the beta-adrenoceptor agonist, clenbuterol (20), induced a rapid (24 hour) and significant reduction of beta-adrenoceptor Bmax (-15%; p < 0.01). Acute treatment with amitryptiline (10), clorgyline (1), fluoxetine (10), nomifensine (10) or maprotiline (20) had no significant effect on [3H]CGP-12177-labelled beta-adrenoceptors, suggesting that rapid down-regulation may not be a general property of antidepressant drugs. None of the antidepressants altered the Bmax of [3H]ketanserin-labelled 5-HT2A receptors on acute treatment. These results show that beta-adrenoceptor down-regulation by clenbuterol and DMI is not dependent on chronic treatment and may, therefore, be a poor correlate of the gradual onset of therapeutic efficacy seen clinically with antidepressant drugs.

Pindolol antagonises G-protein activation at both pre- and postsynaptic serotonin 5-HT1A receptors: a.

The arylalkylamine, pindolol, may potentiate the clinical actions of antidepressant agents. Although it is thought to act via blockade of 5-HT1A autoreceptors, its efficacy at these sites remains controversial. Herein, we evaluated the actions of pindolol at 5-HT1A autoreceptors and specific populations of postsynaptic 5-HT1A receptors employing [35S]GTPgammaS autoradiography, a measure of receptor-mediated G-protein activation. Both 8-OH-DPAT (1 microM) and 5-HT (10 microM) elicited a pronounced increase in [35S]GTPyS binding in the dorsal raphe nucleus, which contains serotonergic cell bodies bearing 5-HT1A autoreceptors. Pindolol abolished their actions. In the dentate gyrus, lateral septum and entorhinal cortex, structures enriched in postsynaptic 5-HT1A receptors, 8-OH-DPAT (1 microM) and 5-HT (10 microM) also elicited a marked increase in [35S]GTPgammaS binding which was likewise blocked by pindolol. The antagonism of 5-HT-induced [35S]GTPgammaS labelling in the dentate gyrus was shown to be concentration-dependent, yielding a pIC50 of 5.82. Pindolol did not, itself, affect [35S]GTPgammaS binding in any brain region examined. In conclusion, these data suggest that, as characterised by [35S]GTPgammaS autoradiography, and compared with 5-HT and 8-OH-DPAT, pindolol possesses low efficacy at both pre- and postsynaptic 5-HT1A receptors.

Labelling of recombinant human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [3H]-S 15535: definition of its binding profile using agonists, antagonists and inverse agonists.

The novel benzodioxopiperazine, 5-HT1A receptor weak partial agonist, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) bound with high affinity and selectivity to membranes of Chinese Hamster Ovary cells stably expressing the human (h) 5-HT1A receptor (Ki = 0.6 nM versus [3H]-8-hydroxy-dipropylamino-tetralin, [3H]-8-OH-DPAT): its affinity at h5-HT1A receptors was more than 70-fold higher than its affinity at > 50 other binding sites. S 15535 was tritiated to high specific activity (50 Ci/mmol) and its binding profile characterised. At 22 degrees C, [3H]-S 15535 associated and dissociated from h5-HT1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a Kd estimate of 3.6 nM. In saturation binding experiments, [3H]-S 15535 displayed a Bmax value for h5-HT1A receptors (1630 fmol/mg), higher than that obtained with the agonist [3H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 microM) reduced the binding of [3H]-S 15535 by only 25% compared with 79% for [3H]-8-OH-DPAT at h5-HT1A receptors. [3H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (Bmax = 820 fmol/mg versus 647 fmol/mg for [3H]-8-OH-DPAT). For both h5-HT1A and rat 5-HT1A receptors, the Ki values for competition binding of 15 serotonergic ligands with [3H]-S 15535 was highly correlated with that of [3H]-8-OH-DPAT. However, important differences were also observed. The agonist, 5-hydroxytryptamine (5-HT), displayed biphasic competition curves with [3H]-S 15535 but not with [3H]-8-OH-DPAT at h5-HT1A receptors. Similarly, the 'antagonists', spiperone, methiothepin and (+)butaclamol, showed biphasic competition isotherms versus [3H]-S 15535 but not [3H]-8-OH-DPAT. When [3H]-S 15535 competition binding experiments were carried out in the presence of GppNHp (100 microM) the 5-HT and 8-OH-DPAT competition curves shifted to the right, whereas the spiperone and methiothepin competition curves shifted to the left. In contrast, in the presence of GppNHp, the competition isotherms for N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-h exanecarboxamide (WAY 100,635) were not altered. Taken together, these data show that (i) [3H]-S 15535 is a highly selective 5-HT1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT1A receptors, (ii) antagonists, such as WAY 100,635, which yield monophasic isotherms in competition with both [3H]-agonists and [3H]-antagonists, are not sensitive to the G-protein coupling state of the receptor, but (iii) spiperone and methiothepin behaved as inverse agonists, their competition isotherms with [3H]-S 15535 being modulated in an opposite manner to those of agonists.

Agonist activity of antimigraine drugs at recombinant human 5-HT1A receptors: potential implications for prophylactic and acute therapy.

The actions of several serotonergic ligands in use or under development for the treatment of migraine headaches were examined at recombinant human 5-HT1A receptors stably expressed in Chinese Hamster Ovary cells. Affinities (K(i)s) at this site were determined in competition binding experiments with [3H]-8-OH-DPAT ([3H](+/-)8-hydroxy-N,N-dipropylaminotetralin), whilst agonist efficacy was measured by stimulation of [35S]-GTP gamma S (guanylyl-5'-[gamma[35S]thio]-triphosphate) binding. Of the prophylactic antimigraine drugs tested, methysergide and lisuride behaved as efficacious agonists (Emax > or = 90% relative to 5-HT) whereas pitozifen and (-)propranolol acted as a partial agonist (60%) and an antagonist, respectively. This suggests that there is no correlation between agonism at 5-HT1A receptors and prophylactic antimigraine action. In contrast, serotonin, dihydroergotamine, sumatriptan, naratriptan and alniditan, which are effective in acute interruption of migraine attacks, each displayed high efficacy (Emax = 100, 100, 92.6, 79.3, 79.1% respectively) and marked affinity (Ki = 18.7, 0.6, 127, 26.4 and 3.0 nM respectively) at 5-HT1A receptors. EC50 values for agonist stimulation of [35S]-GTP gamma S binding correlated with respective Ki values at 5-HT1A receptors (r = 0.93) and the stimulation of [35S]-GTP gamma S binding by these compounds was antagonised by the selective 5-HT1A antagonist WAY 100,635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide; 100 nM). These data suggest that agonism at 5-HT1A receptors may be involved in some actions of drugs used in acute antimigraine therapy. In comparison with the above compounds, novel ligands targeted at 5-HT1B/1D receptors, such as GR125,743 (N-[4-methoxy-3-(4-methyl-piperazin-1-yl)phenyl] -3-methyl-4-(4-pyridyl)benzamide) and GR 127,935 (N-[4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-2'-methyl-4'-(5-m ethyl-1, 2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide), only weakly activated [35S]-GTP gamma S binding (32.4 and 32.1% efficacy) and displayed moderate affinity at 5-HT1A receptors (Kis 53.1 and 49.8 nM) suggesting that they constitute useful tools to differentiate 5-HT1A and 5-HT1B/1D receptor-mediated actions. In conclusion, the present data indicates that several antimigraine agents exhibit marked 5-HT1A receptor activity and that although this is unlikely to be important for prophylactic action it may be relevant to the ancilliary properties of drugs used for acute migraine treatment.

Comparison of hippocampal G protein activation by 5-HT(1A) receptor agonists and the atypical antipsychotics clozapine and S16924.

This study employed [(35)S]guanosine 5'- O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to compare the actions of antipsychotic agents known to stimulate cloned, human 5-HT(1A) receptors with those of reference agonists at postsynaptic 5-HT(1A) receptors. In rat hippocampal membranes, the following order of efficacy was observed (maximum efficacy, E(max), values relative to 5-HT=100): (+)8-OH-DPAT (85), flesinoxan (62), eltoprazine (60), S14506 (59), S16924 (48), buspirone (41), S15535 (22), clozapine (22), ziprasidone (21), pindolol (7), p-MPPI (0), WAY100,635 (0), spiperone (0). Despite differences in species and tissue source, the efficacy and potency (pEC(50)) of agonists (with the exception of clozapine) correlated well with those determined previously at human 5-HT(1A) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast, clozapine was more potent at hippocampal membranes. The selective antagonists p-MPPI and WAY100,635 abolished stimulation of binding by (+)8-OH-DPAT, clozapine and S16924 (p-MPPI), indicating that these actions were mediated specifically by 5-HT(1A) receptors. Clozapine and S16924 also attenuated 5-HT- and (+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding, consistent with partial agonist properties. In [(35)S]GTPgammaS autoradiographic studies, 5-HT-induced stimulation, mediated through 5-HT(1A) receptors, was more potent in the septum (pEC(50) approximately 6.5) than in the dentate gyrus of the hippocampus (pEC(50) approximately 5) suggesting potential differences in coupling efficiency or G protein expression. Though clozapine (30 and 100 microM) did not enhance [(35)S]GTPgammaS labelling in any structure, S16924 (10 micro M) modestly increased [(35)S]GTPgammaS labelling in the dentate gyrus. On the other hand, both these antipsychotic agents attenuated 5-HT (10 microM)-stimulated [(35)S]GTPgammaS binding in the dentate gyrus and septum. In conclusion, clozapine, S16924 and ziprasidone act as partial agonists for G protein activation at postsynaptic 5-HT(1A) receptors in the hippocampus. These data support a role of postsynaptic 5-HT(1A) receptors in the functional profiles of certain antipsychotic agents.