Differential modulation by GTPgammaS of agonist and inverse agonist binding to h5-HT(1A) receptors revealed by [3H]-WAY100,635

1. The interaction of serotonergic ligands at human (h) 5-HT(1A) receptors expressed in Chinese hamster ovary cells was examined with the selective 'neutral' 5-HT(1A) antagonist [(3)H]-WAY100,635. Its binding was saturable (K(D)=0.056 nM) with a B(max) (3.65 pmol mg(-1)) significantly higher than that of two other selective 5-HT(1A) radioligands: the partial agonist, [(3)H]-S15535 (2.77 pmol mg(-1)) and the agonist, [(3)H]-8-OH-DPAT (2.02 pmol mg(-1)). 2. The influence of GTPgammaS (100 microM) on the binding affinity of 15 serotonergic agonists, partial agonists, antagonists and inverse agonists was investigated in competition binding experiments with [(3)H]-WAY100,635. 3. Agonists, including 5-HT, 8-OH-DPAT and buspirone, displayed biphasic isotherms which shifted to the right in the presence of GTPgammaS. In contrast, isotherms of the inverse agonists, methiothepin, (+)butaclamol and spiperone, were shifted to the left in the presence of GTPgammaS. Unlabelled WAY100,635 was the only ligand that was unaffected by GTPgammaS, consistent with 'neutral' antagonist properties. 4. The magnitude of affinity changes induced by GTPgammaS for 13 ligands was highly correlated (r = 0.98) with their efficacy (positive and negative) previously determined by [(35)S]GTPgammaS binding. 5. In contrast, the napthylpiperazine derivative and high efficacy agonist, S14506, displayed only a modest GTPgammaS shift, in accordance with previous indications of 'atypical' binding properties of this ligand. A further full agonist, S14671, which is chemically closely-related to S14506, also displayed a minimal GTPgammaS shift, underpinning this observation. 6. In conclusion, [(3)H]-WAY100,635 constitutes a useful neutral antagonist radioligand for the characterization of drug actions at h5-HT(1A) receptors. GTPgammaS-induced affinity changes of agonist and inverse agonist competition isotherms generally correlate well with ligand efficacy, with the notable exception of two chemically-similar agents, S14506 and S14671, which are efficacious agonists, yet relatively insensitive to h5-HT(1A) receptor/G-protein coupling changes.     

5-HT(1A) receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

1. It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT(1A) receptors. 2. Saturation studies using [(3)H]-8-OH-DPAT and [(3)H]-MPPF revealed a single, high affinity site (K(D)approximately 1 nM) in HEK293 cells expressing human 5-HT(1A) receptors and rat cortex. In recombinant cells, [(3)H]-MPPF labelled 3 - 4 fold more sites than [(3)H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [(3)H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT(1A) receptors but did not bind to native tissue 5-HT(1A) receptors. These data suggest that, in transfected HEK293 cells, human 5-HT(1A) receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. 3. Receptor agonists inhibited [(3)H]-MPPF binding from recombinant 5-HT(1A) receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [(3)H]-8-OH-DPAT and [(3)H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [(3)H]-MPPF and [(3)H]-8-OH-DPAT in a monophasic manner. 4. Functional evaluation of compounds, using [(35)S]-GTPgammaS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. 5. [(3)H]-8-OH-DPAT : [(3)H]-MPPF pK(i) difference correlated well with functional intrinsic activity (r=0.86) as did [(3)H]-8-OH-DPAT : [(3)H]-spiperone pK(i) difference with functional intrinsic activity (r=0.96). 6. Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT(1A) receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT(1A) receptors in which only the high agonist affinity state was detectable.     

Correlation between low/high affinity ratios for 5-HT(1A) receptors and intrinsic activity

G protein-coupled receptors exist in G protein-coupled and -uncoupled forms that exhibit high and low affinity for agonists, respectively. Consequently, affinity differences of a compound for the high vs. the low affinity state of a receptor have been used to estimate its intrinsic activity at that receptor. We examined the affinity of a series of compounds for 5-hydroxytryptamine(1A) (5-HT(1A)) receptor sites labeled with 0.2 nM [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) (high affinity), or with 0.25 nM [3H]4-(2'-methoxy-)-phenyl-1-[2'-(N-2"-pyridyl)-p-fluorobenzamido] eth yl-piperazine ([3H]p-MPPF) in the presence of 100 microM guanylylimidodiphosphate (Gpp(NH)p) (low affinity) in rat hippocampal membranes. For a variety of 5-HT(1A) receptor ligands, the low/high affinity ratio (ranging from 110 for 5-HT to 0.12 for spiperone) was in good agreement with their reported intrinsic activity. Positive rank correlations were found between low/high affinity ratios and intrinsic activities (E(max) values) reported in the literature. The high efficacy 5-HT(1A) receptor agonists, 1[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)piperaz ine (S-14506) and dihydroergotamine, however, had similar, high affinity for both G protein-coupled and -uncoupled forms of the receptor. The Hill coefficients for both compounds were markedly higher than 1.0, suggesting that positive cooperativity could be responsible for the unexpected results. The 5-HT(1A) receptor agonist activity of dihydroergotamine and S-14506, assessed by measuring the inhibition of forskolin-stimulated cAMP accumulation, was blocked completely by pertussis toxin, reinforcing the suggested involvement of an inhibitory G protein in their effects. Taken together, the results suggest that, although the low/high affinity ratio of a ligand for 5-HT(1A) receptors generally covaries with its intrinsic activity, dihydroergotamine and S-14506 may interact with 5-HT(1A) receptors in a manner different from that of other 5-HT(1A) receptor agonists. Their effects, however, appear to be G(i) protein-dependent.     

(3)H]-8-OH-DPAT binding in the rat brain raphe area: involvement of 5-HT(1A) and non-5-HT(1A) receptors

1. The 5-HT(1A) agonist 8-OH-DPAT has been shown to have additional 5-HT uptake inhibiting properties. The present work was undertaken to examine further the binding of [(3)H]-8-OH-DPAT in the raphe area of the rat brain, a region rich in 5-HT(1A) receptors and 5-HT uptake sites. 2. 5-HT inhibited [(3)H]-8-OH-DPAT binding in a biphasic manner (pK(i1): 8.82+/-0.01, pK(i2): 6.07+/-0.05, n=4) with the low affinity site representing 36+/-4% of the total population. A biphasic inhibition curve was found also with the 5-HT(1A) antagonist, WAY 100635 (pK(i1): 8.65+/-0.17, pK(i2): 4.26+/-0.38, n=3). In the presence of 1 microM WAY 100635 to mask 5-HT(1A) receptors, 5-HT inhibited [(3)H]-8-OH-DPAT binding in a monophasic manner (pK(i): 6.04+/-0.07, n=3). 3. The affinities of various compounds for sites labelled by [(3)H]-8-OH-DPAT in the presence of 1 microM WAY 100635 and for sites labelled by [(3)H]-citalopram (a selective 5-HT uptake inhibitor) were determined. There was a significant correlation between pK(i) values at 5-HT uptake sites and at non-5HT(1A) sites labelled by [(3)H]-8-OH-DPAT (r=0.80, P<0. 001, n=17), suggesting these latter sites to be 5-HT uptake sites. 4. Whereas the affinities of R(+) and S(-) enantiomers of 8-OH-DPAT for the 5-HT uptake site are similar, R(+)8-OH-DPAT has 10 times higher affinity for the non-5-HT(1A) site than S(-)8-OH-DPAT and was considered as an outlier in the correlation. It is suggested that [(3)H]-8-OH-DPAT labels other, as yet unknown binding sites in the raphe.     

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-HT_1A 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-HT_1A receptor (K_i = 0.6 nM versus [³H]-8-hydroxy-dipropylamino-tetralin, [³H]-8-OH-DPAT): its affinity at h5-HT_1A 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° C, [³H]-S 15535 associated and dissociated from h5-HT_1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a K_d estimate of 3.6 nM. In saturation binding experiments, [³H]-S 15535 displayed a B_max value for h5-HT_1A receptors (1630 fmol/mg), higher than that obtained with the agonist [³H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 μM) reduced the binding of [³H]-S 15535 by only 25% compared with 79% for [³H]-8-OH-DPAT at h5-HT_1A receptors. [³H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (B_max = 820 fmol/mg versus 647 fmol/mg for [³H]-8-OH-DPAT). For both h5-HT_1A and rat 5-HT_1A receptors, the K_i 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-HT_1A 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 μM) 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)cyclohexanecarboxamide (WAY 100,635) were not altered. Taken together, these data show that (i) [³H]-S 15535 is a highly selective 5-HT_1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT_1A 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. Received: 12 September 1997 / Accepted: 1 December 1997     

Histamine H3 and dopamine D2 receptor-mediated [35S]GTPgamma[S] binding in rat striatum: evidence for additive effects but lack of interactions

The interactions in the rat striatum between H(3) receptors (H(3)Rs) and D(2) receptors (D(2)Rs) were investigated with the [(35)S]GTPgamma[S] binding assay. The H(3)R agonist (R)alpha-methylhistamine increased [(35)S]GTPgamma[S] binding to striatal membranes with an EC(50)=14+/-5 nM and a maximal effect of +19+/-1%. This effect was inhibited by the H(3)R antagonist ciproxifan with a K(i)=1.0+/-0.3 nM. The D(2)R agonist quinpirole increased [(35)S]GTPgamma[S] binding to the same membranes with an EC(50)=1.5+/-0.5 microM and a maximal effect of +28+/-2%. Its effect was blocked by haloperidol with a K(i)=0.3+/-0.1 nM. The maximal effects of the H(3)R and D(2)R agonists were additive (+46+/-3%). However, D(2)R ligands did not modify the effects of H(3)R ligands and vice versa. Ciproxifan behaved as an H(3)R inverse agonist and decreased [(35)S]GTPgamma[S] binding. Haloperidol had no effect and did not change the inverse agonist effect of ciproxifan. Administrations for 10 days of ciproxifan (1.5mg/kg/day) or haloperidol (0.5mg/kg/day) did not change the effects of quinpirole and (R)alpha-methylhistamine, respectively. These data suggest that striatal H(3)Rs and D(2)Rs do not interact through their coupling to G-proteins. However, a hyperactivity of histaminergic and dopaminergic neurons being observed in schizophrenia, the additive activations of H(3)Rs and D(2)Rs suggest that they cooperate to generate some schizophrenic symptoms. Such a postsynaptic mechanism may underlie the antipsychotic-like effects of H(3)R inverse agonists and supports their therapeutic interest, alone or as adjunctive treatment with neuroleptics.     

(3)H]-SB-269970--A selective antagonist radioligand for 5-HT(7) receptors

Binding of the 5-HT(7) receptor antagonist radioligand [(3)H]-SB-269970 to human 5-HT(7(a)) receptors expressed in HEK293 cell membranes (h5-HT(7(a))/293) and to guinea-pig cerebral cortex membranes, was characterized and compared with [(3)H]-5-CT binding. [(3)H]-SB-269970 (1 nM) showed full association with h5-HT(7(a))/293 membranes after 40 min. Specific binding at equilibrium represented >90% of total binding and was fully reversible by methiothepin (10 microM), full dissociation occurring by 100 min. The association (k(+1)) and dissociation (k(-1)) rate constants were 0.05 nM(-1)min(-1) and 0.05 min(-1) respectively, giving a K(D) (k(-1)/k(+1)) of 1.0 nM. [(3)H]-SB-269970 bound saturably and apparently monophasically to both h5-HT(7(a))/293 and guinea-pig cortex membranes, with K(D) values of 1.25+/-0.05 and 1.7+/-0.3 nM respectively. The B(max) for [(3)H]-SB-269970 to both h5-HT(7(a))/293 and guinea-pig cortex membranes (5780+/-380 and 125+/-8.2 fmoles mg protein(-1) respectively) was similar to that for [(3)H]-5-CT (6190+/-940 and 143+/-19 fmoles mg protein(-1) respectively). These data suggest that, in each tissue, both radioligands labelled the same population of receptors, which appear to be present in an agonist high affinity state. The profile of compound inhibition of [(3)H]-SB-269970 binding to h5-HT(7(a))/293 and guineapig cortex membranes correlated well (corr. coeff. 0.98) with those for [(3)H]-5-CT binding and were consistent with the profiles reported previously for the human 5-HT(7(a)) and guinea-pig cortex 5-HT(7) receptors using [(3)H]-5-CT. Hill slopes for inhibition of [(3)H]-SB-269970 and [(3)H]-5-CT binding were close to 1, consistent with binding to a single receptor population in both tissues. [(3)H]-SB-269970 represents the first selective 5-HT(7) antagonist radioligand, which should aid further characterization of 5-HT(7) receptors in recombinant and native tissues and help establish their role in brain function.     

Short-term inverse-agonist treatment induces reciprocal changes in delta-opioid agonist and inverse-agonist binding capacity

This study assessed the effects of short-term treatment (30-min) with inverse agonists on receptor protein levels and on the ability of agonists, inverse agonists, and neutral antagonists to bind to the human delta-opioid receptor (h delta OR). Incubation of human embryonic kidney 293s cells stably expressing h delta OR with the inverse agonist ICI174864 (1 microM) induced reciprocal changes in agonist and inverse-agonist binding. The total number of binding sites recognized by the agonists [(3)H]bremazocine and [(3)H][D-Pen(2),D-Pen(5)]-enkephalin was reduced by 33 and 57%, respectively, whereas binding capacity for the radiolabeled inverse-agonist [(3)H]Tyr-TicY[CH(2)NH]Cha-Phe-OH increased by 44%. In contrast, total receptor protein and sites labeled by neutral antagonists [(3)H]naltrindole and [(3)H]Tyr-D-Tic-Phe-Phe-OH remained unchanged. Pertussis toxin (PTX) and 5-guanylylimidodiphosphate (GppNHp) mimicked the outcome of ICI174864 pretreatment in promoting the loss of agonist binding sites. The lack of an additive effect on [(3)H]bremazocine binding when these three agents were combined indicates that inverse agonists may, in part, share the mechanism by which GppNHp and PTX reduce agonist binding capacity. Spontaneous recovery of maximal agonist binding capacity after inverse-agonist treatment was slow, suggesting a decrease in the isomerization rate between agonist- and inverse agonist-preferring conformations. Overall, the data presented are consistent with the idea that h delta ORs exist in multiple states capable of discriminating among ligands of different levels of efficacy and show that, after short-term treatment with an inverse agonist, the receptor ability to adopt conformations preferentially induced by agonist ligands is reduced.     

Modulation of 5-hydroxytryptamine efflux from rat cortical synaptosomes by opioids and nociceptin

The modulation of [(3)H]-5-hydroxytryptamine ([(3)H]-5-HT) efflux from superfused rat cortical synaptosomes by delta, kappa, mu and ORL(1) opioid receptor agonists and antagonists was studied. Spontaneous [(3)H]-5-HT efflux was reduced (20% inhibition) by either 0.5 microM tetrodotoxin or Ca(2+)-omission. Ten mM K(+)-evoked [(3)H]-5-HT overflow was largely Ca(2+)-dependent (90%) and tetrodotoxin-sensitive (50%). The delta receptor agonist, deltorphin-I, failed to modulate the K(+)-evoked neurotransmitter efflux up to 0.3 microM. The kappa and the mu receptor agonists, U-50,488 and endomorphin-1, inhibited K(+)-evoked [(3)H]-5-HT overflow (EC(50)=112 and 7 nM, respectively; E(max)=28 and 29% inhibition, respectively) in a norBinaltorphimine- (0.3 microM) and naloxone- (1 microM) sensitive manner, respectively. None of these agonists significantly affected spontaneous [(3)H]-5-HT efflux. The ORL(1) receptor agonist nociceptin inhibited both spontaneous (EC(50)=67 nM) and K(+)-evoked (EC(50)=13 nM; E(max)=52% inhibition) [(3)H]-5-HT efflux. The effect of NC was insensitive to naloxone (up to 10 microM), but was antagonized by [Nphe(1)]nociceptin(1-13)NH(2) (a novel selective ORL(1) receptor antagonist; pA(2)=6.7) and by naloxone benzoylhydrazone (pA(2)=6.3). The ORL(1) ligand [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) also inhibited K(+) stimulated [(3)H]-5-HT overflow (EC(50)=64 nM; E(max)=31% inhibition), but its effect was partially antagonized by 10 microM naloxone. It is concluded that the ORL(1) receptor is the most important presynaptic modulator of neocortical 5-HT release within the opioid receptor family. This suggests that the ORL(1)/nociceptin system may have a powerful role in the control of cerebral 5-HT-mediated biological functions.     

Dihydroergotamine and its metabolite, 8'-hydroxy-dihydroergotamine,as 5-HT1A receptor agonists in the rat brain

1 In addition to stopping migraine attacks, dihydroergotamine (DHE) is an efficient drug for migraine prophylaxis. Whether 5-HT(1A) receptors could contribute to the latter action was assessed by investigating the effects of DHE and its metabolite, 8'-OH-DHE, on these receptors in the rat brain. 2 Membrane binding assays with [(3)H]8-OH-DPAT and [(3)H]WAY 100635 as radioligands showed that both DHE (IC(50)=28-30 nM) and 8'-OH-DHE (IC(50)=8-11 nM) are high-affinity 5-HT(1A) receptor ligands. 3 Both DHE and 8'-OH-DHE enhanced the specific binding of [(35)S]GTP-gamma-S to the dorsal raphe nucleus and the hippocampus in brain sections, but to a lower extent than 5-carboxamido-tryptamine (5-CT) in the latter area. 4 Both DHE (EC(50)=10.9+/-0.3 nM) and 8'-OH-DHE (EC(50)=30.4+/-0.8 nM) inhibited the firing of serotoninergic neurons in the dorsal raphe nucleus within brain stem slices. 5 Intracellular recording showed that 8'-OH-DHE was more potent than DHE to hyperpolarize CA1 pyramidal cells in rat hippocampal slices. 6 Both the stimulatory effects of DHE and 8'-OH-DHE on [(35)S]GTP-gamma-S binding and their electrophysiological effects were completely prevented by the selective 5-HT(1A) receptor antagonist WAY 100635. 7 As expected of 5-HT(1A) receptor partial agonists, DHE and 8'-OH-DHE prevented any subsequent hyperpolarization of CA1 pyramidal cells by 5-HT or 5-CT. 8 Through their actions at 5-HT(1A) auto- (in the dorsal raphe nucleus) and hetero-(notably in the hippocampus) receptors, DHE, and even more its metabolite 8'-OH-DHE, can exert both an inhibitory influence on neuronal excitability and anxiolytic effects which might contribute to their antimigraine prophylactic efficiency.     

Pharmacological characterization of the 5-HT1A, 5-HT2 and 5-HT3 receptors in the bovine ciliary muscle

We aimed to investigate the effects of serotonin (5-hydroxytryptamine, 5-HT) on the bovine ciliary muscle and subsequently to characterize and identify the subtypes of 5-HT receptors involved in the serotonin-evoked contractility muscle. The binding of [3H]ketanserin, [3H]granisetron and [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was analyzed. All labelled compounds bound with high affinity to a single site in the membrane preparations studied. The affinity (K(d)) of the binding site was 7.5+/-1.2 nM for [3H]ketanserin, 6.9+/-0.8 nM for [3H]granisetron and 4.4+/-0.31 nM for [3H]8-OH-DPAT. The density of receptors (B(max)) was 1062+/-43.0 fmol/mg protein for [3H]ketanserin, 566+/-2.32 fmol/mg protein for [3H]granisetron and 205+/-4.63 fmol/mg protein for [3H]8-OH-DPAT. The serotonin-induced contraction appeared to be competitively antagonized by ketanserin (0.1, 1 and 10 microM) and ondansetron (0.1, 10 and 100 microM) which produced a pA(2) value of 8.5+/-0.12 and 8.0+/-0.19, respectively. 8-OH-DPAT and 5-carboxamidotryptamine (5-CT) proved to be completely ineffective. We conclude that serotonin induces bovine ciliary muscle contraction via 5-HT(2) and 5-HT(3) receptors while the 5-HT(1A) receptors, although present, do not mediate the contractile response.     

Aging alters in a region-specific manner serotonin transporter sites and 5-HT(1A) receptor-G protein interactions in hamster brain

Key proteins regulating serotonergic activity, specifically the serotonin transporter and 5-HT(1A) receptor, were examined in the midbrain raphe nuclei of young (3-4 months) and old (17-19 months) hamsters (N=7-10/group). An age-related decrease in the maximal density of serotonin transporter sites labelled with [(3)H]paroxetine (fmol/mg protein, Old: 396+/-13; Young: 487+/-27) was observed in the dorsal raphe nucleus (DRN) but not the median raphe nucleus (MRN), without affecting the affinity of [(3)H]paroxetine. In the DRN and MRN, the stimulation of [(35)S]GTP gamma S binding by the 5-HT(1A) receptor agonist 8-OH-DPAT, or the number of 5-HT(1A) receptor sites labeled with [(3)H] MPPF, was not different in old versus young animals. Thus in the DRN, aging decreased serotonin transporter sites without changing 5-HT(1A) receptor activation of G proteins or 5-HT(1A) receptor density. In the CA(1) region of hippocampus, 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding was increased in the older animals (% above basal, Old: 141+/-21; Young: 81+/-17) without changing specific [(3)H] MPPF binding sites, suggesting that the capacity of 5-HT(1A) receptors to activate G proteins is enhanced. Aging also appears to enhance this capacity in the dentate gyrus, because this region exhibited a constant level of 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding in spite of an age-related decrease in the number of [(3)H] MPPF binding sites (fmol/mg protein, Old: 203+/-21; Young: 429+/-51).     

5-hydroxytryptamine interaction with the nicotinic acetylcholine receptor

The present study examines the interaction of the neurotransmitter 5-hydroxytryptamine (5-HT) with muscle-type nicotinic acetylcholine receptors. 5-HT inhibits the initial rate of [125I]alpha-bungarotoxin binding to Torpedo acetylcholine receptor membranes (IC(50)=8.5+/-0.32 mM) and [3H]5-HT can be photoincorporated into acetylcholine receptor subunits, with labeling of the alpha-subunit inhibitable by both agonists and competitive antagonists. Within the agonist-binding domain, [3H]5-HT photoincorporates into alphaTyr(190), alphaCys(192) and alphaCys(193). Functional studies using the human clonal cell line TE671/RD, show that 5-HT is a weak inhibitor (IC(50)=1.55+/-0.25 mM) of acetylcholine receptor activity. In this regard, agonist-response profiles in the absence and presence of 5-HT indicate a noncompetitive mode of inhibition. In addition, 5-HT displaces high affinity [3H]thienylcyclohexylpiperidine binding to the desensitized Torpedo acetylcholine receptor channel (IC(50)=1.61+/-0.07 mM). Collectively, these results indicate that 5-HT interacts weakly with the agonist recognition site and inhibits receptor function noncompetitively by binding to the acetylcholine receptor channel.     

Blockade of voltage-sensitive Na(+) channels by the 5-HT(1A) receptor agonist 8-OH-DPAT: possible significance for neuroprotection

The present study was undertaken to determine whether 5-hydroxytryptamine(1A) (5-HT(1A)) receptor agonists interact with voltage-sensitive Na(+) or N- and P/Q-type Ca(2+) channels to reduce the influx of Na(+) and/or Ca(2+). The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) inhibited both [3H]batrachotoxinin binding to neurotoxin site 2 of the Na(+) channel in rat cortical membranes (IC(50)=5.1 microM) and veratridine-stimulated Na(+) influx into rat synaptosomes (EC(50)=20. 8 microM). The 5-HT(1A) receptor agonist flesinoxan and the 5-HT(1A) receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635) also displaced [3H]batrachotoxinin binding with similar affinities to 8-OH-DPAT, but were much less effective in reducing veratridine-stimulated Na(+) influx. All three serotonergic agents also increased [3H]saxitoxin binding to neurotoxin site 1 of the Na(+) channel. In contrast, none of these agents interacted with radioligand binding to N- or P/Q-type Ca(2+) channels. These data show that 8-OH-DPAT directly interacts with voltage-sensitive Na(+) channels to reduce Na(+) influx so providing an additional mechanism to explain how it functions as a neuroprotectant.     

5-CT stimulation of adenylyl cyclase activity in guinea-pig hippocampus: evidence for involvement of 5-HT7 and 5-HT1A receptors.

1. A number of compounds, including the selective 5-HT7 receptor antagonist SB-258719, were investigated for their effect on [3H]-5-carboxamidotryptamine (5-CT) radioligand binding and 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes, in order to confirm the presence of functionally coupled 5-HT7 receptors in this tissue. 2. The [3H]-5-CT radioligand binding profile was consistent with binding predominantly to 5-HT7 receptors. The affinity of SB-258719 (pKi 7.2+/-0.1) was similar to its reported human 5-HT7 receptor affinity. 3. In the adenylyl cyclase functional assay, 5-CT was a potent and full agonist compared to 5-HT, whereas 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) was a partial agonist (intrinsic activity 0.4+/-0.1). The rank order of potency for agonists (5-CT>5-HT approximately 8-OH-DPAT) was consistent with activation of 5-HT7 receptors. SB-258719 (5 microM) and methiothepin (1 microM) surmountably antagonized the response to 5-CT, consistent with competitive antagonism. The pKB for SB-258719 (7.2+/-0.1) was in good agreement with its reported antagonist potency at the human cloned 5-HT7 receptor. 4. In the functional assay, WAY-100635 (100 nM) and cyanopindolol (1 microM) induced a biphasic 5-CT response curve, consistent with selective antagonism of a component of the response to 5-CT. The estimated pKB values for WAY-100635 and cyanopindolol (9.6 and 8.4 respectively) were in good agreement with their reported 5-HT1A receptor affinities. 5. The data are consistent with the presence of 5-HT7 receptors in guinea-pig hippocampus which are positively coupled to adenylyl cyclase. In addition, 5-HT7 receptor-mediated stimulation of adenylyl cyclase activity in this tissue appears to be augmented by a mechanism involving 5-HT1A receptor activation.     

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

1. Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT(1A) serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). 2. Ligand binding studies were used to determine dissociation constants for agonist binding to the 5-HT(1A) receptor: (a) K(i) values for agonists were determined in competition versus the binding of the agonist [(3)H]-8-OH DPAT. Competition data were all fitted best by a one-binding site model. (b) K(i) values for agonists were also determined in competition versus the binding of the antagonist [(3)H]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (K(h)) and lower affinity (K(l)) sites were determined. 3. The ability of the agonists to activate the 5-HT(1A) receptor was determined using stimulation of [(35)S]-GTPgammaS binding. Maximal effects of agonists (E(max)) and their potencies (EC(50)) were determined from concentration/response curves for stimulation of [(35)S]-GTPgammaS binding. 4. K(l)/K(h) determined from ligand binding assays correlated with the relative efficacy (relative E(max)) of agonists determined in [(35)S]-GTPgammaS binding assays. There was also a correlation between K(l)/K(h) and K(l)/EC(50) for agonists determined from ligand binding and [(35)S]-GTPgammaS binding assays. 5. Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of K(l)/K(h) for predicting the relative efficacy of agonists.     

Oleamide is a selective endogenous agonist of rat and human CB1 cannabinoid receptors

1. The ability of the endogenous fatty acid amide, cis-oleamide (ODA), to bind to and activate cannabinoid CB(1) and CB(2) receptors was investigated. 2. ODA competitively inhibited binding of the nonselective cannabinoid agonist [(3)H]CP55,940 and the selective CB(1) antagonist [(3)H]SR141716A to rat whole-brain membranes with K(i) values of 1.14 microm (0.52-2.53 microm, Hill slope=0.80, n=6) and 2.63 microm (0.62-11.20 microm, Hill slope=0.92, n=4), respectively. AEA inhibited [(3)H]CP55,940 binding in rat whole-brain membranes with a K(i) of 428 nm (346-510 nm, Hill slope=-1.33, n=3). 3. ODA competitively inhibited [(3)H]CP55,940 binding in human CB(1) (hCB(1)) cell membranes with a K(i) value of 8.13 microm (4.97-13.32 microm, n=2). In human CB(2) transfected (hCB(2)) HEK-293T cell membranes, 100 microm ODA produced only a partial (42.5+/-7%) inhibition of [(3)H]CP55,940 binding. 4. ODA stimulated [(35)S]GTPgammaS binding in a concentration-dependent manner (EC(50)=1.64 microm (0.29-9.32 microm), R(2)=0.99, n=4-9), with maximal stimulation of 188+/-9% of basal at 100 microm. AEA stimulated [(35)S]GTPgammaS binding with an EC(50) of 10.43 microm (4.45-24.42 microm, R(2)=1.00, n=3, 195+/-4% of basal at 300 microm). Trans-oleamide (trans-ODA) failed to significantly stimulate [(35)S]GTPgammaS binding at concentrations up to 100 microm. 5. ODA (10 microm)-stimulated [(35)S]GTPgammaS binding was reversed by the selective CB(1) antagonist SR141716A (IC(50)=2.11 nm (0.32-13.77 nm), R(2)=1.00, n=6). 6. The anatomical distribution of ODA-stimulated [(35)S]GTPgammaS binding in rat brain sections was indistinguishable from that of HU210. Increases of similar magnitude were observed due to both agonists in the striatum, cortex, hippocampus and cerebellum. 7. ODA (10 microm) significantly inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in mouse neuroblastoma N1E 115 cells (P=0.02, n=11). ODA-mediated inhibition was completely reversed by 1 microm SR141716A (P<0.001, n=11) and was also reversed by pretreatment with 300 ng ml(-1) pertussis toxin (P<0.001, n=6). 8. These data demonstrate that ODA is a full cannabinoid CB(1) receptor agonist. Therefore, in addition to allosteric modulation of other receptors and possible entourage effects due to fatty acid amide hydrolase inhibition, the effects of ODA may be mediated directly via the CB(1) receptor.     

Histamine H3-receptor-mediated [35S]GTP gamma[S] binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors

Constitutive activity of the recombinant and native rat and human H(3) receptors (H(3)Rs) was studied using H(3)R-mediated [(35)S]GTPgamma[S] binding and [(3)H]-arachidonic acid release. Ciproxifan, an inverse agonist at the rat H(3)R (rH(3)R), decreased [(3)H]arachidonic acid release from CHO cells expressing moderate densities (approximately 200 - 300 fmol mg(-1) protein) of the human H(3)R (hH(3)R). This effect occurred with the same magnitude than at the rH(3)R. The expression of the hH(3)R was associated with an increase in [(35)S]GTPgamma[S] binding to membranes of CHO cells. Ciproxifan decreased [(35)S]GTPgamma[S] binding to membranes of CHO (hH(3)R) cells. Both effects were correlated to receptor density and revealed that constitutive activity of the hH(3)R, although lower than that of the rH(3)R in this assay, was again observed at physiological densities (<500 fmol mg(-1) protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (K(i)=45 nM), but also as an inverse agonist (EC(50)=15 nM). Constitutive activity of the hH(3)R was also evidenced using inhibition of [(35)S]GTPgamma[S] binding by unlabelled GTPgammaS. The expression of the hH(3)R generated a high affinity binding for GTPgammaS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist. [(35)S]GTPgamma[S] binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H(3)R, whose effects were blocked by proxyfan, a neutral antagonist. [(35)S]GTPgamma[S] binding was also decreased by an A(1)-adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D(2)/D(3) dopamine, H(1) and H(2) histamine, alpha(2)-adrenergic and delta opioid receptors. In conclusion, the present study shows that the recombinant rat and human H(3) receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H(3)Rs is one of the highest among G-protein-coupled receptors present in rat brain.     

Selectivity of serotonergic drugs for multiple brain serotonin receptors. Role of [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB), a 5-HT2 agonist radioligand

The affinities of putative serotonin receptor agonists and antagonists for 5-HT1A, 5-HT1B, 5-HT1C, and 5-HT2 receptors were assayed using radioligand binding assays. The 5-HT1 sites were labeled with the agonist radioligands [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin [3H]-8-OH-DPAT, [3H]-5-HT, and [3H]mesulergine. The 5-HT2 receptor was labeled with the antagonist radioligand [3H]ketanserin or the agonist radioligand [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB). The apparent 5-HT1 receptor selectivity of agonist compounds was found to be 50- to 100-fold higher when the 5-HT2 receptor affinity was determined using the antagonist radioligand [3H]ketanserin than when the agonist radioligand [3H]DOB was used. Quipazine, a putative specific 5-HT2 agonist, appeared to be only 3-fold more potent at 5-HT2 than at 5-HT1A receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, quipazine was determined to be 100-fold more potent at 5-HT2 receptors than at 5-HT1A receptors. 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a putative specific 5-HT1B receptor agonist was apparently 10-fold more potent at 5-HT1B receptors than at 5-HT2 receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, TFMPP was found to be equipotent at 5-HT1B and 5-HT2 receptors. Using the 5-HT2 antagonist radioligand [3H]ketanserin, a similar pattern of underestimating 5-HT2 receptor selectivity and/or overestimating 5-HT1A or 5-HT1B receptor selectivity was observed for a series of serotonin receptor agonists. Antagonist receptor selectivity was not affected significantly by the nature of the 5-HT2 receptor assay used. These data indicate that, by using an antagonist radioligand to label 5-HT2 receptors and agonist radioligands to label 5-HT1 receptors, the 5-HT1 receptor selectivity may be overestimated. This may be an especially severe problem in serotonin drug development as drugs that interact potently with 5-HT2 receptors have been reported to be psychoactive and/or hallucinogenic.     

Native rat hippocampal 5-HT1A receptors show constitutive activity

Previous studies have shown that human 5-hydroxytryptamine (5-HT)1A receptors stably expressed in transfected cell lines show constitutive G-protein activity, as revealed by the inhibitory effect of inverse agonists, such as spiperone, on basal guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding. In the present study, we evaluated the constitutive activity of native rat 5-HT1A receptors in hippocampal membranes. Using anti-Galphao-antibody capture coupled to scintillation proximity assay under low sodium (30 mM) conditions, we observed high basal [35S]GTPgammaS binding to Galphao subunits (defined as 100%). Under these conditions, 5-HT and the prototypic selective 5-HT1A agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] both stimulated [35S]GTPgammaS binding to Galphao to a similar extent, raising binding to approximately 130% of basal with pEC50 values of 7.91 and 7.87, respectively. The 5-HT1A-selective neutral antagonist [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) could block these effects in a competitive manner with pKb values (5-HT, 9.57; (+)-8-OH-DPAT, 9.52) that are consistent with its pKi value at r5-HT1A receptors (9.33). In this native receptor system, spiperone and methiothepin reduced basal [35S]GTPgammaS binding to Galphao in a concentration-dependent manner to 90% of basal with pIC50 values of 7.37 and 7.98, respectively. The inhibition of basal [35S]GTPgammaS binding induced by maximally effective concentrations of spiperone (10 microM) or methiothepin (1 microM) was antagonized by WAY100,635 in a concentration-dependent manner (pKb, 9.52 and 8.87, respectively), thus indicating that this inverse agonism was mediated by 5-HT1A receptors. These data provide the first demonstration that native rat serotonin 5-HT1A receptors can exhibit constitutive activity in vitro.