Ritanserin blocks CaV1.2 channels in rat artery smooth muscles: electrophysiological, functional, and computational studies.

CaV1.2 channel blockers or 5-HT2 receptor antagonists constitute effective therapy for Raynaud's syndrome. A functional link between the inhibition of 5-HT2 receptors and CaV1.2 channel blockade in arterial smooth muscles has been hypothesized. Therefore, the effects of ritanserin, a nonselective 5-HT2 receptor antagonist, on vascular CaV1.2 channels were investigated through electrophysiological, functional, and computational studies. Ritanserin blocked CaV1.2 channel currents (ICa1.2) in a concentration-dependent manner (Kr = 3.61 µM); ICa1.2 inhibition was antagonized by Bay K 8644 and partially reverted upon washout. Conversely, the ritanserin analog ketanserin (100 µM) inhibited ICa1.2 by ~50%. Ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (Ki = 1.58 µM) without affecting the slope of inactivation and the activation curve, and decreased ICa1.2 progressively during repetitive (1 Hz) step depolarizations (use-dependent block). The addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. Furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by Bay K 8644. Ritanserin and ketanserin were docked at a region of the CaV1.2 α1C subunit nearby that of Bay K 8644; however, only ritanserin and Bay K 8644 formed a hydrogen bond with key residue Tyr-1489. In conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of CaV1.2 channels, which was achieved preferentially in the inactivated and/or resting state of the channel. This novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of Raynaud's syndrome.

Deconstructing Lipid Kinase Inhibitors by Chemical Proteomics.

Diacylglycerol kinases (DGKs) regulate lipid metabolism and cell signaling through ATP-dependent phosphorylation of diacylglycerol to biosynthesize phosphatidic acid. Selective chemical probes for studying DGKs are currently lacking and are needed to annotate isoform-specific functions of these elusive lipid kinases. Previously, we explored fragment-based approaches to discover a core fragment of DGK-α (DGKα) inhibitors responsible for selective binding to the DGKα active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGKα inhibitors to identify structural regions mediating off-target activity. We tested the activity of a fragment (RLM001) derived from a nucleotide-like region found in the DGKα inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGKα as well as >60 native ATP-binding proteins (kinases and ATPases) detected in cell proteomes. Equipotent inhibition of activity-based probe labeling by RLM001 supports a contiguous ligand-binding site composed of C1, DAGKc, and DAGKa domains in the DGKα active site. Given the lack of available crystal structures of DGKs, our studies highlight the utility of chemical proteomics in revealing active-site features of lipid kinases to enable development of inhibitors with enhanced selectivity against the human proteome.

The Ligand Binding Landscape of Diacylglycerol Kinases.

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

Molecular dynamics of serotonin and ritanserin interacting with the 5-HT2 receptor.

A three-dimensional model of the serotonin (5-hydroxytrytamine; 5-HT) 5-HT2 receptor was constructed from the amino acid sequence by molecular graphics techniques, molecular mechanics energy calculations and molecular dynamics simulations. The receptor model has 7 alpha helical segments which form a membrane-spanning duct with a putative ligand binding site. Most of the synaptic domains and the ligand binding site were surrounded by negative electrostatic potentials, suggesting that positively charged ligands are attracted to the receptor by electrostatic forces. The cytoplasmic domains, except the C-terminal tail, had mainly positive electrostatic potentials. The molecular dynamics of the receptor-ligand complex was examined in 100 ps simulations with 5-HT or ritanserin at a postulated binding site. During the simulations the helices moved from an initial circular arrangement into a more oval arrangement, and became slightly tilted relative to each other. The protonated ligands neutralized the negative electrostatic potentials around Asp 120 and Asp 155 in the central core of the receptor. 5-HT had only weak interactions with Asp 155 but strong interactions with Asp 120 during the simulations, with the amino group of 5-HT tightly bound to the carboxylic side chain of Asp 120. Ritanserin showed similarly strong interactions with Asp 120 and Asp 155 during the simulations.

Molecular modeling of serotonin, ketanserin, ritanserin and their 5-HT2C receptor interactions.

Molecular modeling techniques were used to build a three-dimensional model of the rat 5-HT2C receptor, which was used to examine receptor interactions for protonated forms of serotonin, ketanserin and ritanserin. Molecular dynamics simulations which were started with the fluoro benzene moiety of ketanserin and ritanserin oriented towards the cytoplasmic side of the receptor model, produced the strongest antagonist-receptor interactions. The fluoro bezene ring(s) of the antagonists interacted strongly with aromatic residues in the receptor model, which predicts slightly different orientations and ligand-receptor interactions of ketanserin and ritanserin at a putative binding site. The model suggests that Asn333 (transmembrane helix 6) is involved in a hydrogen-bonding interaction with ketanserin, but not with ritanserin. The model also also suggests that the position corresponding to Cys362 (transmembrane helix 7) may be an important determinant for specifying 5-HT2A receptor selectivity in ketanserin binding.

Effects of ritanserin on haloperidol-induced dopamine (D2) receptor up-regulation in the rat.

Rats were treated with vehicle, ritanserin (5 mg/kg, i.p.), haloperidol (1 mg/kg, i.p.) or both ritanserin and haloperidol for 19 days to determine whether chronic administration of the serotonin (5HT2) antagonist ritanserin affects D2 receptor up-regulation produced by haloperidol. Brain sections were prepared for D2 and 5HT2 receptor autoradiography with [3H]spiperone and [3H]ketanserin, respectively. Ritanserin significantly reduced 5HT2 receptors to 80% of vehicle in the sulcal area of the frontal cortex but had no effect on D2 receptors. Haloperidol significantly increased striatal and n. accumbens D2 receptors with no effect on 5HT2 receptors. In ritanserin/haloperidol-treated rats, D2 receptors were significantly increased along with significant decreases in 5HT2 receptors of the frontal cortex. These results suggest 5HT2 receptor antagonism by ritanserin does not significantly affect D2 receptor up-regulation produced by haloperidol.

BIMT 17, a 5-HT1A receptor agonist/5-HT2A receptor antagonist, directly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex.

BIMT 17 (1-[2-[4-(3-trifluoromethyl phenyl) piperazin-1-yl] ethyl] benzimidazol- [1H]-2-one), a 5-HT1A receptor agonist/5-HT2A receptor antagonist (see Borsini et al., accompanying paper), in a dose range of 1-10 mg/kg i.v., dose-dependently inhibited the electrical activity of rat medial prefronto-cortical neurons, whereas buspirone, in a dose range of 0.1-1000 micrograms/kg, increased it. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and 1-[2-(2-thenoylamino)ethyl]-4-[1-(7-methoxynaphthyl)] piperazine (S 14671) presented biphasic patterns of response; they increased electrical activity at doses in the range of 0.1-10 micrograms/kg and 0.1-3 micrograms/kg i.v. respectively, and reduced it at high doses, 30-300 micrograms/kg and 10-30 micrograms/kg i.v., respectively. The inhibitory effect of BIMT 17 on the firing rate of neurons in the frontal cortex was antagonized by the 5-HT1A antagonists tertatolol and WAY 100135, and was still present after destruction of serotonin (5-HT) containing neuronal endings by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 150 micrograms/rat, given intraventricularly), which reduced the cortical 5-HT content by 85%. This destruction of 5-HT neurons, while suppressing the ability of 8-OH-DPAT to inhibit the firing rate at high doses, did not change the excitatory action of this compound at low doses. The addition of ritanserin, a 5-HT2A receptor antagonist, potentiated both the excitatory and inhibitory effects of 8-OH-DPAT on neuronal electrical activity. Direct microiontophoretic application (100 nA/20 s) of 5-HT and BIMT 17, but not that of 8-OH-DPAT, onto medial prefronto-cortical neurons, decreased the firing rate of these neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for presynaptic location of inhibitory 5-HT1D beta-like autoreceptors in the guinea-pig brain cortex.

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K+ were determined in superfused synaptosomes and slices, preincubated with [3H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [3H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT1A and 5-HT2C sites) to guinea-pig cortical synaptosomes and membranes. 5-HT receptor agonists inhibited the K(+)-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl] -1H-indole-3-yl] ethylamine (L-694,247) > 5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) > or = 5-HT > sumatriptan > or = 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT1D alpha and 5-HT1D beta receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA2: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT1D alpha, but not the 5-HT1D beta, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [3H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [3H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin > 5-CT > 5-methoxytryptamine > 5-HT > or = sumatriptan > or = oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [3H]5-HT binding to synaptosomes and membranes. In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [3H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT1D beta-like.

Serotonin-2 and dopamine-1 binding components of clozapine in frontal cortex and striatum in the human brain visualized by positron emission tomography.

The specific binding of N-methyl-11C-clozapine in the human brain was studied in five healthy volunteers with positron emission tomography (PET). Four of the volunteers were reexamined after treatment with the dopamine D1 and D2 receptor antagonist flupenthixol, and all five volunteers were reexamined after pretreatment with the serotonin2 receptor antagonist ritanserin. The examinations after flupenthixol and ritanserin treatment were performed on different occasions. In the flupenthixol part of the study, two of the subjects were given an oral dose of 1 mg flupenthixol 2-3 h before the posttreatment study with PET. The other two subjects received 0.5 mg orally three times during the 24 h preceding the posttreatment PET study, with the last dose being administered < or = 4 h before the scan. All five ritanserin-treated subjects followed the same dosing regimen. During the 5 days preceding the posttreatment PET study, they were given a 10-mg tablet of ritanserin in the evening. The last dose was administered 2-1/2 hours before the study. Both flupenthixol and ritanserin pretreatment were associated with decreased binding of N-methyl-11C-clozapine in dorsolateral and medial frontal cortical regions. These results support previous findings that clozapine has affinity for both dopamine D1 and serotonin 5-HT2 receptors in the human frontal cortex. No consistent change of binding was observed in striatal regions following flupenthixol or ritanserin pretreatment. The clinical aspects of this feature are discussed, both with respect to efficacy and side effects.

Ritanserin pharmacokinetics in abstinent chronic alcoholics.

Ritanserin is a thymosthenic agent with an extensive hepatic metabolism and long elimination half life in healthy volunteers. It has been used in abstinent chronic alcoholic patients showing an interesting performance in this condition. Ritanserin pharmacokinetics has only been evaluated in single dose healthy volunteer studies and, on the other hand, chronic use of the drug in alcoholic patients during withdrawal period could be anticipated. Therefore, the objective of the present study, is to assess the cumulating kinetics of the serotonin antagonist during chronic administration. Ten abstinent alcoholic patients were included in an open study. The drug was administered at a daily dose of 10 mg for 28 days and the active phase was preceded by a seven-day wash out period with placebo. Blood samples were taken on the 1st, and 28th, day of treatment, 24 hours after taking the drug. Urine samples were taken during the night before the second and 28th dose. Plasma and urine ritanserin concentration was measured by high performance liquid chromatography. Very large variations in initial and steady state concentrations (CV = 65 and 52% respectively) were found, which is reflected in the large variability of the calculated pharmacokinetic parameters. Ritanserin cumulation factor (R) was 6.9 +/- 8.3 (mean+SEM). Two patients showing extensive accumulation had prolonged elimination half lives (433 and 279 hours) that are explained mostly by an expansion of the volume of distribution and, to a lesser extent, by diminished clearance.

Serotonergic and dopaminergic distinctions in the behavioral pharmacology of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD).

RATIONALE: After decades of social stigma, hallucinogens have reappeared in the clinical literature demonstrating unique benefits in medicine. The precise behavioral pharmacology of these compounds remains unclear, however. OBJECTIVES: Two commonly studied hallucinogens, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD), were investigated both in vivo and in vitro to determine the pharmacology of their behavioral effects in an animal model. METHOD: Rabbits were administered DOI or LSD and observed for head bob behavior after chronic drug treatment or after pretreatment with antagonist ligands. The receptor binding characteristics of DOI and LSD were studied in vitro in frontocortical homogenates from naïve rabbits or ex vivo in animals receiving an acute drug injection. RESULTS: Both DOI- and LSD-elicited head bobs required serotonin(2A) (5-HT(2A)) and dopamine(1) (D(1)) receptor activation. Serotonin(2B/2C) receptors were not implicated in these behaviors. In vitro studies demonstrated that LSD and the 5-HT(2A/2C) receptor antagonist, ritanserin, bound frontocortical 5-HT(2A) receptors in a pseudo-irreversible manner. In contrast, DOI and the 5-HT(2A/2C) receptor antagonist, ketanserin, bound reversibly. These binding properties were reflected in ex vivo binding studies. The two hallucinogens also differed in that LSD showed modest D(1) receptor binding affinity whereas DOI had negligible binding affinity at this receptor. CONCLUSION: Although DOI and LSD differed in their receptor binding properties, activation of 5-HT(2A) and D(1) receptors was a common mechanism for eliciting head bob behavior. These findings implicate these two receptors in the mechanism of action of hallucinogens.

Binding affinity of sarpogrelate, a new antiplatelet agent, and its metabolite for serotonin receptor subtypes.

We analyzed the displacement activity of sarpogrelate and its active metabolite (M-1) in the radiolabeled ligand binding to various 5-hydroxytryptamine (5-HT) receptor subtypes using rat brain cortical membranes. Sarpogrelate was shown to have the same affinity as ritanserin for 5-HT2A receptors, with a Ki value of 8.39 nM. The active metabolite of sarpogrelate, M-1, was more active than sarpogrelate itself and of ritanserin, with a Ki value of 1.70 nM. Both sarpogrelate and M-1 had no affinity for 5-HT1A receptors, but these substances, at a concentration of 10 microM, displaced the specific binding to the 5-HT1B receptors of [125I]iodocyanopindolol, resulting in Ki values of 0.881 and 0.859 microM, respectively. The Ki values of sarpogrelate and M-1 are almost the same as that of ritanserin, a specific 5-HT2 receptor antagonist. Sarpogrelate and M-1, as well as ritanserin, are shown to have very low affinity for 5-HT1B receptors. Both sarpogrelate and M-1 had no affinity for 5-HT3 receptor subtypes. In the 5-HT4 receptor binding experiments, sarpogrelate exhibited almost no affinity, while M-1, at the concentration of 10 microM, displaced the binding activity, resulting in a Ki value of 0.838 microM. Both drugs had a weak antagonistic effect on a 5-HT4 receptor-mediated function, i.e., the 5-HT-induced relaxation of rat isolated esophageal tunica muscularis mucosae. In conclusion, sarpogrelate and M-1 have high affinity for 5-HT2A receptors with a relatively high selectivity.

Excitatory responses to serotonin (5-HT) in neurons of the rat piriform cortex: evidence for mediation by 5-HT1C receptors in pyramidal cells and 5-HT2 receptors in interneurons.

As a prerequisite to pharmacological analysis of the excitatory effects of serotonin (5-HT) on piriform pyramidal cells and interneurons, this study first examined the physiological characteristics of these two cell types. Intracellular recordings confirmed that the subpopulation of 5-HT-activated cells located at the border of layers II and III are indeed interneurons. Voltage clamp recordings in pyramidal cells showed that the increase in excitability produced by 5-HT in these cells was the result of voltage- and Ca(2+)-dependent outward currents with the characteristics of IM and IAHP. Pharmacological studies were designed to discriminate 5-HT2 from 5-HT1C responses in interneurons and pyramidal cells of piriform cortex. The 5-HT antagonist spiperone, which has a much higher affinity for 5-HT2 receptors than for 5-HT1C receptors, blocked the excitatory effect of 5-HT at lower concentrations in interneurons (IC50 = 31 nM) than in pyramidal cells (IC50 = 2.1 microM). Similarly, ritanserin, a drug which also has a higher affinity for 5-HT2 than 5-HT1C receptors, blocked the effect of 5-HT at lower concentrations in interneurons (IC50 = 400 nM) than in pyramidal cells (IC50 = 8.1 microM). In contrast, LY 53857, an antagonist with higher affinity for 5-HT1C than for 5-HT2 receptors, blocked the effect of 5-HT at lower concentrations in pyramidal cells (IC50 = 26 nM) than in interneurons (IC50 = 364 nM). The 5-HT1C partial agonist/5-HT2 antagonist mCPP produced agonist-like effects in only 66% of pyramidal cells tested indicating that not all pyramidal cells may express 5-HT1C receptors. In that both spiperone and ritanserin have higher affinity for 5-HT2 receptors than for 5-HT1C receptors and LY 53857 has a higher affinity for 5-HT1C receptors than for 5-HT2 receptors, these data suggest that in piriform cortex excitatory effects of 5-HT are mediated by 5-HT1C receptors in pyramidal cells an by 5-HT2 receptors in interneurons.

The ameliorating effect of the water layer of Fructus Schisandrae on cycloheximide-induced amnesia in rats: interaction with drugs acting at neurotransmitter receptors.

Our previous study indicated that the water layer present in Fructus Schisandra(FS(w)) at 10 and 25 mg kg(-1)significantly counteracted cycloheximide (CXM)-induced amnesia. Therefore, the mechanism of action of the ameliorating effect of FS(w)on CXM-induced amnesia in the passive avoidance task was investigated in rats. The ameliorating effect of FS(w)on CXM-induced amnesia was depressed by scopolamine. The serotonin releaser, p -chloroamphetamine significantly antagonized the ameliorating effect of FS(w)on CXM-induced amnesia. Furthermore, the ameliorating effect was also inhibited by the 5-HT(1A)receptor agonist 8-OH-DPAT, but potentiated by the 5-HT(2)receptor antagonist ritanserin. Finally, the GABA(A)receptor antagonist bicuculline blocked the ameliorating effect of FS(w). These results suggest that the beneficial effect of FS(w)on CXM-induced amnesia is amplified by treatment with serotonergic 5-HT(2)receptor antagonists, but reduced by serotonergic 5-HT(1A)receptor agonists as well as GABA(A)and cholinergic receptor antagonists.