Yeast Platforms for Production and Screening of Bioactive Derivatives of Rauwolscine.

Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.

Yohimbine ameliorates liver inflammation and fibrosis by regulating oxidative stress and Wnt/ß-catenin pathway.

BACKGROUND AND PURPOSE: Chronic liver injury, caused by various aetiologies, causes recurrent tissue damage, culminating in decreased liver regenerative ability and resulting in fibrosis followed by cirrhosis. In this study, the anti-fibrotic activity of Yohimbine hydrochloride (YHC) was investigated using various in vitro models and in vivo models. METHODS: To assess the anti-inflammatory, antioxidant, and anti-fibrotic effects of YHC, lipopolysaccharide or TGF-ß induced differentiation or lipid-induced oxidative-stress models were employed using HLECs, HSC-LX2, and HepG2 cells. Further, thioacetamide (TAA) induced hepatic inflammation/fibrosis models were utilized to validate the YHC's anti-fibrotic activity in rats. RESULTS: Inflammation/differentiation experiments in HLECs and HSC-LX2 revealed that YHC treatment significantly (p < 0.001) mitigated the lipopolysaccharide or TGF-ß induced upregulation of inflammatory and fibrotic markers expression respectively. In addition, YHC dose-dependently reduced the TGF-ß induced migration and palmitic acid-induced oxidative stress in HepG2 cells. Further, TAA administration (5 weeks) in vivo rat model showed increased inflammatory marker levels/expression, oxidative stress, and pathological abnormalities. Additionally, TAA administration (9 weeks) elevated the fibrotic marker expression, collagen deposition in liver tissues, and shortened longevity in rats. Treatment with YHC dose-dependently mitigated the TAA-induced abnormalities in both inflammation and fibrosis models and improved the survival of the rats. Further mechanistic approaches revealed that TAA administration elevated the JNK, Wnt components and ß-catenin expression in hepatic stellate cells and animal tissues. Further treatment with YHC significantly modulated the JNK/Wnt/ß-catenin signaling. Moreover, the ß-catenin nuclear translocation results showed that ß-catenin levels were significantly elevated in the nuclear fraction of TAA control samples and reduced in YHC-treated samples. CONCLUSION: Yohimbine treatment significantly improved inflammation and fibrosis by inhibiting differentiation, oxidative stress, and collagen deposition by partly modulating the JNK/Wnt/ß-catenin pathway. These results might serve as a foundation for proposing yohimbine as a potential lead compound for liver fibrosis.

Combined In Vivo Microdialysis and PET Studies to Validate [11C]Yohimbine Binding as a Marker of Noradrenaline Release.

The noradrenaline system attracts attention for its role in mood disorders and neurodegenerative diseases but the lack of well-validated methods impairs our understanding when assessing its function and release in vivo. This study combines simultaneous positron emission tomography (PET) and microdialysis to explore if [11C]yohimbine, a selective antagonist radioligand of the α2 adrenoceptors, may be used to assess in vivo changes in synaptic noradrenaline during acute pharmacological challenges. Anesthetised Göttingen minipigs were positioned in a head holder in a PET/CT device. Microdialysis probes were placed in the thalamus, striatum and cortex and dialysis samples were collected every 10 min. Three 90 min [11C]yohimbine scans were acquired: at baseline and at two timepoints after the administration of amphetamine (1-10 mg/kg), a non-specific releaser of dopamine and noradrenaline, or nisoxetine (1 mg/kg), a specific noradrenaline transporter inhibitor. [11C]yohimbine volumes of distribution (VT) were obtained using the Logan kinetic model. Both challenges induced a significant decrease in yohimbine VT, with time courses reflecting their different mechanisms of action. Dialysis samples revealed a significant increase in noradrenaline extracellular concentrations after challenge and an inverse correlation with changes in yohimbine VT. These data suggest that [11C]yohimbine can be used to evaluate acute variations in synaptic noradrenaline concentrations after pharmacological challenges.

Distribution of α2-Adrenergic Receptors in the Living Human Brain Using [11C]yohimbine PET.

The neurofunctional basis of the noradrenergic (NA) system and its associated disorders is still very incomplete because in vivo imaging tools in humans have been missing up to now. Here, for the first time, we use [11C]yohimbine in a large sample of subjects (46 healthy volunteers, 23 females, 23 males; aged 20-50) to perform direct quantification of regional alpha 2 adrenergic receptors' (α2-ARs) availability in the living human brain. The global map shows the highest [11C]yohimbine binding in the hippocampus, the occipital lobe, the cingulate gyrus, and the frontal lobe. Moderate binding was found in the parietal lobe, thalamus, parahippocampus, insula, and temporal lobe. Low levels of binding were found in the basal ganglia, the amygdala, the cerebellum, and the raphe nucleus. Parcellation of the brain into anatomical subregions revealed important variations in [11C]yohimbine binding within most structures. Strong heterogeneity was found in the occipital lobe, the frontal lobe, and the basal ganglia, with substantial gender effects. Mapping the distribution of α2-ARs in the living human brain may prove useful not only for understanding the role of the NA system in many brain functions, but also for understanding neurodegenerative diseases in which altered NA transmission with specific loss of α2-ARs is suspected.

Dexmedetomidine reduces IL-4 and IgE expression through downregulation of theTLR4/NF-κB signaling pathway to alleviate airway hyperresponsiveness in OVA mice.

BACKGROUND: Airway hyperresponsiveness (AHR) is a clinical manifestation of airflow limitation due to abnormal tracheal and bronchial sensitivity and is the main basis for the diagnosis of asthma. Patients with AHR are at high risk of perioperative tracheal and bronchospasm, which can lead to hypoxaemia and haemodynamic instability and, in severe cases, to a life-threatening 'silent lung'. It is therefore important to reduce the incidence or intensity of AHR episodes in the perioperative period. The inflammatory response is key to the development and progression of AHR. HYPOTHESIS/PURPOSE: Based on the modulatory role of dexmedetomidine (DEX) in the inflammatory response, we hypothesised that dexmedetomidine (DEX) attenuates inflammatory properties by inhibiting the toll-like receptor 4 (TLR4)/nuclear factor (NF-κB) signalling pathway and can reduce the respiratory parameters of mechanical ventilation in ovalbumin-induced allergic airway hyperresponsiveness. STUDY DESIGN: BABL/C mice were divided into control and OVA groups (ovalbumin-induced allergy. Ten mice in all OVA models were randomly selected for in vivo invasive lung function monitoring to analyse airway resistance parameters and demonstrate successful model establishment. The remaining OVA mice were treated with dexmedetomidine 25 µg/kg for 5 days (OVA + DEX group) or dexmedetomidine 25 µg/kg + yohimbine 1 mg/kg for 5 days (OVA + DEX + yohimbine). After treatment, bronchoalveolar lavage fluid (BAL) and peripheral blood (ELISA) and lung tissue (H&E and PAS) were collected for analysis of inflammatory factors, and lung tissue was verified by PCR for genes and proteins that do correlate with inflammatory mediators. RESULTS: All airway resistance parameters were increased in OVA mice by invasive lung function monitoring. Proximal airway resistance (parameter Rn) and total respiratory resistance (parameter Rrs) were attenuated after dexmedetomidine intervention treatment. Dexmedetomidine reduced total inflammatory cell count and inflammatory infiltration of lung tissue in BALF and down-regulated IL-4 and IgE levels in BALF and peripheral blood, as shown by Giemsa, H&E, PAS staining and ELISA; this mechanism of action was found to be related to the TLR4/NFκB pathway, but not to TLR4/NFκB, as measured by PCR. CONCLUSION: Dexmedetomidine reduces hyperresponsiveness and airway inflammatory responses. This mechanism of action may be related to the TLR4/NFκB signalling pathway. Overall conclusions are presented in.

The affinity and selectivity of α-adrenoceptor antagonists, antidepressants and antipsychotics for the human α2A, α2B, and α2C-adrenoceptors and comparison with human α1 and ß-adrenoceptors.

α2-Adrenoceptors, subdivided into α2A, α2B, and α2C subtypes and expressed in heart, blood vessels, kidney, platelets and brain, are important for blood pressure, sedation, analgesia, and platelet aggregation. Brain α2C-adrenoceptor blockade has also been suggested to be beneficial for antipsychotic action. However, comparing α2-adrenoceptor subtype affinity is difficult due to significant species and methodology differences in published studies. Here, 3 H-rauwolscine whole cell binding was used to determine the affinity and selectivity of 99 α-antagonists (including antidepressants and antipsychotics) in CHO cells expressing human α2A, α2B, or α2C-adrenoceptors, using an identical method to ß and α1-adrenoceptor measurements, thus allowing direct human receptor comparisons. Yohimbine, RX821002, RS79948, and atipamezole are high affinity non-selective α2-antagonists. BRL44408 was the most α2A-selective antagonist, although its α1A-affinity (81 nM) is only 9-fold greater than its α2C-affinity. MK-912 is the highest-affinity, most α2C-selective antagonist (0.15 nM α2C-affinity) although its α2C-selectivity is only 13-fold greater than at α2A. There are no truely α2B-selective antagonists. A few α-ligands with significant ß-affinity were detected, for example, naftopidil where its clinical α1A-affinity is only 3-fold greater than off-target ß2-affinity. Antidepressants (except mirtazapine) and first-generation antipsychotics have higher α1A than α2-adrenoceptor affinity but poor ß-affinity. Second-generation antipsychotics varied widely in their α2-adrenoceptor affinity. Risperidone (9 nM) and paliperidone (14 nM) have the highest α2C-adrenoceptor affinity however this is only 5-fold selective over α2A, and both have a higher affinity for α1A (2 nM and 4 nM, respectively). So, despite a century of yohimbine use, and decades of α2-subtype studies, there remains plenty of scope to develop α2-subtype selective antagonists.

Modeling [11C]yohimbine PET human brain kinetics with test-retest reliability, competition sensitivity studies and search for a suitable reference region.

Previous work introduced the [11C]yohimbine as a suitable ligand of central α2-adrenoreceptors (α2-ARs) for PET imaging. However, reproducibility of [11C]yohimbine PET measurements in healthy humans estimated with a simplified modeling method with reference region, as well as sensitivity of [11C]yohimbine to noradrenergic competition were not evaluated. The objectives of the present study were therefore to fill this gap. METHODS: Thirteen healthy humans underwent two [11C]yohimbine 90-minute dynamic scans performed on a PET-MRI scanner. Seven had arterial blood sampling with metabolite assessment and plasmatic yohimbine free fraction evaluation at the first scan to have arterial input function and test appropriate kinetic modeling. The second scan was a simple retest for 6 subjects to evaluate the test-retest reproducibility. For the remaining 7 subjects the second scan was a challenge study with the administration of a single oral dose of 150 µg of clonidine 90 min before the PET scan. Parametric images of α2-ARs distribution volume ratios (DVR) were generated with two non-invasive models: Logan graphical analysis with Reference (LREF) and Simplified Reference Tissue Method (SRTM). Three reference regions (cerebellum white matter (CERWM), frontal white matter (FLWM), and corpus callosum (CC)) were tested. RESULTS: We showed high test-retest reproducibility of DVR estimation with LREF and SRTM regardless of reference region (CC, CERWM, FLWM). The best fit was obtained with SRTMCC (r2=0.94). Test-retest showed that the SRTMCC is highly reproducible (mean ICC>0.7), with a slight bias (-1.8%), whereas SRTMCERWM had lower bias (-0.1%), and excellent ICC (mean>0.8). Using SRTMCC, regional changes have been observed after clonidine administration with a significant increase reported in the amygdala and striatum as well as in several posterior cortical areas as revealed with the voxel-based analysis. CONCLUSION: The results add experimental support for the suitability of [11C]yohimbine PET in the quantitative assessment of α2-ARs occupancy in vivo in the human brain. Trial registration EudraCT 2018-000380-82.

Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays.

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

Lipokines and Thermogenesis.

Adaptive thermogenesis is a catabolic process that consumes energy-storing molecules and expends that energy as heat in response to environmental changes. This process occurs primarily in brown and beige adipose tissue. Thermogenesis is regulated by many factors, including lipid derived paracrine and endocrine hormones called lipokines. Recently, technologic advances for identifying new lipid biomarkers of thermogenic activity have shed light on a diverse set of lipokines that act through different pathways to regulate energy expenditure. In this review, we highlight a few examples of lipokines that regulate thermogenesis. The biosynthesis, regulation, and effects of the thermogenic lipokines in several families are reviewed, including oloeylethanolamine, endocannabinoids, prostaglandin E2, and 12,13-diHOME. These thermogenic lipokines present potential therapeutic targets to combat states of excess energy storage, such as obesity and related metabolic disorders.

Quantification of microdosed oral yohimbine and its major metabolite in human plasma in the picogram range.

Aim: Pharmacokinetics after oral microdosing of the anticipated CYP2D6 substrate yohimbine and its metabolite 11-OH-yohimbine is potentially useful for drug-drug interaction trials and profiling of CYP2D6 enzyme activity. Materials & methods: We developed an ultrasensitive ultra performance liquid chromatography coupled to tandem mass spectrometry assay for quantification of yohimbine and its main metabolite 11-OH-yohimbine in plasma with a linear calibration range of 5-2500 pg/ml and validated it according to US FDA's and EMA's guidelines. Sample preparation was performed using fast liquid-liquid extraction. The assay was applied for the determination of concentrations of yohimbine and 11-OH-yohimbine in plasma after oral administration of 50 µg yohimbine to two subjects. Conclusion: Ultrasensitive quantification of yohimbine and its metabolite enables the determination of their concentrations in plasma after microdosing which would be applicable to use in CYP2D6 phenotyping.

Radioligand binding analysis of α 2 adrenoceptors with [11C]yohimbine in brain in vivo: Extended Inhibition Plot correction for plasma protein binding.

We describe a novel method of kinetic analysis of radioligand binding to neuroreceptors in brain in vivo, here applied to noradrenaline receptors in rat brain. The method uses positron emission tomography (PET) of [11C]yohimbine binding in brain to quantify the density and affinity of α 2 adrenoceptors under condition of changing radioligand binding to plasma proteins. We obtained dynamic PET recordings from brain of Spraque Dawley rats at baseline, followed by pharmacological challenge with unlabeled yohimbine (0.3 mg/kg). The challenge with unlabeled ligand failed to diminish radioligand accumulation in brain tissue, due to the blocking of radioligand binding to plasma proteins that elevated the free fractions of the radioligand in plasma. We devised a method that graphically resolved the masking of unlabeled ligand binding by the increase of radioligand free fractions in plasma. The Extended Inhibition Plot introduced here yielded an estimate of the volume of distribution of non-displaceable ligand in brain tissue that increased with the increase of the free fraction of the radioligand in plasma. The resulting binding potentials of the radioligand declined by 50-60% in the presence of unlabeled ligand. The kinetic unmasking of inhibited binding reflected in the increase of the reference volume of distribution yielded estimates of receptor saturation consistent with the binding of unlabeled ligand.

Impaired responsiveness of platelets to epinephrine due to α2A adrenoreceptor deficiency in Male Chinese.

Epinephrine is known as a weak, but important, agonist for platelet activation. It has been reported that the responsiveness of platelets to epinephrine was markedly impaired in 6% of Caucasians and in 16% of Japanese. The purpose of this study was to screen and characterize this abnormality in healthy Taiwanese Chinese volunteers. We used aggregometry, flow cytometry and platelet function analyzer (PFA)-100 system to assess in 50 healthy male volunteers the responsiveness of platelets to epinephrine stimulation. Using α2A adrenoceptor antagonist BRL44408 maleate competition and a [(3)H]yohimbin binding assay, we evaluated α2A adrenoceptors on platelets. The aggregation of platelets after stimulation with 10 µM of epinephrine indicated two distinct groups of study participants: 24 (48.0%) good- and 26 (52.0%) impaired-responders to epinephrine. Flow cytometric analysis of platelets after stimulated with 1 µM epinephrine showed that glycoprotein (GP) IIb/IIIa and P-selectin expression of epinephrine good- and impaired-responders were 27.1 ± 11.0% vs. 9.9 ± 5.4% (p = 0.003) and 12.2 ± 6.2% vs. 3.6 ± 3.5% (p < 0.001), respectively. The PFA-100 system showed that epinephrine-impaired-responders had a longer collagen-epinephrine induced closure time. Good-responder platelets incubated with BRL44408 maleate had an impaired response to epinephrine stimulation. [(3)H]yohimbine binding studies showed fewer α2A adrenoreceptors on the platelets of epinephrine-impaired-responders than on those of good-responders. The prevalence of impaired responsiveness to epinephrine was high and probably due to α2A adrenoreceptor deficiency in male Taiwanese Chinese.

Acute Vagal Nerve Stimulation Lowers α2 Adrenoceptor Availability: Possible Mechanism of Therapeutic Action.

BACKGROUND: Vagal nerve stimulation (VNS) emerged as an anti-epileptic therapy, and more recently as a potential antidepressant intervention. OBJECTIVE/HYPOTHESIS: We hypothesized that salutary effects of VNS are mediated, at least in part, by augmentation of the inhibitory effects of cortical monoaminergic neurotransmission at appropriate receptors, specifically adrenoceptors. Our objective was to measure the effect of acute VNS on α2 adrenoceptor binding. METHODS: Using positron emission tomography (PET), we measured changes in noradrenaline receptor binding associated with acute VNS stimulation in six anesthetized Göttingen minipigs. We used the selective α2 adrenoceptor antagonist [11C]yohimbine, previously shown to be sensitive to competition from the receptor's endogenous ligands, as a surrogate marker of monoamine release. PET records were acquired 4-6 weeks after the implant of a VNS electrode in minipigs before and within 30 min of the initiation of 1 mA stimulation. Kinetic analysis with the Logan graphical linearization generated tracer volumes of distribution for each condition. We used an averaged value of the distribution volume of non-displaceable ligand (VND), to calculate binding potentials for selected brain regions of each animal. RESULTS: VNS treatment markedly reduced the binding potential of yohimbine in limbic, thalamic and cortical brain regions, in inverse correlation with the baseline binding potential. CONCLUSION: The result is consistent with release of noradrenaline by antidepressant therapy, implying a possible explanation for the antidepressant effect of VNS.

Amphetamine challenge decreases yohimbine binding to α2 adrenoceptors in Landrace pig brain.

RATIONALE: The noradrenaline (NA) system is implicated in neurodegenerative and psychiatric disorders; however, our understanding is impaired by the lack of well-validated radioligands to assess NA function and release. Yohimbine, an α2 adrenoceptor antagonist, has recently been developed as a carbon-11 [11C]-labeled radioligand for positron emission tomography (PET) imaging studies. OBJECTIVES: Here we explore the hypothesis that yohimbine can be used as an in vivo tracer of NA receptor binding and release during amphetamine challenges in Landrace pigs. METHODS: Pigs underwent baseline PET scans with [11C]yohimbine and were then challenged with 10 mg/kg d-amphetamine 20 min prior to a second [11C]yohimbine scan. Using the Logan analysis model, volumes of distribution were calculated from fits of the kinetic data 25-90 min post-yohimbine injection. RESULTS: Amphetamine decreased [11C]yohimbine volume of distribution in the brain regions under investigation, including the thalamus, caudate nucleus, and cortical regions. CONCLUSION: These data suggest that the binding of [11C]yohimbine to α2 adrenoceptors may be displaceable by increases in synaptic concentrations of the endogenous ligand, NA, and possibly dopamine, suggesting the possibility that [11C]yohimbine may be used as a surrogate marker of NA release in vivo.

Central and peripheral anti-inflammatory effects of maprotiline on carrageenan-induced paw edema in rats.

OBJECTIVE: To explore the site of action of maprotiline, as an atypical antidepressant, on carrageenan-induced paw edema. SUBJECTS: Male Wistar rats were used. METHODS: Firstly, the anti-inflammatory effect of systemic maprotiline (12.5, 25 and 50 mg kg(-1)) was assessed using a paw edema model. Secondly, different doses of maprotiline were administrated intracerebroventricularly, intrathecally and locally before carrageenan challenge. Finally, we tried to reverse the anti-inflammatory effect of maprotiline by propranolol (10 mg kg(-1)), prazosin (4 mg kg(-1)), yohimbine (10 mg kg(-1)), naloxone (4 mg kg(-1)) and mifepristone (5 mg kg(-1)). RESULTS: Systemic, intracerebroventricular and subplantar application of maprotiline significantly inhibited peripheral edema, but intrathecal maprotiline did not alter the degree of paw swelling. The applied antagonists failed to change the anti-inflammatory activity of maprotiline. CONCLUSION: These results demonstrate that maprotiline has a potent anti-inflammatory effect and this effect is linked to the peripheral and supraspinal actions of the drug.

[Studies on predict of absorption of corynanthine, yohimbine, ajmalicine and ajmaline across human intestinal epithelial by using human Caco-2 cells monolayers].

OBJECTIVE: To predict the absorption of corynanthine (COR), yohimbine (YOH), ajmalicine (AMC) and ajmaline (AML) as chemical constituents of some traditional Chinese medicines in human intestinal epithelial. METHOD: By using Caco-2 (the human colonic adenocarcinoma cell lines) cell monolayers as a human intestinal epithelial cell model, the permeability of COR, YOH, AMC and AML were studied from apical side (AP side) to basolateral side (BL side) or from BL side to AP side. The four alkaloids were measured by high performance liquid chromatography (HPLC) coupled with UV detector. Transport parameters and apty) and atenolol (a control substance of poor permeability). The relationship between P(app) and log D values of four alkaloids was investigated by using drugs ADMET predict software. RESULT: The P(app) values of COR, YOH, AMC and AML were (1.863 +/- 0.055) x 10(-5), (1.540 +/- 0.082) x 10(-5), (2.522 +/- 0.246) x 10(-5) and (1.155 +/- 0.099) x 10(-5) cm x s(-1) from AP side to BL side, and (2.390 +/- 0.017) x 10(-5), (1.987 +/- 0.154) x 10(-5), (1.374 +/- 0.260) x 10(-5) and (2.418 +/- 0.124) x 10(-5) cm x s(-1) from BL side to AP side, respectively, which P(app) values were identical with that of propranolol [(2.23 +/- 0.10) x 10(-5) cm x s(-1) from AP to BL side]. The ratio of P(app B --> A)/P(app A -->B) of COR, YOH, AMC and AML were 1.28, 1.29, 0.54 and 2.09, respectively, which suggested that the efflux transport of AML was 2.09 times higher more than its influx transport. CONCLUSION: COR, YOH, AMC and AML can be transported and absorbed across the human Caco-2 cells monolayers, and they belong to completely absorbed compounds. AML may have been involved in efflux mechanism in Caco-2 cells monolayers model from the BL to AP side direction. The oil-water partition coefficient play key roles in the transport and absorption of the four alkaloids.

Identification of a key amino acid of the human 5-HT(2B) serotonin receptor important for sarpogrelate binding.

Based on radio-ligand binding and molecular modeling studies, sarpogrelate shows a moderate selectivity for 5-HT(2B) versus 5-HT(2A) receptors. To confirm the modeling data of sarpogrelate to 5-HT(2B) receptors predicting interaction of sarpogrelate towards Asp135 in helix 3 of 5-HT(2B) receptors, we constructed and characterized the mutation of this residue by site-directed mutagenesis. The Asp135Ala mutant did not exhibit any affinity for [(3)H]rauwolscine. Therefore, it was not possible to find sarpogrelate affinity to the mutant using [(3)H]rauwolscine. The mutation also abolished agonist-stimulated inositol phosphates formation. These results provide evidence that Asp135 is important for the interaction between 5-HT(2B) receptors and sarpogrelate.

Molecular cloning and heterologous expression of an alpha-adrenergic-like octopamine receptor from the silkworm Bombyx mori.

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.

Long-lasting effects of yohimbine on the ejaculatory function in male dogs.

Previous studies have demonstrated that systemic administration of a low dose of the alpha2-adrenoceptor antagonists stimulates the ejaculatory response of male dogs, when this function is analyzed using the amount of ejaculated semen in response to genital stimulation. The present study was designed to further examine the features of the stimulatory effects of the alpha2-adrenoceptor antagonists on ejaculation, especially the duration of action. Treatment with yohimbine (0.1 mg/ kg, i.p.) to male dogs, at 0.5, 1, 3, or 5 h before the testing, produced a significant stimulatory effects on the ejaculatory response elicited by manual penile stimulation; the amount of ejaculated semen was increased and the onset of ejaculation was shortened following each treatment. However, such effects were not observed in the treatment with yohimbine at 8 and 24 h before the testing, indicating that the ejaculatory stimulation induced by yohimbine lasted for a relative long period. By contrast, the stimulatory effects of RX821002 (0.1 mg/kg, i.p.), a selective alpha2-adrenoceptor antagonist, on ejaculation were observed only for 1 h after administration. To determine the contribution of the alpha2-adrenoceptor blockade for the long-lasting effect of yohimbine, we tested whether yohimbine can prevent the ejaculatory inhibition induced by clonidine, an alpha2-adrenoceptor agonist. The ejaculatory inhibition (a decrease in the amount of ejaculated semen and a delay onset of ejaculation) elicited by clonidine (0.05 mg/kg, i.p.; 1 h before testing) was completely blocked by pretreatment with yohimbine at 1 or 5 h before the testing, whereas the pretreatment with the drug at 24 h before the testing did not affect the clonidine-induced ejaculatory inhibition. These results indicate that yohimbine-induced ejaculatory stimulation is continued for a relative long period (at least 5 h after administration), and this long-lasting effects may be related to the alpha2-adrenoceptor blocking property of the drug.

Synthesis and biological studies of yohimbine derivatives on human alpha2C-adrenergic receptors.

A series of yohimbine derivatives was synthesized and evaluated for binding affinity at the human alpha(2C)-adrenergic receptors expressed in Chinese hamster ovary cells. It has been found that compound 5 shows a higher affinity for alpha(2C)-AR than the parent compound yohimbine 1, thereby illustrating that the nature of the linkers affect binding potencies on these receptors.