Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase ß phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase ß. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

Is the 5-hydroxytryptamine 7 Receptor Constitutively Active in the Vasculature? A Study in Veins/Vein.

ABSTRACT: The 5-hydroxytryptamine 7 (5-HT 7 ) receptor is reported to have considerable constitutive activity when transfected into cells. Constitutive activity-receptor activity in the absence of known agonist-is important for understanding the contributions of a receptor to (patho)physiology. We test the hypothesis that the 5-HT 7 receptor possesses constitutive activity in a physiological situation. Isolated veins from male and female Sprague Dawley rats were used as models for measuring isometric force; the abdominal vena cava possesses a functional 5-HT 7 receptor that mediates relaxation, whereas the small mesenteric vein does not. Compounds reported to act as inverse agonists were investigated for their ability to cause contraction (moving a constitutively active relaxant receptor to an inactive state, removing relaxation). Compared with a vehicle control, clozapine, risperidone, ketanserin, and SB269970 caused no contraction in the isolated male abdominal vena cava. By contrast, methiothepin caused a concentration-dependent contraction of the male but not female abdominal vena cava, although with low potency (-log EC 50 [M] = 5.50 ± 0.45) and efficacy (∼12% of contraction to endothelin-1). Methiothepin-induced contraction was not reduced by the 5-HT 7 receptor antagonist (SB269970, 1 µM, not active in the vena cava). These same compounds showed little to no effect in the isolated mesenteric vein. We conclude that the 5-HT 7 receptor in the isolated veins of the Sprague Dawley rat does not possess constitutive activity. We raise the question of the physiological relevance of constitutive activity of this receptor important to such diverse physiological functions as sleep, circadian rhythm, temperature, and blood pressure regulation.

Methiothepin Increases Chemotherapy Efficacy against Resistant Melanoma Cells.

We previously reported that methiothepin, a small molecule known as a nonselective serotonin 5-HT receptor antagonist, inhibited the doxorubicin efflux activity of the Hedgehog receptor Ptch1 and enhanced the cytotoxic, pro-apoptotic, anti-proliferative, and anti-clonogenic effects of doxorubicin on adrenocortical carcinoma cells. Here, we show that methiothepin also inhibits doxorubicin efflux and increases doxorubicin cytotoxicity in melanoma cells which endogenously overexpress Ptch1. Melanoma patients having the BRAFV600E mutation are treated with vemurafenib, an inhibitor of BRAFV600E, often in combination with trametinib, an inhibitor of MEK. Almost all patients ultimately acquire resistance to the treatment leading to disease progression. Here, we report that methiothepin overcomes the resistance of BRAFV600E melanoma cells by enhancing the cytotoxicity of vemurafenib and trametinib on these cells leading to melanoma cells death. We observe that the addition of methiothepin to vemurafenib prevents migration of resistant melanoma cells more efficiently than vemurafenib alone. Our results provide an additional proof that Ptch1 drug efflux inhibition increases the effectiveness of anti-cancer treatments and overcomes resistance of melanoma cells expressing Ptch1.

Tumor-suppressive function of methiothepin in human placental choriocarcinoma cells.

Placental choriocarcinoma is a malignant trophoblastic tumor associated with placentation. During placentation, complicated molecular networks are mediated by endocrine and paracrine signals. Serotonin neurotransmitters have been identified in the transmembrane region of human placental choriocarcinoma (HPC) cells as tumor promoters; therefore, their antagonists have anti-cancer properties. Although methiothepin, a serotonin receptor antagonist and FDA-approved psychotropic agent, has shown multi-pharmacological functions in various disease models, its anti-tumorigenic activity and mechanisms underlying its action against HPC are unknown. Therefore, we identified the anti-cancer effects of methiothepin in JEG3 and JAR HPC cells. Methiothepin attenuated mitochondrial function and induced endoplasmic reticular stress, reducing oxidative phosphorylation and causing metabolic shifting in HPC cells. Furthermore, methiothepin showed synergistic pharmacological effects with paclitaxel in HPC cells. Our results highlight the robust tumor-suppressive function of methiothepin in HPC. Our findings provide new insights into the repositioning of methiothepin from a psychotropic agent to novel anti-cancer agents, especially against HPC.

Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor.

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.

Methiothepin mesylate causes apoptosis of human prostate cancer cells by mediating oxidative stress and mitochondrial dysfunction.

Prostate cancer is difficult to treat if it metastasizes to other organs. The development of prostate cancer independent of androgen is closely related to the action of neuroendocrine products. Serotonin promotes cell growth in various cancers, and antagonists for serotonin receptors are known to inhibit proliferation and induce cell death in various carcinomas. However, little is known about how antagonists for serotonin receptor function in prostate cancer. We verified apoptotic cell death in prostate cancer cell lines after treatment with methiothepin mesylate (MET), an antagonist for serotonin receptor 5-HT1. MET induced hydrogen peroxide (H2O2) production and mitochondrial Ca2+ overload. Moreover, MET induced changes in the expression of proteins associated with endoplasmic reticulum stress, autophagy, and mitochondrial membrane potential. MET also promoted phosphorylation of JNK, which induced cell death mediated by oxidant production, as evidenced by the JNK inhibitor and oxidant scavenger. Finally, MET has the potential to prevent metastasis by inhibiting the migration of prostate cancer cells. Thus, we show that MET is a potentially novel anticancer agent that can suppress the development of prostate cancer caused by neuroendocrine differentiation.

Proteins or RNA synthesis inhibitors suppressed induction of amnesia developing under impairment of memory reconsolidation by serotonin receptors antagonist.

Studies have shown that retrieval of long-term memory can cause memory reconsolidation, and impaired reconsolidation leads to amnesia development. However, the mechanisms of amnesia induction due to impaired memory reconsolidation remains poorly described. Using experiments involving grape snails trained to conditioned food aversion, we studied the role of translation and transcription processes and the role of serotonin receptors in the mechanisms of amnesia induction. We found that administration of a serotonin receptor antagonist or a protein synthesis inhibitor before the administration of a reminder using a conditioned food stimulus induced amnesia development, whereas injections of mRNA synthesis inhibitor did not affect memory safety. Moreover, combined injections of an antagonist of serotonin receptor and inhibitors of protein or mRNA synthesis before reminder administration completely prevented amnesia development. In addition, inhibitors of protein or mRNA synthesis prevented amnesia development 3 h but not 9 h after the administration of a serotonin receptor antagonist/reminder. We hypothesize that the mechanisms of amnesia induction caused by impaired memory reconsolidation depend on protein and mRNA syntheses within a certain time window, similar to the mechanisms of induction of other long-term plastic brain rearrangements.

Administration of Protein Synthesis Inhibitor before Reminder Reverses Amnesia Induced by Memory Reconsolidation Impairment with 5-HT Receptors Antagonist.

Administration of 5-HT receptor antagonist to snails trained in conditioned food aversion prior to reminding of the conditioning stimulus caused amnesia. At the early period of amnesia (day 3), injections of protein synthesis inhibitor cycloheximide without reminder or reminder alone were ineffective. At the same time, injections of the inhibitor combined with reminder led to memory recovery; this effect in most animals persisted for at least 10 days. In the rest snails, aversive responses to presentations of the conditioning stimulus persisted for 2 days. Cycloheximide injection and reminder in 10 days after induction of amnesia did not affect its development or caused a transient memory recovery (2 days). We hypothesized that amnesia is an active process unfolding in time. One of mechanism of this process is reminder-induced and protein synthesis-depended reactivation of amnesia. Inhibitor of protein synthesis disturbed this reactivation and led to recovery of the initial memory of conditioned food aversion.

NMDA or 5-HT receptor antagonists impair memory reconsolidation and induce various types of amnesia.

Elucidation of amnesia mechanisms is one of the central problems in neuroscience with immense practical application. Previously, we found that conditioned food presentation combined with injection of a neurotransmitter receptor antagonist or protein synthesis inhibitor led to amnesia induction. In the present study, we investigated the time course and features of two amnesias: induced by impairment of memory reconsolidation using an NMDA glutamate receptor antagonist (MK-801) and a serotonin receptor antagonist (methiothepin, MET) on snails trained with food aversion conditioning. During the early period of amnesia (<10th day), the unpaired presentation of conditioned stimuli (CS) or unconditioned stimuli (US) in the same training context did not have an effect on both types of amnesia. Retraining an on 1st or 3rd day of amnesia induction facilitated memory formation, i.e. the number of CS + US pairings was lower than at initial training. On the 10th or 30th day after the MET/reminder, the number of CS + US pairings did not change between initial training and retraining. Retraining on the 10th or 30th day following the MK-801/reminder in the same or a new context of learning resulted in short, but not long-term, memory, and the number of CS + US pairings was higher than at the initial training. This type of amnesia was specific to the CS we used at initial training, since long-term memory for another kind of CS could be formed in the same snails. The attained results suggest that disruption of memory reconsolidation using antagonists of serotonin or NMDA glutamate receptors induced amnesias with different abilities to form long-term memory during the late period of development.

Targeting the multidrug transporter Patched potentiates chemotherapy efficiency on adrenocortical carcinoma in vitro and in vivo.

One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug-like molecule enhances the cytotoxic, pro-apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched-expressing cancers.

Gating of long-term potentiation (LTP) in the thalamocortical auditory system of rats by serotonergic (5-HT) receptors.

The neuromodulator serotonin (5-hydroxytryptamine, 5-HT) plays an important role in controlling the induction threshold and maintenance of long-term potentiation (LTP) in the visual cortex and hippocampus of rodents. Serotonergic fibers also innervate the rodent primary auditory cortex (A1), but the regulation of A1 plasticity by 5-HT receptors (5-HTRs) is largely uncharted. Thus, we examined the role of several, predominant 5-HT receptor classes (5-HT1ARs, 5-HT2Rs, and 5-HT3Rs) in gating in vivo LTP induction at A1 synapses of adult, urethane-anesthetized rats. Theta-burst stimulation (TBS) applied to the medial geniculate nucleus resulted in successful LTP induction of field postsynaptic potentials (fPSPs) generated by excitation of thalamocortical and intracortical A1 synapses. Local application (by reverse microdialysis in A1) of the broad-acting 5-HTR antagonist methiothepin suppressed LTP at both thalamocortical and intracortical synapses. In fact, rather than LTP, TBS elicited long-term depression during methiothepin application, an effect that was mimicked by the selective 5-HT2R antagonist ketanserin, but not the 5-HT1AR blocker WAY 100635. Interestingly, antagonism of 5-HT3Rs by granisetron selectively blocked LTP at thalamocortical, but not intracortical A1 synapses. Further, in the absence of TBS, granisetron application resulted in a pronounced increase in fPSP amplitude, suggesting that 5-HT3Rs play an important role in regulating baseline (non-potentiated) transmission at A1 synapses. Together, these results indicate that activation of 5-HT2Rs and 5-HT3Rs, but not 5-HT1ARs, exerts a clear, facilitating effect on LTP induction at A1 synapses, allowing 5-HT to act as a powerful regulator of long-term plasticity induction in the fully matured A1 of mammalian species.

Elucidation of the profound antagonism of contractile action of phenylephrine in rat aorta effected by an atypical sympathomimetic decongestant

Vasoconstrictive properties of sympathomimetic drugs are the basis of their widespread use as decongestants and possible source of adverse responses. Insufficiently substantiated practice of combining decongestants in some marketed preparations, such are those containing phenylephrine and lerimazoline, may affect the overall contractile activity, and thus their therapeutic utility. This study aimed to examine the interaction between lerimazoline and phenylephrine in isolated rat aortic rings, and also to assess the substrate of the obtained lerimazoline-induced attenuation of phenylephrine contraction. Namely, while lower concentrations of lerimazoline (10⁻⁶ M and especially 10⁻⁷ M) expectedly tended to potentiate the phenylephrine-induced contractions, lerimazoline in higher concentrations (10⁻⁴ M and above) unexpectedly and profoundly depleted the phenylephrine concentration-response curve. Suppression of NO with NO synthase (NOS) inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME; 10⁻⁴ M) or NO scavanger OHB₁₂ (10⁻³ M), as well as non-specific inhibition of K⁺-channels with tetraethylammonium (TEA; 10⁻³ M), have reversed lerimazoline-induced relaxation of phenylephrine contractions, while cyclooxygenase inhibitor indomethacin (10⁻⁵ M) did not affect the interaction between two vasoconstrictors. At the receptor level, non-selective 5-HT receptor antagonist methiothepin reversed the attenuating effect of lerimazoline on phenylephrine contraction when applied at 3×10⁻⁷ and 10⁻⁶ M, but not at the highest concentration (10⁻⁴ M). Neither the 5-HT1D-receptor selective antagonist BRL 15572 (10⁻⁶ M) nor 5-HT₇ receptor selective antagonist SB 269970 (10⁻⁶ M) affected the lerimazoline-induced attenuation of phenylephrine activity. The mechanism of lerimazoline-induced suppression of phenylephrine contractions may involve potentiation of activity of NO and K⁺-channels and activation of some methiothepin-sensitive receptors, possibly of the 5-HT(2B) subtype.

Anti-allodynic effect of mangiferin in neuropathic rats: Involvement of nitric oxide-cyclic GMP-ATP sensitive K+ channels pathway and serotoninergic system.

The neurobiology of neuropathic pain is caused by injury in the central or peripheral nervous system. Recent evidence points out that mangiferin shows anti-nociceptive effect in inflammatory pain. However, its role in inflammatory and neuropathic pain and the possible mechanisms of action are not yet established. The purpose of this study was to determine the possible anti-allodynic effect of mangiferin in rats with spinal nerve ligation (SNL). Furthermore, we sought to investigate the possible mechanisms of action that contribute to these effects. Mechanical allodynia to stimulation with the von Frey filaments was measured by the up and down method. Intrathecal administration of mangiferin prevented, in a dose-dependent fashion, SNL-induced mechanical allodynia. Mangiferin-induced anti-allodynia was prevented by the intrathecal administration of L-NAME (100µg/rat, non-selective nitric oxide synthase inhibitor), ODQ (10µg/rat, inhibitor of guanylate-cyclase) and glibenclamide (50µg/rat, channel blocker of ATP-sensitive K+ channels). Moreover, methiothepin (30µg/rat, non-selective 5-HT receptor antagonist), WAY-100635 (6µg/rat, selective 5-HT1A receptor antagonist), SB-224289 (5µg/rat, selective 5-HT1B receptor antagonist), BRL-15572 (4µg/rat, selective 5-HT1D receptor antagonist) and SB-659551 (6µg/rat, selective 5-HT5A receptor antagonist), but not naloxone (50µg/rat, non-selective opioid receptor antagonist), were able to prevent mangiferin-induced anti-allodynic effect. These data suggest that the anti-allodynic effect induced by mangiferin is mediated at least in part by the serotoninergic system, involving the activation of 5-HT1A/1B/1D/5A receptors, as well as the nitric oxide-cyclic GMP-ATP-sensitive K+ channels pathway, but not by the opioidergic system, in the SNL model of neuropathic pain in rats.

The potential role of serotonergic mechanisms in the spinal oxytocin-induced antinociception.

The role of oxytocin (OXT) in pain modulation has been suggested. Indeed, hypothalamic paraventricular nuclei (PVN) electrical stimuli reduce the nociceptive neuronal activity (i.e., neuronal discharge associated with activation of Aδ- and C-fibers) of the spinal dorsal horn wide dynamic range (WDR) cells and nociceptive behavior. Furthermore, raphe magnus nuclei lesion reduces the PVN-induced antinociception, suggesting a functional interaction between the OXT and the serotoninergic system. The present study investigated in Wistar rats the potential role of spinal serotonergic mechanisms in the OXT- and PVN-induced antinociception. In long-term secondary mechanical allodynia and hyperalgesia induced by formalin or extracellular unitary recordings of the WDR cells we evaluated the role of 5-hydroxytryptamine (5-HT) effect on the OXT-induced antinociception. All drugs were given intrathecally (i.t.). OXT (1×10-5-1×10-4nmol) or 5-HT (1×10-3-1×10-1nmol) prevented the formalin-induced sensitization, an effect mimicked by PVN stimulation. Moreover, administration of OXT (1×10-5nmol) plus 5-HT (1×10-3nmol) at ineffective doses, produced antinociception. This effect was antagonized by: (i) d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT (oxytocin receptor antagonist; 2×10-2nmol); or (ii) methiothepin (a non-specific 5-HT1/2/5/6/7 receptor antagonist; 80nmol). Similar results were obtained with PVN stimulation plus 5-HT (5×10-5nmol). In WDR cell recordings, the PVN-induced antinociception was enhanced by i.t. 5-HT and partly blocked when the spinal cord was pre-treated with methiothepin (80nmol). Taken together, these results suggest that serotonergic mechanisms at the spinal cord level are partly involved in the OXT-induced antinociception.

Age-Dependent Switch of the Role of Serotonergic 5-HT1A Receptors in Gating Long-Term Potentiation in Rat Visual Cortex In Vivo.

The rodent primary visual cortex (V1) is densely innervated by serotonergic axons and previous in vitro work has shown that serotonin (5-HT) can modulate plasticity (e.g., long-term potentiation (LTP)) at V1 synapses. However, little work has examined the effects of 5-HT on LTP under in vivo conditions. We examined the role of 5-HT on LTP in V1 elicited by theta burst stimulation (TBS) of the lateral geniculate nucleus in urethane-anesthetized (adult and juvenile) rats. Thalamic TBS consistently induced potentiation of field postsynaptic potentials (fPSPs) recorded in V1. While 5-HT application (0.1-10 mM) itself did not alter LTP levels, the broad-acting 5-HT receptor antagonists methiothepin (1 mM) resulted in a clear facilitation of LTP in adult animals, an effect that was mimicked by the selective 5-HT1A receptor antagonist WAY 100635 (1 mM). Interestingly, in juvenile rats, WAY 100635 application inhibited LTP, indicative of an age-dependent switch in the role of 5-HT1A receptors in gating V1 plasticity. Analyses of spontaneous electrocorticographic (ECoG) activity in V1 indicated that the antagonist-induced LTP enhancement was not related to systematic changes in oscillatory activity in V1. Together, these data suggest a facilitating role of 5-HT1A receptor activation on LTP in the juvenile V1, which switches to a tonic, inhibitory influence in adulthood.

Different components of conditioned food aversion memory.

Memory reconsolidation processes and protein kinase Mzeta (PKMzeta) activity in memory maintenance and reorganization are poorly understood. Therefore, we examined memory reconsolidation and PKMzeta activity during the maintenance and reorganization of a conditioned food aversion memory among snails. These processes were specifically evaluated after administration of a serotonin receptor antagonist (methiothepin), NMDA glutamate receptor antagonist (MK-801), protein synthesis inhibitor (cycloheximide; CYH), or PKMzeta inhibitor (zeta inhibitory peptide; ZIP) either 2 or 10 days after aversion training. Two days post-training, injections of MK-801 or CYH, combined with a conditioned stimulus reminder, caused amnesia development, and a second training 11 days after this induction did not lead to long-term memory formation. Interestingly, MK-801 or CYH injections and the reminder 10 days after training did not affect memory retrieval. Methiothepin and the reminder, or ZIP without the reminder, at 2 and 10 days after training led to memory impairment, while a second training 11 days after amnesia induction resulted in memory formation. These results suggest that the maintenance of a conditioned food aversion involves two different components with variable dynamics. One component could be characterized by memory strengthening over time and involve N-methyl-D-aspartate receptors and protein synthesis reconsolidation at early, but not late, training stages. The other memory component could involve serotonin-dependent reconsolidation and Mzeta-like kinase activity at both early and late stages after learning. Deficiencies within these two components led to various forms of memory impairment, which differed in terms of the formation of a conditioned food aversion during the second training.

Dynamics of the Development of Amnesia Caused by Disruption of Memory Reconsolidation by Neurotransmitter Receptors Antagonists.

The dynamics of amnesia development under conditions of memory reconsolidation disruption by serotonin receptor antagonist methiothepin or NMDA glutamate receptor antagonist MK-801 was studied in snails trained in conventional food aversion. In 2 days after training, injection of methiothepin or MK-801 before reminder induced amnesia development. During repeated training in 3 days after amnesia induction, the skill was formed more rapidly than during the initial training. During repeated training in 10 days after administration of methiothepin and reminder, the dynamics of habit formation was similar to that during initial learning. At the same time, repeated training in 10 days after MK-801 administration and reminder did not result in long-term memory formation. Disruption of reconsolidation of conditioned food aversion memory by antagonists of serotonin or NMDA glutamate receptors led to the development of different types of amnesia that had similar strengthening gradient at the early stages, but differed by the possibility of memory formation during re-training at the late stage.

Pharmacological characteristics of Artemisia vulgaris L. in isolated porcine basilar artery.

ETHNOPHARMACOLOGICAL RELEVANCE: In Vietnamese traditional herbalism, there are conflicting opinions about the effect of Artemisia vulgaris L. (AVL, English name: mugwort) on hypertension. Some ethnic doctors recommend the use of AVL for treatment of hypertension, whereas others advise against it. The purpose of this study was to clarify the pharmacological characteristics of AVL in isolated arteries to explain the conflicts surrounding the use of AVL for treatment of hypertension. MATERIALS AND METHODS: We initially performed a functional study using an organ bath system to investigate the effect of AVL extract on isolated porcine basilar artery. We then measured the change in intracellular free Ca(2+) concentration elicited by AVL using cultured smooth muscle cells loaded with the Ca(2+) indicator fluo-4. Finally, using HPLC, we determined the active components in AVL. RESULTS AND DISCUSSION: AVL induced vasoconstriction at resting tension, and endothelial removal enhanced this effect significantly. Pretreatment with PD123319 (an AT2 receptor antagonist), Nω-nitro-L-arginine (a nitric oxide synthase inhibitor), or both, also enhanced this effect. AVL-induced contraction was competitively inhibited by methiothepin (a 5-HT1 and 5-HT2 receptor antagonist) in the presence of ketanserin (a 5-HT2 receptor antagonist). Removal of extracellular calcium with nifedipine (an L-type Ca(2+) channel blocker) or ruthenium red (a ryanodine receptor blocker) significantly reduced AVL-induced contraction, whereas losartan (an AT1 receptor antagonist) and diphenhydramine (a H1 receptor antagonist) had no effect on this contraction. AVL increased the intracellular free Ca(2+) concentration in cultured cells, and this increment was inhibited by methiothepin. HPLC analysis revealed that the retention time of the first peak in the AVL profile was similar to that of the 5-HT standard, and that addition of 5-HT to the AVL sample enhanced this peak. On the other hand, AVL induced endothelium-independent relaxation under precontracted conditions with 60mM KCl. Captopril (an angiotensin converting enzyme inhibitor), atenolol (a ß1 receptor antagonist) and cimetidine (a H2 receptor antagonist) had no effect on this relaxation. In Ca(2+)-free 60mM KCl-containing solution, pretreatment with AVL significantly inhibited CaCl2-induced contraction. CONCLUSION: For the first time, the present study has demonstrated that AVL has two opposite effects, contraction and relaxation, on isolated artery, which may help to explain the conflicting indications for AVL in traditional herbalism. 5-HT is a significant factor affecting artery contraction in the presence of AVL.

Role of spinal 5-HT5A, and 5-HT1A/1B/1D, receptors in neuropathic pain induced by spinal nerve ligation in rats.

Serotonin (5-HT) participates in pain modulation by interacting with different 5-HT receptors. The role of 5-HT5A receptor in neuropathic pain has not previously studied. The purpose of this study was to investigate: A) the role of 5-HT5A receptors in rats subjected to spinal nerve injury; B) the expression of 5-HT5A receptors in dorsal spinal cord and dorsal root ganglia (DRG). Neuropathic pain was induced by L5/L6 spinal nerve ligation. Tactile allodynia in neuropathic rats was assessed with von Frey filaments. Western blot methodology was used to determine 5-HT5A receptor protein expression. Intrathecal administration (on day 14th) of 5-HT (10-100 nmol) or 5-carboxamidotryptamine (5-CT, 0.03-0.3 nmol) reversed nerve injury-induced tactile allodynia. Intrathecal non-selective (methiothepin, 0.1-0.8 nmol) and selective (SB-699551, 1-10 nmol) 5-HT5A receptor antagonists reduced, by ~60% and ~25%, respectively, the antiallodynic effect of 5-HT (100 nmol) or 5-CT (0.3 nmol). Moreover, both selective 5-HT1A and 5-HT1B/1D receptor antagonists, WAY-100635 (0.3-1 nmol) and GR-127935 (0.3-1 nmol), respectively, partially diminished the antiallodynic effect of 5-HT or 5-CT by about 30%. Injection of antagonists, by themselves, did not affect allodynia. 5-HT5A receptors were expressed in the ipsilateral dorsal lumbar spinal cord and DRG and L5/L6 spinal nerve ligation did not modify 5-HT5A receptor protein expression in those sites. Results suggest that 5-HT5A receptors reduce pain processing in the spinal cord and that 5-HT and 5-CT reduce neuropathic pain through activation of 5-HT5A and 5-HT1A/1B/1D receptors. These receptors could be an important part of the descending pain inhibitory system.

Serotonin Receptor Agonist 5-Nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry.

UNLABELLED: Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development. IMPORTANCE: Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-nonyloxytryptamine (5-NT) impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.