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.

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.

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.

Analgesia induced by 2- or 100-Hz electroacupuncture in the rat tail-flick test depends on the anterior pretectal nucleus.

AIMS: The anterior pretectal nucleus (APtN) and electroacupuncture (EA) activate descending mechanisms to modulate nociceptive inputs in the spinal dorsal horn. This study examines qualitatively whether mechanisms in the APtN participate in the EA-induced analgesia in rats. MAIN METHODS: The tail-flick test was utilized to examine the changes produced by non-selective antagonists of serotonergic (methysergide, 37 pg), muscarinic (atropine, 10 ng) and opioid (naloxone, 10 ng) receptors; selective antagonists against µ (CTOP, 6.4 µg), δ (ICI174,864, 6.9 µg) or κ (nor-BNI, 7.3 µg); 5HT1 (methiothepin, 0.47 µg), 5HT2 (ketanserin, 5.4 µg), or 5HT3 (MDL 72222, 15.7 µg); and GABAA (bicuculline, 150 ng) receptors injected into the dorsal (d) or ventral (v) APtN on the antinociception induced by a 20-min EA applied at 2- or 100-Hz frequency to the Zusanli and Sanyinjiao acupoints. KEY FINDINGS: The 2-Hz EA-induced analgesia was blocked by naloxone, CTOP or atropine, was less intense after bicuculline, was shorter after methysergide or methiothepin in dAPtN, and was less intense after methysergide, methiothepin and bicuculline in vAPtN. The 100-Hz EA-induced analgesia was less intense after methysergide, methiothepin and CTOP in vAPtN, and remained unchanged after injection of the antagonists into the dAPtN. SIGNIFICANCE: The 2-Hz EA-induced analgesia utilizes cholinergic muscarinic, µ-opioid, GABAA and 5-HT1 mechanisms in the dAPtN and µ-opioid and 5-HT1 mechanisms in the vAPtN, while 100-Hz EA-induced analgesia utilizes µ-opioid and 5-HT1 mechanisms in the vAPtN but does not utilize them in the dAPtN.

Serotonin receptors are involved in the spinal mediation of descending facilitation of surgical incision-induced increase of Fos-like immunoreactivity in rats.

BACKGROUND: Descending pronociceptive pathways may be implicated in states of persistent pain. Paw skin incision is a well-established postoperative pain model that causes behavioral nociceptive responses and enhanced excitability of spinal dorsal horn neurons. The number of spinal c-Fos positive neurons of rats treated intrathecally with serotonin, noradrenaline or acetylcholine antagonists where evaluated to study the descending pathways activated by a surgical paw incision. RESULTS: The number of c-Fos positive neurons in laminae I/II ipsilateral, lamina V bilateral to the incised paw, and in lamina X significantly increased after the incision. These changes: remained unchanged in phenoxybenzamine-treated rats; were increased in the contralateral lamina V of atropine-treated rats; were inhibited in the ipsilateral lamina I/II by 5-HT1/2B/2C (methysergide), 5-HT2A (ketanserin) or 5-HT1/2A/2C/5/6/7 (methiothepin) receptors antagonists, in the ipsilateral lamina V by methysergide or methiothepin, in the contralateral lamina V by all the serotonergic antagonists and in the lamina X by LY 278,584, ketanserin or methiothepin. CONCLUSIONS: We conclude: (1) muscarinic cholinergic mechanisms reduce incision-induced response of spinal neurons inputs from the contralateral paw; (2) 5-HT1/2A/2C/3 receptors-mediate mechanisms increase the activity of descending pathways that facilitates the response of spinal neurons to noxious inputs from the contralateral paw; (3) 5-HT1/2A/2C and 5-HT1/2C receptors increases the descending facilitation mechanisms induced by incision in the ipsilateral paw; (4) 5-HT2A/3 receptors contribute to descending pronociceptive pathways conveyed by lamina X spinal neurons; (5) alpha-adrenergic receptors are unlikely to participate in the incision-induced facilitation of the spinal neurons.

Am5-HT7: molecular and pharmacological characterization of the first serotonin receptor of the honeybee (Apis mellifera).

The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT(7) receptor family. Expression of the Am5-HT(7) receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT(7) is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC(50) = 1.1-1.8 nm). The Am5-HT(7) receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.

Heterologous desensitization is evoked by both agonist and antagonist stimulation of the human 5-HT(7) serotonin receptor.

Previously, we demonstrated that human serotonin (5-HT) 5-HT(7) receptors display marked constitutive activity. Here, we tested if the constitutive activation of adenylyl cyclase by 5-HT(7) receptors influenced both the desensitization properties of transfected 5-HT(7) receptors and the ability of endogenous G(s)-coupled receptors to activate adenylyl cyclase. Using membranes from stably transfected HEK293 cells expressing the recombinant human 5-HT(7) receptor splice variants (5-HT(7(a)), 5-HT(7(b)) and 5-HT(7(d))), we compared the effects of 1-h or 24-h preincubation of the agonist 5-HT, partial inverse agonists mesulergine and SB269970, and full inverse agonists clozapine and methiothepin on subsequent activation of adenylyl cyclase by both 5-HT through transfected 5-HT(7) receptors and the endogenous G(s)-coupled beta-adrenoceptors and prostaglandin receptors of HEK293 cells. The data show that stable expression of 5-HT(7) receptors is sufficient to attenuate adenylyl cyclase activation by endogenous G(s)-coupled receptors. Interestingly, preincubation with inverse agonists not only failed to result in the predicted resensitization of all receptor mediated adenylyl cyclase activation, but some inverse agonists further attenuated (desensitized) beta-adrenoceptor and prostaglandin-stimulated adenylyl cyclase activation similar to long-term agonist exposure by 5-HT. These effects were not correlated with inverse agonist efficacy, were not accompanied by receptor down-regulation and appear to be mediated by a protein kinase A (PKA) independent mechanism. It is concluded that the human 5-HT(7) receptor mediates heterologous desensitization of endogenous G(s)-coupled receptors through an unknown and potentially novel mechanism.

Serotonin receptor antagonists discriminate between PKA- and PKC-mediated plasticity in aplysia sensory neurons.

Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

Clozapine directly relaxes bovine retinal arteries.

PURPOSE: It was suggested that clozapine might be helpful in the development of new antiglaucoma agents, as it combines lowering the intraocular pressure after topical instillation with vasodilation. This study aimed to evaluate and characterize the vasodilatory effect of clozapine in isolated bovine retinal arteries (BRAs). METHODS: Retinal arteries were isolated from bovine eyes and mounted in the organ bath of a small vessel myograph. RESULTS: Cumulative addition of clozapine (1 nM to 10 microM) caused a concentration-dependent relaxation of the BRAs. Removal of the endothelium, inhibition of nitric oxide synthase and of soluble guanylyl cyclase reduced the clozapine response, whereas cyclooxygenase inhibition had no influence. A Ca2+ channel activator, a 5-hydroxytryptamine receptor antagonist, and an adenosine receptor antagonist failed in affecting the clozapine-induced relaxations. CONCLUSIONS: Clozapine relaxes bovine retinal arteries. Endothelium-derived NO seems to be involved, whereas prostanoids, calcium entry blockade, 5-HT7 receptor stimulation, and adenosine receptor stimulation do not.

Differential inverse agonist efficacies of SB-258719, SB-258741 and SB-269970 at human recombinant serotonin 5-HT7 receptors.

Recombinant 5-hydroxytryptamine 5-HT7 receptors are known to express constitutive, i.e., agonist-independent activity. Nonselective ligands, like methiothepin, ritanserin or clozapine behave as full inverse agonists at 5-HT7 receptors. The aim of the present study was to evaluate the degree of inverse agonist activity of three selective 5-HT7 receptor antagonists ((R)-3,N-dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl)propyl]benzene sulfonamide or SB-258719, R-(+)-1-(toluene-3-sulfonyl)-2-[2-(4-methylpiperidin-1-yl)ethyl]-pyrrolidine or SB-258741 and (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolidine-1-sulfonyl)-phenol or SB-269970) in the same model. cAMP accumulation was measured in intact Chinese hamster ovary (CHO) cells expressing human recombinant 5-HT7a receptors. In these cells, 5-HT stimulated cAMP levels and a series of ligands antagonized the effect of 5-HT with a 5-HT7 receptor-like profile. SB-258719 had no inverse agonist activity, SB-258741 behaved as a partial inverse agonist and SB-269970 was a quasi-full inverse agonist (as compared to methiothepin). The inverse agonist effect of SB-269970 was antagonized in a concentration-dependent manner by SB-258719. The widespread spectrum of inverse agonist activities shown by these compounds should help assessing the physiological relevance of constitutive 5-HT7 receptor activity in native tissues.

Mechanisms involved in the loss of excitatory post-stimulus responses by inflammation.

AIM: Electrical stimulation of colonic muscles elicits a response during the stimulation period, and a transient excitation after the stimulus. Post-stimulus or "rebound" excitation has been linked to pathways involving inhibitory neurotransmitters, prostaglandins and substance P but the mechanism is incompletely understood. Because rabbit colitis is characterized by a loss of inhibitory neurotransmission we hypothesized it might affect the rebound response. Therefore we characterized rebound responses in non-inflamed and inflamed tissue by comparing the effect of antagonists/blockers of putative (nitric oxide [NO], ATP, substance P, prostaglandins) and new (serotonin) neurotransmitters. METHODS: Strips from rabbits with colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) were subjected to electrical field stimulation. Because rebound responses are more prominent under nonadrenergic noncholinergic (NANC) conditions, the effect of specific antagonists (N(omega)-nitro-L-arginine methyl ester (L-NAME), indomethacin, SR140333, methiothepin) on the rebound response was compared under normal and NANC conditions. RESULTS: NANC-conditions increased rebound responses in non-inflamed strips, but this effect was reduced or abolished in inflamed strips. Rebound responses were reduced by pretreatment with the NO-synthase inhibitor, L-NAME, under NANC conditions in non-inflamed strips but not affected in inflamed tissue. In contrast, the P(2) purine receptor antagonist, suramin, did not affect rebound responses in inflamed and non-inflamed strips. The effect of the cyclo-oxygenase inhibitor (COX), indomethacin, on rebound responses was reversed from excitatory to inhibitory by inflammation. Under NANC conditions rebound contractions were also reduced by the neurokinin-1 (NK(1)) antagonist, SR140333, both in normal and inflamed strips. The most pronounced reduction in rebound responses in inflamed and non-inflamed strips under normal conditions was observed with the 5-hydroxytryptamin (1,2) (5-HT(1,2)) antagonist, methiothepin. CONCLUSION: Rebound responses are mainly non-cholinergic and involve NO, substance P, serotonin and inhibitory prostaglandins. In inflamed tissue the nitrergic pathway is absent, excitatory prostaglandins prevail and the cholinergic and tachykinergic components are relatively more important. However there remains an important serotonergic contribution. Our data suggest that inflammation damages different neural pathways to a different extent and is most selective for nitrergic pathways.

5-HT7 receptors are involved in mediating 5-HT-induced activation of rat primary afferent neurons.

The purpose of this study was to determine whether the 5-hydroxytryptamine7 (5-HT7) receptor is expressed by nociceptor-like neurons in the rat PNS and whether 5-HT activates these nociceptors via the 5-HT7 receptor subtype. Using a polyclonal antibody and the method of immunofluorescence staining, we demonstrated that the 5-HT7 receptor appears predominately on "nociceptor-like" neurons of the rat lumbar dorsal root ganglia. Using immunocytochemical methods, we showed that the immunoreactivity of the 5-HT7 receptor antibody complex is localized in the superficial layers of the spinal cord dorsal horn, which corresponds with laminae I, IIouter and IIinner. Furthermore, we demonstrated that noxious stimulation produced by knee injection of 5-HT or a 5-HT7 agonist dose-dependently increases c-Fos production of the rat spinal cord dorsal horn. This effect was significantly inhibited by the preinjection of a 5-HT7 antagonist. We conclude that the 5-HT7 receptor is expressed by rat primary afferent nociceptors which terminate in the superficial layers of the spinal cord dorsal horn and that the 5-HT7 receptor subtype is involved in nociceptor activation by 5-HT.

Cell proliferation in carcinoid valve disease: a mechanism for serotonin effects.

BACKGROUND AND AIM OF THE STUDY: Elevated serum serotonin is associated with carcinoid heart disease, the hallmark of which is valvular thickening. Yet, the mechanistic role of serotonin in carcinoid heart disease is poorly understood. We postulated that serotonin has a direct mitogenic effect on cardiac valvular subendocardial cells, and that this effect is mediated by serotonin receptors. METHODS: The dose-dependent proliferative effects of serotonin (10(-8) to 10(-4)M) on cultured porcine aortic valve cells via a [3H]thymidine assay were determined in vitro. Serotonin receptor antagonist studies in culture were also performed using methiotepin, a 5HT1b antagonist, and ketanserin, a 5HT2 receptor antagonist, to determine the mechanism of serotonin action. The ex-vivo proliferation level in human carcinoid (n = 26) and normal valves (n = 10) was compared using proliferating cell nuclear antigen (PCNA) staining, a marker for proliferation. Identification and localization of specific 5HT receptor was assessed by immunostaining for serotonin receptors in the valves. RESULTS: Serotonin increased valvular proliferation in vitro in a dose-dependent manner (10-fold increase) (p <0.001), and this mitogenic effect was inhibited by methiotepin but not ketanserin. In human carcinoid heart valves the level of proliferation was 35-fold higher than in normal human valves (p <0.001). 5HT1b receptors were found only in the carcinoid valves, and not in the normal valves. CONCLUSION: Serotonin is a powerful mitogen for valvular subendocardial cells. The mitogenic effect is at least partly mediated via 5HT1b receptors. Subendothelial cell proliferation is significantly elevated in human carcinoid valves in vivo. The data suggest a mechanism whereby serotonin may contribute to valvular proliferation in carcinoid heart disease.

Interaction between the serotoninergic and dopaminergic systems in d-fenfluramine-induced activation of c-fos and jun B genes in rat striatal neurons.

To test for the relative contributions of the dopaminergic and serotoninergic systems in the striatum to the effects of d-fenfluramine, an indirect serotonin receptor agonist, we assessed the expression of Fos/Jun proteins induced by d-fenfluramine given alone or in the presence of dopaminergic or serotoninergic agents. To determine the neuronal targets of d-fenfluramine in the striatum, we identified the phenotypes of striatal neurons in which d-fenfluramine induced Fos expression. Our results demonstrated that d-fenfluramine evokes nuclear expression of Fos/Jun B proteins in the striatum, and that the Fos expression was dose-dependent and accompanied by transient induction of c-fos mRNA. Fos expression was blocked by p-chloroamphetamine, a serotoninergic neurotoxin. Pretreatment with SCH 23390, a D1-dopamine receptor antagonist, led to a marked decrease in Fos/Jun B expression in the caudoputamen, but not in the cortex, whereas pretreatment with methiothepin, a nonselective serotonin 5-HT1 receptor antagonist, blocked Fos expression completely in the cortex and only partially in the caudoputamen. The expression of Fos/Jun B in the striatum occurred mainly in dynorphin-containing neurons and in a subpopulation of striatal interneurons that exhibited NADPH-diaphorase activity. Most of the enkephalin-containing neurons of the striatum did not show Fos/Jun B staining. These results suggest that the mechanism by which d-fenfluramine induces c-fos and jun B expression in the rat caudoputamen depends at least in part on activation of the dopaminergic system by serotonin.

Monoaminergic long-term facilitation of GABA-mediated inhibitory transmission at cerebellar synapses.

Long-term facilitation of neurotransmission by monoaminergic systems is implicated in the cellular mechanism of memory and learning-related processes at invertebrate synapses. Using whole-cell recording and rat cerebellar slices, we have examined whether mammalian monoamine-containing neurons play analogous roles in synaptic plasticity, and our results suggest that serotonin and noradrenaline are critically involved in short- and long-term modulation of GABAergic transmission in the cerebellar cortex. Exogenously applied serotonin and noradrenaline selectively induced a short-term enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells, their effect subsiding in 30 min. Successive amine applications converted this effect to long-term facilitation lasting more than 2 h. During the monoamine-induced short- and long-term facilitation, spontaneously occurring miniature inhibitory synaptic responses increased in frequency, without significant changes in their mean amplitude and amplitude distribution, as well as the GABA receptor sensitivity of Purkinje cells. The actions of the two amines on the inhibitory transmission were mimicked by forskolin and blocked by kinase inhibitors, H-7, H-89 and Rp-adenosine 3',5'-cyclic monophosphothioate. Thus, serotonin and noradrenaline are likely to activate cyclic-AMP- and protein kinase-dependent pathways in GABAergic interneurons, thereby reinforcing the inhibitory transmission on to Purkinje cells. Repetitive electrical stimulation within the molecular layer mimicked the facilitatory effect induced by exogenous monoamines: namely, neural stimulation selectively elicited long-lasting enhancement of GABAergic transmission in a manner sensitive to the monoamine receptor antagonists, methiothepin and propranolol, and an uptake inhibitor, imipramine. Synaptically released monoamines thus appear to induce cyclic-AMP- and protein kinase-dependent long-term facilitation of cerebellar GABAergic transmission, thereby providing a likely mechanism of synaptic plasticity associated with motor coordination within the mammalian cerebellar system.

Native 5-HT1B receptors expressed in OK cells display dual coupling to elevation of intracellular calcium concentrations and inhibition of adenylate cyclase.

The opossum kidney (OK) cell line has been shown previously to express endogenous 5-HT1B receptors which negatively couple to adenylate cyclase. Since other Gi-linked receptors have been shown to inhibit adenylate cyclase and to elevate intracellular calcium concentrations ([Ca2+]i), studies were initiated to determine whether native opossum 5-HT1B receptors could also display dual coupling to these signal transduction mechanisms. Saturation studies using [125I](-)-iodocyanopindolol ([125I]CYP) to radiolabel the 5-HT1B receptor in OK cell membranes (in the presence of 3 microM (-)-isoproterenol to mask beta-adrenergic receptors) yielded an equilibrium dissociation constant (pKd) of 10.04 and binding site density (Bmax) of 55 fmol/mg protein. Exposure of intact OK cells to 5-HT, CP 93,129, a selective rodent 5-HT1B receptor agonist, and (+/-)-cyanopindolol, a mixed 5-HT1A/1B receptor agonist/antagonist, produced concentration-dependent inhibitions of forskolin (3 MM)-stimulated cAMP accumulation (FSCA; Emax=90-95%) and elevations of [Ca2+]i (Emax approximately 200 nM increase above basal levels). Agonist potencies (pEC50) ranged from 9.7 to 8.1 and were comparable between the two second messenger assays, although slightly higher agonist potencies (approximately three-fold) were observed in the cAMP assay. GR 127,935, a selective 5-HT1B/1D receptor antagonist, behaved as a strong partial agonist in both the cAMP and calcium assays, with an intrinsic activity of 0.7 relative to 5-HT. Methiothepin, a nonselective 5-HT receptor antagonist, competitively antagonized the inhibitory cAMP response elicited by CP 93,129, yielding an apparent pKb value of 7.3. Methiothepin (10 microM) completely antagonized the stimulatory calcium response evoked by a saturating concentration of CP 93,129 (100 nM). Pertussis toxin pretreatment blocked the CP 93,129-induced inhibition of FSCA and elevation of [Ca2+]i in OK cells, indicating the involvement of Gi/o proteins in transducing these second messenger responses. The agonist properties of (+/-)-cyanopindolol and GR 127,935 observed in both second messenger assays suggests that a large degree of receptor reserve may be present, even though 5-HT1B receptor expression is low in OK cells. The OK cell line continues to serve as a model system to investigate 5-HT1B receptor-mediated signaling events.

P2-receptor-mediated inhibition of serotonin release in the rat brain cortex.

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.

Chronic antidepressant exposure enhances 5-hydroxytryptamine7 receptor-mediated cyclic adenosine monophosphate accumulation in rat frontocortical astrocytes.

In rat frontal cortical astrocytes, 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine and 5-methoxytryptamine elicited a concentration-dependent increase of cyclic AMP accumulation, with EC50 values of 137 +/- 7 nM (5-HT), 10.4 +/- 3.9 nM (5-carboxamidotryptamine) and 57.8 +/- 13.6 nM (5-methoxytryptamine). Accumulation of cyclic AMP stimulated by 5-HT (10 microM) was inhibited by methiothepin (IC50 = 21 +/- 13 nM) but not by pindolol, SCH 23390, ICS 205-930 or GR 113808. These pharmacological effects on cyclic AMP formation in astrocytes are consistent with those observed in 5-HT7 receptor binding studies. Indeed, reverse transcription-polymerase chain reaction analysis revealed the presence of 5-HT7 mRNA in astrocytes. Chronic exposure of astrocytes to mianserin (0.05-4 microM) or amitriptyline (1-8 microM) for 3 days produced a concentration-dependent enhancement of 5-HT-stimulated cyclic AMP accumulation, with EC50 values of 0.47 +/- 0.04 microM (mianserin) and 2.5 +/- 0.3 microM (amitriptyline). The enhancement of cyclic AMP accumulation was time dependent, reaching maximal levels within 2 days. Mianserin exposure (2 microM, for 3 days) resulted in a comparable enhancement of cyclic AMP accumulation mediated by 5-carboxamidotryptamine and 5-methoxytryptamine, whereas this treatment had no effect on cyclic AMP production induced by G protein-specific stimulators and catalytic subunit-selective stimulators. The mianserin-induced enhancement of 5-HT-stimulated cyclic AMP accumulation was decreased by methiothepin (IC50 = 15 +/- 8 nM) and significantly attenuated by pretreatment with 5-HT7 receptor antisense oligonucleotides, suggesting that chronic mianserin exposure produces an increase in 5-HT7 receptor activity. Chronic exposure to maprotiline, setiptiline or clomipramine (5 microM, for 3 days) mimicked the effect of mianserin. The enhancement of 5-HT7 receptor activity after chronic antidepressant exposure may be a mechanism underlying the therapeutic effects.

5-HT1-like receptor-mediated contraction in the human internal mammary artery.

We wished to characterize the 5-hydroxytryptamine (5-HT) receptors mediating vasoconstriction in the human internal mammary artery (IMA). Segments of the IMA obtained from patients undergoing coronary by-pass surgery were suspended in an organ bath and exposed to 5-HT and sumatriptan (SUM), a 5-HT1-like receptor agonist, in the presence and absence of potassium chloride (KCl) and angiotensin II. 5-HT induced concentration-dependent contractions in all quiescent and pre-contracted preparations. SUM induced small contractions in 70% of quiescent IMA rings, whereas it elicited marked and concentration-dependent contractions in all of the preparations given a moderate tone by a threshold concentration of KCl and angiotensin II. The efficacy of SUM was higher in precontracted arteries. Concentration-effect curves (CEC) of 5-HT and SUM were not affected by the 5-HT3-receptor antagonist tropisetron (1 microM). The nonselective antagonist, methiothepin (30 nM), shifted the CEC of SUM to the right. 5-HT2A-receptor antagonist, ketanserin (1 microM) inhibited responses to 5-HT, whereas it affected only the responses to the smaller concentrations of SUM. When methiothepin (30 nM) was applied in the presence of ketanserin (1 microM), a further inhibition in the responses to 5-HT was observed. These results suggest that 5-HT1-like receptors mediate the contractile action of SUM and contribute to that of 5-HT in IMA.