Activation Of 5-HT1B Receptors Research Articles

Presynaptic 5-HT1B receptor-mediated serotonergic inhibition of glutamate transmission in the bed nucleus of the stria terminalis

Activation of neurons in the bed nucleus of the stria terminalis (BNST) plays a critical role in stress and anxiety-related behaviors. Previously, we have shown that serotonin (5-HT) can directly modulate BNST neuronal excitability by an action at postsynaptic receptors. In this study we built upon that work to examine the effects of 5-HT on excitatory neurotransmission in an in vitro rat BNST slice preparation. Bath application of 5-HT reversibly reduced the amplitude of evoked excitatory postsynaptic currents (eEPSCs). These effects were mimicked by the 5-HT1B/D receptor agonist, sumatriptan, and by the 5-HT1B receptor selective agonist, CP93129. Conversely, the effects of 5-HT and sumatriptan could be blocked by the 5-HT1B receptor-selective antagonist, GR55562. In contrast, the 5-HT1A receptor agonist 8-OH DPAT or antagonist WAY 100635 could not mimic or block the effect of 5-HT on eEPSCs. Together, these data suggest that the 5-HT-induced attenuation of eEPSCs was mediated by 5-HT1B receptor activation. Moreover, sumatriptan had no effect on the amplitude of the postsynaptic current elicited by pressure applied AMPA, suggesting a possible presynaptic locus for the 5-HT1B receptor. Furthermore, 5-HT, sumatriptan and CP93129 all increased the paired pulse ratio of eEPSCs while they concomitantly decreased the amplitude of eEPSCs, suggesting that these agonists act to reduce glutamate release probability at presynaptic locus. Consistent with this observation, sumatriptan decreased the frequency of miniature EPSCs, but had no effect on their amplitude. Taken together, these results suggest that 5-HT suppresses glutamatergic neurotransmission in the BNST by activating presynaptic 5-HT1B receptors to decrease glutamate release from presynaptic terminals. This study illustrates a new pathway by which the activity of BNST neurons can be indirectly modulated by 5-HT, and suggests a potential new target for the development of novel treatments for depression and anxiety disorders.

Lack of serotonin 5-HT2Breceptor alters proliferation and network volume of interstitial cells of Cajalin vivo

Normal gastrointestinal motility requires intact networks of interstitial cells of Cajal (ICC). Interstitial cells of Cajal numbers are maintained by a balance between cell loss factors and survival/trophic/growth factors. Activation of 5-HT(2B) receptors expressed on ICC increases ICC proliferation in vitro. It is not known whether 5-HT(2B) receptors on ICC are activated in vivo. The aims of this study were to investigate if adult ICC proliferate, whether the proliferation of ICC in vivo is affected by knocking out the 5-HT(2B) receptor, and if alterations in proliferation affect ICC networks. Proliferating ICC were identified by immunoreactivity for Ki67 in both the myenteric and deep muscular plexus regions of the jejunum in mice with a targeted insertion of a neomycin resistance cassette into the second coding exon of the htr2b receptor gene. Adult ICC do proliferate. The number of proliferating ICC was lower in the myenteric plexus region of Htr2b(-/-) compared to Htr2b(+/+) mice. The volume of Kit-positive ICC was 30% lower in the myenteric plexus region and 40% lower in the deep muscular plexus region in Htr2b(-/-) mice where the number of ICC was also reduced. Interstitial cells of Cajal proliferate in adult mice and activation of 5-HT(2B) receptors results in increased proliferation of ICC in vivo. Furthermore, lack of 5-HT(2B) receptor signaling reduces the density of ICC networks in mature mice. These data suggest that 5-HT(2B) receptor signaling is required for maintenance of ICC networks, adding 5-HT to the growing number of factors shown to regulate ICC networks.

Serotonergic drugs and valvular heart disease

Background: The serotonin (5-HT) releasers (±)-fenfluramine and (+)-fenfluramine were withdrawn from clinical use owing to increased risk of valvular heart disease. One prevailing hypothesis (i.e., the ‘5-HT hypothesis’) suggests that fenfluramine-induced increases in plasma 5-HT underlie the disease. Objective: Here, we critically evaluate the possible mechanisms responsible for fenfluramine-associated valve disease. Methods: Findings from in vitro and in vivo experiments performed in our laboratory are reviewed. The data are integrated with existing literature to address the validity of the 5-HT hypothesis and suggest alternative explanations. Conclusions: The overwhelming majority of evidence refutes the 5-HT hypothesis. A more likely cause of fenfluramine-induced valvulopathy is activation of 5-HT2B receptors on heart valves by the metabolite norfenfluramine. Future serotonergic medications should be designed to lack 5-HT2B agonist activity.

P.1.06 Ketanzerine improves cognitive performance in ovariectomized rats with scopolamine-induced amnesia

Using a number of agonist and antagonist compounds, we attempted to characterize the responses and receptors involved in the effects of 5-hydroxytryptamine (5-HT) in the in situ blood perfused rat mesentery. An intra-arterial (i.a.) bolus injection of 5-HT increased mesenteric perfusion pressure in a dose-dependent way but did not change the systemic blood pressure. The selective 5-HT2 receptor agonists α-methyl-5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (m-CPP) and (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI), caused a local vasoconstrictor effect in the autoperfused vascular mesenteric bed. Intra-arterial injection of 5-carboxamidotryptamine (5-CT) and 1-(m-chlorophenyl)-biguanide (m-CPBG) did not modify the mesenteric perfusion pressure. The vasoconstrictor effect elicited by 5-HT and α-methyl-5-HT was significantly decreased by ritanserin and by a selective 5-HT2B/2C receptor antagonist, N-3-pyridinyl-3,5-dihydro-5-methyl-benzo[1,2-b:4,5-b′]dipyrrole-1(2H)-carboxamide hydrochloride (SB 206553), but was not modified by prazosin, propranolol, indomethacin or enalapril pretreatment. Our data suggest that the vasoconstrictor serotonergic response induced in the in situ autoperfused rat mesenteric vascular bed is mainly mediated by activation of 5-HT2B and/or 5-HT2C receptors.

Fluvoxamine exerts anorexic effect in 5-HT2C receptor mutant mice with heterozygous mutation of β-endorphin gene

Serotonin (5-hydroxytryptamine; 5-HT) 2C receptors and the downstream melanocortin pathway are suggested to mediate the anorexic effects of m-chlorophenylpiperazine (mCPP) and fenfluramine. We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor, together with pharmacological inactivation of 5-HT2C receptors exert feeding suppression through activation of 5-HT1B receptors in mice. Here, we report that fluvoxamine exerted anorexic effects in 5-HT2C receptor mutant mice with heterozygous mutation of beta-endorphin gene (2CREnd mice), whereas fluvoxamine had no effect on food intake in age-matched wild-type mice and 5-HT2C receptor mutant mice, which are associated with decreases in hypothalamic proopiomelanocortin (POMC) expression. mCPP suppressed food intake in 5-HT2C receptor mutant mice, 2CREnd mice and age-matched wild-type mice. These results suggest that fluvoxamine-induced feeding suppression requires a perturbation of 5-HT2C receptor and beta-endorphin signalling plus functional hypothalamic POMC activity, whereas mCPP-induced feeding suppression does not always require functional 5-HT2C receptor, beta-endorphin, and POMC activity in mice.

Pre- and Postsynaptic Serotoninergic Excitation of Globus Pallidus Neurons

The basal ganglia (BG) play a critical role in the pathogenesis and pathophysiology of Parkinson's disease (PD). Recent studies indicate that serotoninergic systems modulate BG activity and may be implicated in the pathophysiology and treatment of PD. The globus pallidus (GP), the rodent homologue of the primate GPe, is the main central nucleus of the basal ganglia, affecting the striatum, the subthalamic nucleus (STN), and BG output structures. We therefore studied the effect of serotonin (5-HT) and specific 5-HT agonists and antagonists on GP neurons from rat brain slices. Using intra- and extracellular recordings of GP neurons we found that serotonin increases the firing rate of GP neurons. Analyzing the effects of specific 5-HT agonists and antagonists on the firing rate of GP neurons showed that the increase in firing rate is due to the activation of 5-HT1B and 5-HT1A receptors. Intracellular recordings in both voltage- and current-clamp modes revealed that serotonin mediates its effect via pre- and postsynaptic mechanisms. The presynaptic effect is mediated by attenuation of gamma-aminobutyric acid release, probably through activation of 5-HT1B receptors. Postsynaptically, serotonin activates a hyperpolarization-activated cation channel, probably via 5-HT1A receptors. Furthermore, serotonin decreases the fast synaptic depression characteristic of the striatal afferent input. The decreased serotonin concentrations in the BG nuclei in PD may contribute to depressed GP activity and enhance the emergence of BG pathological synchronous oscillations. We therefore suggest that future therapeutics of PD should be directed toward restoration of normal serotonin levels in BG nuclei.

NADPH oxidase activity is necessary for phrenic motor facilitation induced by 5HT2B receptor activation

Acute intermittent hypoxia (AIH) elicits phrenic long‐term facilitation (pLTF), a form of respiratory plasticity that requires activation of spinal 5HT2 receptors (Mahamed and Mitchell, 2006). AIH‐induced pLTF requires NADPH oxidase‐derived ROS formation (MacFarlane et al., FASEB J., 2007). Spinal 5‐HT receptor activation in vivo is sufficient to induce phrenic motor facilitation by an NADPH oxidase‐dependent mechanism, but the specific 5‐HT receptor subtypes involved are not known. In this study, we tested 1) whether spinal 5‐HT2B receptor activation is sufficient to elicit phrenic motor facilitation (without hypoxia), and 2) whether phrenic motor facilitation following 5‐HT2B receptor activation requires spinal NADPH oxidase activity. In anesthetized, paralyzed and ventilated rats with constant arterial PO2 and PCO2, episodic intrathecal (i.t.) injections of 5‐HT (1mM) at C4 (5μl, 3 injections, 5min intervals) or the selective 5‐HT2B receptor agonist BW723C86 (1mM) both elicited significant phrenic motor facilitation at 60min post‐injections. Pre‐treatment with a single i.t. injection of apocynin (600μM, 12μl, i.t.), an NADPH oxidase inhibitor, blocked phrenic motor facilitation induced by both 5‐HT and BW723C86. Thus, episodic spinal 5‐HT2B receptor activation is sufficient to elicit phrenic motor facilitation in vivo by a mechanism that requires spinal NADPH oxidase activity. These findings sugest that 5‐HT2B receptors contribute to serotonin‐dependent phrenic motor facilitation (including AIH‐induced pLTF) by an NADPH oxidase dependent mechanism.

Ejaculatory response induced by a 5-HT 2 receptor agonist m-CPP in rats: Differential roles of 5-HT 2 receptor subtypes

It has been reported that systemic administration of m-CPP (1-[3-chlorophenyl] piperazine hydrochloride), a 5-HT 2 receptor agonist, produces a 5-HT 2C receptor-mediated penile erections and self-grooming in rats. In the present study, we examined the ability of m-CPP to induce ejaculation in rats and determined which 5-HT 2 receptor subtypes may be involved in the m-CPP-induced ejaculation. The ejaculatory response was assessed by weighing the seminal materials accumulated over 30 min. In Experiment 1, systemic administration of m-CPP (0.1–3.0 mg/kg, i.p.) produced a dose-dependent increase in both the incidence of ejaculation and the weight of the seminal materials. The inverted U-shaped dose-response effects of m-CPP on penile erection and genital grooming were also observed, with maximum effects at 0.6 mg/kg. Pretreatment with SB242084 (0.1 and 0.3 mg/kg, i.p.), a selective 5-HT 2C receptor antagonist, dose-dependently attenuated the ejaculatory response induced by m-CPP (3.0 mg/kg). The proejaculatory effect of m-CPP was also attenuated by ketanserin (0.3 and 1.0 mg/kg, i.p.), a 5-HT 2A receptor antagonist, whereas SB204741 (0.1 and 0.3 mg/kg, i.p.), a selective 5-HT 2B receptor antagonist, significantly potentiated the m-CPP-induced ejaculatory response. Penile erection and genital grooming induced by m-CPP (0.3 mg/kg, i.p.) was only blocked by SB242084. In Experiment 2 (termed as corset test), in rats fitted with a corset at the thoracic level to prevent the loss of seminal materials by genital grooming, the proejaculatory effect of m-CPP was more efficiently detected than in the non-fitted animals: the ED 50 value for inducing ejaculation was reduced to less than 50% of the ED 50 in non-fitted animals. In this test, the proejaculatory effect of m-CPP (0.6 mg/kg, i.p.) was completely blocked by SB242084 (0.3 mg/kg, i.p.), whereas ketanserin (0.3 mg/kg, i.p.) or SB204741 (0.3 mg/kg, i.p.) did not affect the m-CPP -induced ejaculation. From these observations, it is suggested that the 5-HT 2 receptor agonist m-CPP at low doses (0.3–1.0 mg/kg) possesses the proejaculatory as well as proerectile effects in rats that are primarily associated with the activation of 5-HT 2C receptors, and that the activation of 5-HT2B receptors may produce an inhibitory effect on ejaculation induced by a high dose (3.0 mg/kg) of m-CPP . Furthermore, the results of the present study also indicate that the corset test employed in this study may be useful for detecting the proejaculatory effect of the compounds.

5‐HT1B receptor‐mediated presynaptic inhibition at the calyx of Held of immature rats

5-hydroxytryptamine (5-HT) inhibits transmitter release via activating GTP-binding proteins, but the target of 5-HT receptors in the nerve terminal is not determined. We addressed this question at the calyx of Held synapse in the brainstem slice of immature rats. Bath-application of 5-HT attenuated the amplitude of nerve-evoked excitatory postsynaptic currents (EPSCs) associated with an increase in the paired-pulse ratio, whereas it had no effect on the amplitude of spontaneous miniature EPSCs. The 5-HT1B receptor agonist CP93129 mimicked the inhibitory effect of 5-HT, but the 5-HT1A agonist (R)-(+)-8-hydroxy-DPAT (8-OHDPAT) had no effect. The 5-HT1B receptor antagonist NAS-181 blocked the inhibitory effect of 5-HT. These results suggest that 5-HT activated 5-HT1B receptors in calyceal nerve terminals, thereby inhibiting transmitter release. In direct whole-cell recordings from calyceal nerve terminals, 5-HT attenuated voltage-dependent Ca2+ currents, but had no effect on voltage-dependent K+ currents. When EPSCs were evoked by presynaptic Ca2+ currents during simultaneous pre- and postsynaptic recordings, the magnitude of the 5-HT-induced inhibition of Ca2+ currents fully explained that of EPSCs. Upon repetitive applications, 5-HT showed tachyphylaxis, with its effect on both EPSCs and presynaptic Ca2+ currents becoming weaker in the second application. 1,2-bis(o-aminophenoxy)ethane-N-N'-N'-N'-tetraacetic acid (BAPTA; 10 mm) loaded into the nerve terminal abolished this tachyphylaxis. The presynaptic inhibitory effect of 5-HT was prominent at postnatal day 5, but became weaker as animals matured. We conclude that activation of 5-HT1B receptors inhibits voltage-gated Ca2+ channels, thereby inhibiting transmitter release at immature calyceal nerve terminals, and that 5-HT1B receptors undergo Ca2+-dependent tachyphylaxis on repetitive activations.

Fluvoxamine, a selective serotonin reuptake inhibitor, and 5-HT2C receptor inactivation induce appetite-suppressing effects in mice via 5-HT1B receptors

Serotonin (5-hydroxytryptamine; 5-HT) 2C receptors and the downstream melanocortin pathway are suggested to mediate the appetite-suppressing effects of 5-HT drugs such as m-chlorophenylpiperazine (mCPP) and fenfluramine. Here, we report that fluvoxamine (3-30 mg/kg), a selective serotonin reuptake inhibitor (SSRI), in the presence of SB 242084 (1-2 mg/kg), a selective 5-HT2C receptor antagonist, exerts appetite-suppressing effects while fluvoxamine or SB 242084 alone has no effect. The appetite-suppressing effects were attenuated in the presence of SB 224289 (5 mg/kg), a selective 5-HT1B receptor antagonist. Moreover, CP 94253 (5-10 mg/kg), a selective 5-HT1B receptor agonist, exerted appetite-suppressing effects and significantly increased hypothalamic pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) gene expression and decreased hypothalamic orexin gene expression. These results suggest that fluvoxamine and inactivation of 5-HT2C receptors exert feeding suppression through activation of 5-HT1B receptors, and that 5-HT1B receptors up-regulate hypothalamic POMC and CART gene expression and down-regulate hypothalamic orexin gene expression in mice.

Sumatriptan provokes coronary artery spasm in patients with variant angina: Possible involvement of serotonin 1B receptor

Serotonin (5HT) can induce coronary artery spasm (CAS) in patients with variant angina (VA). We have previously reported that 5HT(1B) and 5HT(2A) receptors gene were expressed in human coronary arterial smooth muscle cells and that isolated coronary artery from a patient with VA showed the supersensitivity to sumatriptan (SMT), a 5HT(1B/1D) receptor agonist. The aim of the present study was to determine whether SMT can provoke CAS directly or indirectly through platelet aggregation in patients with VA. We evaluated the effects of intracoronary infusion of graded concentrations of SMT on coronary arteries in 9 patients, including 5 documented VA and 4 participants with atypical chest pain as control. SMT provoked CAS in all patients with VA. SMT could not induce CAS in control. SMT (10(-4) M) caused significant contractions (%diameter of baseline; median [interquartile range], 0 [0-18.4]% in VA, as compared with control (proximal segments; 92.6 [77.9-118.9]%, p<0.05 vs. VA, distal segments; 92.9 [65.3-158.5]%, p<0.01 vs. VA). In control, minor dilation occurred at SMT concentration up to 10(-5) M. SMT could induce in vitro platelet aggregation neither in healthy subjects nor in patients with VA. These findings suggest that activation of 5HT(1B) receptor by SMT can induce CAS directly in patients with VA without platelet activation. This is the first report directly demonstrating the effect of 5HT(1B) receptor activation on human coronary arteries in vivo.

P.1.d.018 Effect of dopamine depletion and 5-HT1B receptors blockade on the behavioural effect of various antidepressant in the mice FST

The importance of calcitonin gene-related peptide (CGRP) in the regulation of vascular tone has been widely documented. Indeed, stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses, which are mediated by CGRP release. These vasodepressor responses are inhibited by clonidine via prejunctional α2A/2C-adrenoceptors, but no study has yet reported the role of prejunctional 5-hydroxytryptamine (5-HT) receptors in this experimental model. Since activation of prejunctional 5-HT1 receptors results in inhibition of neurotransmitter release, this study sets out to investigate as an initial approach the role of 5-HT1B receptors in the inhibition of the vasodepressor sensory outflow in pithed rats. Male Wistar pithed rats were pretreated with hexamethonium (2 mg/kg.min) followed by i.v. continuous infusions of methoxamine (20 µg/kg.min), and then by saline (0.02 ml/min) or CP-93,129 (a rodent 5-HT1B receptor agonist; 0.1, 1 and 10 µg/kg.min). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T9-T12) resulted in frequency-dependent decreases in diastolic blood pressure. The infusions of CP-93,129, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous α-CGRP (0.1, 0.18, 0.31, 0.56 and 1 μg/kg). This inhibition by CP-93,129 was abolished by the antagonists GR127935 (5-HT1B/1D) or SB224289 (5-HT1B), but not by BRL15572 (5-HT1D). The above results suggest that CP-93,129-induced inhibition of the vasodepressor (perivascular) sensory outflow in pithed rats is mainly mediated by activation of prejunctional 5-HT1B receptors.

Triptans Induce Vasoconstriction of Human Arteries and Veins from the Thoracic Wall

A common side effect of migraine treatment with triptans is chest symptoms. The origin of these symptoms is not known. The aim of the present study was to examine the vasocontractile effect of triptans in human arteries and veins from the thoracic wall and in coronary artery bypass grafts. In vitro pharmacology experiments showed that the 5-hydroxytryptamine (5-HT) type 1B and 1D receptor agonists, eletriptan, naratriptan, rizatriptan, sumatriptan, and zolmitriptan, induced vasoconstriction in the thoracic blood vessels from 38% to 57% of the patients. 5-carboxamidotryptamine (5-CT) and sumatriptan elicited a vasoconstriction that was antagonized by the 5-HT1B receptor antagonist SB224289, whereas the 5-HT1D receptor antagonist BRL115572 had no effect. 5-HT induced a contraction that was inhibited by the 5-HT2A receptor antagonist ketanserin. 5-HT2A, 5-HT1B, and 5-HT1D receptor mRNA levels were detected by real-time PCR in all blood vessels studied. In conclusion, triptans induce vasoconstriction in arteries and veins from the thoracic wall, most likely by activation of 5-HT1B receptors. This response could be observed in only 38% to 57% of the patients, which may provide an explanation for why a similar number of patients experience chest symptoms as a side effect of migraine treatment with triptans.

In vivo effects of local activation and blockade of 5-HT1B receptors on globus pallidus neuronal spiking

Several morphological works have shown that the globus pallidus (GP) contains the highest density of 5-HT1B receptors within the telencephalon. However, the role of these receptors in the spiking of GP neurons in vivo is unknown. In the present work, we use single-unit extracellular recordings in the anesthetized rat to analyze changes in the firing rate of GP neurons evoked by local activation and blockade of 5-HT1B receptors. Intrapallidal administration of serotonin, or the serotonin uptake inhibitor fluoxetine, predominantly produced an excitatory effect in the basal firing rate of GP neurons. The 5-HT1B receptor agonist, L-694,247, caused a dose-dependent excitatory effect on most pallidal neurons tested. Blockade of 5-HT1B receptors by intrapallidal application of methiothepin predominantly caused inhibition in GP neurons firing rate. Moreover, methiothepin diminished the excitatory effect evoked by L-694,247. Furthermore, local serotonin did not evoke significant changes in the basal firing rate of GP neurons in unilateral striatal lesioned rats. Taken all together, these results suggest that serotonin 5-HT1B receptors significantly contribute to the control of spiking of the rat GP neurons, and that the 5-HT1B receptors exerting this control are most likely localized in the striato-pallidal pathway.

Activation of the JAK/STAT pathway in vascular smooth muscle by serotonin

Serotonin (5-hydroxytryptamine, 5-HT) is a vasoconstrictor and mitogen whose levels are elevated in diabetes. Previous studies have shown the presence of 5-HT2A, 5-HT2B, and 5-HT1B receptors in vascular smooth muscle cells (VSMCs). There are currently no data regarding 5-HT2B and 5-HT1B receptor activation of the JAK/STAT pathway in VSMCs and resultant potential alterations in 5-HT signaling in diabetes. Therefore, we tested the hypothesis that 5-HT differentially activates the JAK/STAT pathway in VSMCs under conditions of normal (5 mM) and high (25 mM) glucose. Treatment of rat VSMCs with 5-HT (10(-6) M) resulted in time-dependent activation ( approximately 2-fold) of JAK2, JAK1, and STAT1, but not STAT3 (maximal at 5 min, returned to baseline by 30 min). The 5-HT2B receptor agonist BW723C86 and the 5-HT1B receptor agonist CGS12066A (10(-9)-10(-5) M, 5-min stimulation) did not activate the JAK/STAT pathway. Treatment with the 5-HT2A receptor antagonist ketanserin (10 nM) inhibited JAK2 activation by 5-HT. Treatment of streptozotocin-induced diabetic rats with ketanserin (5 mg.kg-1.day-1) reduced activation of JAK2 and STAT1 but not STAT3 in endothelium-denuded thoracic aorta in vivo. 5-HT (10(-6) M) treatment resulted in increased cell proliferation and increased DNA synthesis, which were inhibited by the JAK2 inhibitor AG490. Further studies with apocynin, diphenyleneiodonium chloride, catalase, and virally transfected superoxide dismutase had no effect at either glucose concentration on activation of the JAK/STAT pathway by 5-HT. Therefore, we conclude that 5-HT activates JAK2, JAK1, and STAT1 via the 5-HT2A receptors in a reactive oxygen species-independent manner under both normal and high glucose conditions.

Selective blockade of serotonin 5-HT2A receptor increases coronary blood flow via augmented cardiac nitric oxide release through 5-HT1B receptor in hypoperfused canine hearts

Serotonin (5-hydroxytryptamine [5-HT]), which induces vasoconstriction via 5-HT2A receptors in smooth muscle cells and vasodilation through activating nitric oxide (NO) synthase (NOS) via 5-HT1B receptors in endothelial cells, possesses divergent effects on regulating vascular tone. These facts lead us to consider that sarpogrelate, a 5-HT2A receptor blocker, may increase coronary blood flow (CBF) via either attenuation of vasoconstriction through 5-HT2A receptor blockade or augmentation of vasodilation by relative stimulation of NOS through 5-HT1B receptor and we tested this hypothesis in ischemic canine hearts. In open chest dogs, coronary perfusion pressure was reduced so that CBF was decreased to 33% of the baseline and kept constant. Thereafter, sarpogrelate was infused selectively into the left anterior descending artery with and without either an inhibitor of NOS (NG-nitro- l-arginine methyl ester ( l-NAME)) or a 5-HT1B receptor antagonist (GR55562). An intracoronary administration of sarpogrelate increased CBF (34.0 ± 4.0 to 44.5 ± 4.4 ml/100 g/min, P < 0.05), along with the cardiac NOx release (3.2 ± 0.6 to 6.8 ± 1.2 nmol/ml, P < 0.05). The increases in both CBF and NOx by sarpogrelate were completely blunted by the co-administration of either l-NAME or GR55562. Interestingly, sarpogrelate increased the cardiac serotonin release (–4.8 ± 3.2 vs. 22.1 ± 1.5 ng/ml, P < 0.05, respectively) in the hypoperfused heart. Immunohistochemical analysis showed that sarpogrelate induced serotonin production in ischemic cardiac myocytes. These results suggest that sarpogrelate increases CBF via augmented cardiac NO production through 5-HT1B receptor activation along with the blockade of 5-HT2A receptors. The increase in cardiac release of serotonin may increase NO production in the ischemic heart.

Differential regulation of chondrogenic differentiation by the serotonin2B receptor and retinoic acid in the embryonic mouse hindlimb.

Retinoic acid (RA) synthesizing and metabolizing enzymes are coordinately expressed with serotonin 2B (5-HT2B) receptors at sites of epithelial-mesenchymal (E-M) interaction in the mouse embryo (Bhasin et al., 1999). The promoter of the 5-HT2B receptor contains potential RA response element (RAREs) as well as an AP-2 site. Because both retinoid and serotonergic signaling have been implicated in the regulation of chondrogenic differentiation, the present study investigated whether these signals may work together to regulate this morphogenetic process in hindlimb bud micromass cultures. Results indicate that 5-HT promotes [35S]sulfate incorporation (chondrogenic differentiation) by activation of 5-HT2B receptors, which use the mitogen activated protein kinase (p42 MAPK) signal transduction pathway, whereas RA dose-dependently inhibits sulfate incorporation and promotes expression of RARbeta, which could lead to inhibition of p38 MAPK. No evidence was found to support the possibility that RA negatively regulates expression of 5-HT2B receptors. Taken together, these results suggest that 5-HT and RA may act as opposing signals to regulate chondrogenic differentiation in the developing hindlimb, possibly mediated by different MAPK signal transduction pathways.

Local perfusion of mCPP into ventromedial hypothalamic nucleus, but not into lateral hypothalamic area and frontal cortex, inhibits food intake in rats

The serotonergic (5-hydroxytryptamine, 5-HT) system is extensively implicated in feeding behavior. In recent years, 5-HT receptors have been classified into 14 subtypes, and activation of 5-HT1B and 5-HT2C receptors inhibits food intake in rats. However, the precise functions in local brain areas of these receptor subtypes are unclear. Frontal cortex (FC), lateral hypothalamic area (LH), or ventromedial hypothalamic nucleus (VMH) are involved in control of feeding behavior. We investigated the effects of 5-HT1B and 5-HT2C receptor stimulations in the three local brain areas on feeding behavior and on 5-HT metabolism. We perfused mCPP, 5-HT(1B/2C) agonist, at multiple doses via a microdialysis probe into the three local brain areas and observed food intake. Extracellular concentrations of 5-HT and 5-HIAA were measured simultaneously. Perfusion of 1 mM mCPP into VMH, but not into LH nor FC at any dose, induced significant reduction of food intake compared with control. The extracellular concentrations of 5-HT were markedly increased in all three areas, but the concentrations of 5-HIAA were not changed by mCPP perfusions. These results indicate that the effects of 5-HT1B or 5-HT2C receptor activation on feeding behaviors depended on the brain regions, and that 5-HT1B or 5-HT2C receptors in VMH, but not in FC or in LH, play important roles in the regulation of food intake. The results also suggested that mCPP acts not only as a 5-HT(1B/2C) agonist, but also as a 5-HT releaser or as a re-uptake inhibitor. Further studies using antagonists should be conducted.

The modulation by 5-HT of glutamatergic inputs from the raphe pallidus to rat hypoglossal motoneurones, in vitro.

Decreases in the activity of 5-HT-containing caudal raphe neurones during sleep are thought to be partially responsible for the resultant disfacilitation of hypoglossal motoneurones. Whilst 5-HT has a direct excitatory action on hypoglossal motoneurones as a result of activation of 5-HT2 receptors, microinjection of 5-HT2 antagonists into the hypoglossal nucleus reduces motor activity to a much lesser extent compared to the suppression observed during sleep suggesting other transmitters co-localised in caudal raphe neurones may also be involved. The aim of the present study was therefore to characterise raphe pallidus inputs to hypoglossal motoneurones. Whole cell recordings were made from hypoglossal motoneurones in vitro. 5-HT evoked a direct membrane depolarisation (8.45 +/- 3.8 mV, P < 0.001) and increase in cell input resistance (53 +/- 40 %, P < 0.001) which was blocked by the 5-HT2 antagonist, ritanserin (2.40 +/- 2.7 vs. 7.04 +/- 4.6 mV). Stimulation within the raphe pallidus evoked a monosynaptic EPSC that was significantly reduced by the AMPA/kainate antagonist, NBQX (22.8 +/- 16 % of control, P < 0.001). In contrast, the 5-HT2 antagonist, ritanserin, had no effect on the amplitude of these EPSCs (106 +/- 31 % of control, P = n.s.). 5-HT reduced these EPSCs to 50.0 +/- 13 % of control (P < 0.001), as did the 5-HT1A agonist, 8-OH-DPAT (52.5 +/- 17 %, P < 0.001) and the 5-HT1B agonist, CP 93129 (40.6 +/- 29 %, P < 0.01). 8-OH-DPAT and CP 93129 increased the paired pulse ratio (1.38 +/- 0.27 to 1.91 +/- 0.54, P < 0.05 & 1.27 +/- 0.08 to 1.44 +/- 0.13, P < 0.01 respectively) but had no effect on the postsynaptic glutamate response (99 +/- 4.4 % and 100 +/- 2.5 %, P = n.s.). They also increased the frequency (P < 0.001), but not the amplitude, of miniature glutamatergic EPSCs in hypoglossal motoneurones. These data demonstrate that raphe pallidus inputs to hypoglossal motoneurones are predominantly glutamatergic in nature, with 5-HT decreasing the release of glutamate from these projections as a result of activation of 5-HT1A and/or 5-HT1B receptors located on presynaptic terminals.

Pharmacological profile of YM348, a novel, potent and orally active 5-HT2C receptor agonist

YM348, (S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine, showed a high affinity for cloned human 5-HT2C receptors (Ki: 0.89 nM). The functional selectivity for 5-HT2C receptors in the 5-HT2 receptor family was the highest among 5-HT2C receptor agonists, including m-chlorophenylpiperazine (mCPP) and Ro60-0175 ((S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine). Oral administration of YM348 induced penile erections and hypolocomotion in rats, being completely inhibited by a selective 5-HT2C receptor antagonist, SB242084 (6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy) pyridin-3-yl carbamoyl] indoline). The dose–response curve for penile erections, unlike that for hypolocomotion, was an inverted U-shape in the dose range of 0.0677–2.03 mg/kg. A selective 5-HT2A receptor antagonist, MDL100907 (R(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol), and a selective 5-HT2B receptor antagonist, RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine), had no effect on the decline in penile erection frequency at 2.03 mg/kg of YM348. YM348 did not affect blood pressure at 2.03 mg/kg. In conclusion, YM348 is a novel, potent and orally active 5-HT2C receptor agonist, and neither the activation of 5-HT2A or 5-HT2B receptors nor a cardiovascular effect is likely to contribute to the inverted U-shape dose–response curve for penile erections.