Activation Of 5-HT2 Receptors Research Articles

The platelet of the patients with ischemic cardiopathy and cardiac valve disease showed a reduction of 8OH-DPAT binding sites

Depression is prospectively associated with increased risk of coronary artery disease in individuals initially free of clinical cardiovascular disease probably by an increased platelet activity. The serotonergic receptors mainly implied in depression are 5-HT1A and 5-HT2 receptors. Activation of 5HT2 receptor induces platelet aggregation. Drugs with 5-HT1A receptor agonist and 5-HT2A receptor antagonist effects reduced the receptor-mediated platelet aggregation. There are only indirect data about 5-HT1A receptors presence in platelet membranes, thus our aims were to study the characteristics of the platelet membranes 5-HT1A binding sites of both healthy volunteers and patients with cardiac valve disease and ischemic cardiopathy. The bound of the 5-HT1A selective agonist 3H-8OH-DPAT to the platelet membranes 5-HT1A binding sites of patients with cardiac valve disease and ischemic cardiopathy were compared with a control group of healthy voluntaries using radioligand binding methods. The patients with cardiovascular disease showed a reduction (-50.40%) (p<0.01) of the 3H-8OH-DPAT bound to the platelet membranes 5-HT1A receptors (1.652+/-0.79 fmol/mg protein) with respect to the control group (3.331+/-0.16 fmol/mg protein). 3H-8OH-DPAT binding to human platelet membranes is saturable, of high affinity, and seems selective for 5-HT1A receptors, and similar to that described in animal brain and in other human cells. Patients with ischemic cardiopathy and cardiac valve disease showed a reduction of the 8OH-DPAT bound to the platelet membranes. Taken together, these findings suggest that the 8OH-DPAT bound to the human platelet membranes is modulated by modifications produced by cardiovascular disease conditions.

MDMA Induces EPSP–Spike Potentiation in Rat Ventral Hippocampus In Vitro Via Serotonin and Noradrenaline Release and Coactivation of 5-HT4 and β1 Receptors

It is well documented that N-methyl-3,4-methylenedioxyamphetamine (MDMA, ecstasy) releases brain serotonin (5-HT; 5-hydroxytryptamine), noradrenaline (NE; norepinephrine), and dopamine, but the consequent effect on brain functioning remains elusive. In this study, we characterized the effects of MDMA on electrically evoked responses in the ventral CA1 region of a rat hippocampal slice preparation. Superfusion with MDMA (10 microM, 30 min) increased the population spike amplitude (PSA) by 48.9+/-31.2% and decreased population spike latency (PSL) by 103+/-139 mus (both: mean+/-SD, n=123; p<0.0001, Wilcoxon test), without affecting field excitatory postsynaptic potential (fEPSP). This effect persisted for at least 1 h after MDMA washout; we have called this EPSP-spike potentiation (ESP) by MDMA, ESP MDMA. Antagonism of GABAergic transmission did not prevent ESP MDMA, suggesting that an increase in excitability of pyramidal cells underlies this MDMA action. Block of serotonin transporter (SERT) with citalopram or 5-HT depletion with (+/-)-p-chlorophenylalanine pretreatment partially inhibited the ESP MDMA. Block of both SERT and NE transporter prevented ESP MDMA, indicating its dependence on release of both 5-HT and NE. ESP MDMA is produced by simultaneous activation of 5-HT4 and beta1 receptors, with a predominant role of 5-HT4 receptors. Block of both 5-HT4 and beta1 receptors revealed an inhibitory component of the MDMA action mediated by 5-HT1A receptor. The concentration range of MDMA which produced ESP MDMA (1-30 microM) corresponds to that commonly reached in human plasma following the ingestion of psychoactive MDMA doses, suggesting that release of both 5-HT and NE, and consequent ESP MDMA may underlie some of the psychoactive effects of MDMA in humans.

Regulation of apoptosis signal‐regulating kinase 1 degradation by Gα13

Apoptosis signal-regulating kinase (ASK1) is a mitogen-activated protein kinase (MAPK) that transduces apoptotic signals from a variety of stresses. We have shown previously that alpha subunits of heterotrimeric G12 and G13 proteins stimulate ASK1 kinase activity and ASK1-dependent apoptosis. Here, we report a novel mechanism of G-protein-dependent regulation of ASK1. We demonstrated that G alpha13 forms a complex with ASK1 in an activation-independent manner. Both N- and C-terminal regulatory domains of ASK1 were essential for the efficient interaction, while its kinase domain was not required. Formation of the G alpha13-ASK1 complex was enhanced by JNK-interacting leucine zipper protein, JLP. Constitutively activated G alpha13Q226L increased ASK1 expression. Short-term activation of a serotonin 5-HT4 receptor that is coupled to G alpha13 also increased ASK1 expression. Importantly, prolonged activation of 5-HT4 receptor in COS-7 cells or prolonged treatment of human umbilical vein endothelial cells with thrombin concomitantly down-regulated both G alpha13 and ASK1. Data showed that G alpha13Q226L reduced the rate of ASK1 degradation, decreased ASK1 ubiquitination, and reduced association of ASK1 with an E3 ubiquitin ligase CHIP, previously shown to mediate ASK1 degradation. Our findings indicate that ASK1 expression levels can be regulated by G alpha13, at least in part via control of ASK1 ubiquitination and degradation.

Dopamine Induces a Slow Afterdepolarization in Lateral Amygdala Neurons

The amygdala and dopaminergic innervation thereunto are considered to cooperatively regulate emotional states and behaviors. The present experiments examined effects of dopamine on lateral amygdala (LA) neuron excitability by whole cell recordings. Bath application of dopamine induced slow afterdepolarization (sADP). This sADP lasted for >5 s, and its magnitude varied in a concentration-dependent manner. Co-application of the D1 receptor antagonist SKF83566 reduced its amplitude. The D1 receptor agonist SKF38393, applied alone, induced sADP of a smaller amplitude. Induction of the full sADP required 5-HT(2) and noradrenalin alpha(1) receptor activation as well. D2 receptor activation or blockade did not affect sADP induction. The calcium channel blocker cadmium or intracellular calcium chelator bis-(o-aminophenoxy)-N,N,N',N' tetraacetic acid (BAPTA) blocked induction of the sADP, which was suggested to be triggered by calcium influx. Under voltage clamp, membrane conductance decreased at the peak of sADP current (I(sADP)). I(sADP) was suppressed by cesium included in pipettes. The I-V curve of the net I(sADP) was shifted as the external concentration of potassium was raised, and the reversal potential was identical to that of potassium, suggesting that dopamine decreases potassium conductance to induce the sADP. The present sADP may serve as a positive-feedback regulator of excitability in LA neurons.

Compensatory airway dilation and additive ventilatory augmentation mediated by dorsomedial medullary 5-hydroxytryptamine 2 receptor activity and hypercapnia

5-HT2 receptor activity in the hypoglossal nucleus and hypercapnia is associated with airway dilation. 5-HT neurons in the medullary raphe and hypercapnia are responsible for tidal volume change. In this study, the effects of 5-HT2 receptors in the dorsomedial medulla oblongata (DMM), which receives projections from the medullary raphe, and hypercapnia on airway resistance and respiratory variables were studied in mice while monitoring 5-HT release in the DMM. A microdialysis probe was inserted into the DMM of anesthetized adult mice. Each mouse was placed in a double-chamber plethysmograph. After recovery from anesthesia, the mice were exposed to stepwise increases in CO(2) inhalation (5%, 7%, and 9% CO(2) in O(2)) at 8-min intervals with a selective serotonin reuptake inhibitor, fluoxetine, or fluoxetine plus a 5-HT2 receptor antagonist, LY-53857 in the DMM. In response to fluoxetine plus LY-53857 coperfusion, specific airway resistance was increased, and tidal volume and minute ventilation were decreased. CO(2) inhalation with fluoxetine plus LY-53857 coperfusion in the DMM largely decreased airway resistance and additively increased minute ventilation. Thus, 5-HT2 receptor activity in the DMM increases basal levels of airway dilation and ventilatory volume, dependent on central inspiratory activity and the volume threshold of the inspiratory off-switch mechanism. Hypercapnia with low 5-HT2 receptor activity in the DMM largely recovers airway dilation and additively increases ventilatory volume. Interaction between 5-HT2 receptor activity in the DMM and CO(2) drive may elicit a cycle of hyperventilation with airway dilation and hypoventilation with airway narrowing.

Intestinal nutrients elicit satiation through concomitant activation of CCK1 and 5-HT3 receptors

Previous studies demonstrate that cholecystokinin type-1 (CCK1) and serotonin type-3 (5-HT3) dependent pathways are independently involved in intestinal nutrient-induced meal termination. In the current study, we employed selective antagonists to investigate the relative contribution of CCK1 and 5-HT3 receptors in mediating the anorexia produced by duodenal infusion of Polycose or Intralipid in rats. Combined administration of 1 mg/kg ondansetron (Ond) and 1 mg/kg devazepide (Dev) reversed 132 mM Polycose-induced suppression to the level of control intake and significantly attenuated 263 mM Polycose-induced suppression greater than either antagonist alone. Similar results were observed when subthreshold doses of Ond (500 μg/kg) and Dev (5 μg/kg) were co-administered prior to 263 mM Polycose infusion. Suppression of intake resulting from 130 mM Intralipid was reversed to the level of control when Ond and Dev were co-administered at both independent effective doses (1 mg/kg each) and subthreshold doses (500 μg/kg and 5 μg/kg, respectively). Finally, combined administration of the antagonists increased sucrose intakes beyond intakes following control or treatment with either antagonist alone when rats were infused with saline. These data demonstrate that intestinal carbohydrates and lipids inhibit food intake through simultaneous CCK1 and 5-HT3 receptor activation and that these receptors appear to completely mediate the Intralipid-induced suppression of intake.

Episodic Stimulation of α1-Adrenoreceptors Induces Protein Kinase C-Dependent Persistent Changes in Motoneuronal Excitability

In vitro long-term facilitation (ivLTF) is a novel form of activity-independent postsynaptic enhancement of AMPA receptor function in hypoglossal (XII) motoneurons that can be induced by intermittent activation of 5-HT2 receptors. In vivo respiratory long-term facilitation (LTF) is characterized by a persistent 5-HT2 receptor-dependent increase in respiratory motor output or ventilation after episodic exposures to hypoxia in adult rats. Here, we demonstrate that ivLTF can also be induced by episodic but not continuous stimulation of alpha1-adrenergic receptors that requires protein kinase C (PKC), but not PKA (protein kinase A), activation. Additionally, we show that in vivo respiratory LTF is also alpha1-adrenergic receptor dependent. We suggest that, in vivo, concurrent episodic activation of 5-HT2 and alpha1-adrenergic receptors is necessary to produce long-lasting changes in the excitability of respiratory motoneurons, possibly involving PKC activation via the G alpha(q)-PLC (phospholipase C) signaling pathway common to both receptor subtypes. Such plasticity of XII motor output may increase upper airway muscle (innervated by XII nerve) tone and improve the likelihood that airway patency will be maintained. Elucidating the mechanism underlying LTF can be of clinical importance to the patients suffering from sleep-disordered breathing.

Dynamics of Protein Kinase A Signaling at the Membrane, in the Cytosol, and in the Nucleus of Neurons in Mouse Brain Slices

The cAMP-dependent protein kinase A (PKA) plays a ubiquitous role in the regulation of neuronal activity, but the dynamics of its activation have been difficult to investigate. We used the genetically encoded fluorescent probe AKAR2 to record PKA activation in the cytosol and the nucleus of neurons in mouse brain slice preparations, whereas the potassium current underlying the slow afterhyperpolarization potential (sAHP) in thalamic intralaminar neurons was used to monitor PKA activation at the membrane. Adenylyl cyclase was stimulated either directly using forskolin or via activation of 5-HT7 receptors. Both stimulations produced a maximal effect on sAHP, whereas in the cytosol, the amplitude of the 5-HT7 receptor-mediated response was half of that after direct adenylyl cyclase stimulation with forskolin. 5-HT7-mediated PKA responses were obtained in 30 s at the membrane, in 2.5 min in the cytosol, and in 13 min in the nucleus. Our results show in morphologically intact mammalian neurons the potential physiological relevance of PKA signal integration at the subcellular level: neuromodulators produce fast and powerful effects on membrane excitability, consistent with a highly efficient functional coupling between adenylyl cyclases, PKA, and target channels. Phosphorylation in the cytosol is slower and of graded amplitude, showing a differential integration of the PKA signal between the membrane and the cytosol. The nucleus integrates these cytosolic signals over periods of tens of minutes, consistent with passive diffusion of the free catalytic subunit of PKA into the nucleus, eventually resulting in a graded modulation of gene expression.

Behavioral and neuropharmacological evidence that serotonin crosses the blood-brain barrier in Coturnix japonica (Galliformes; Aves)

This study was carried out aiming to reach behavioral and neuropharmacological evidence of the permeability of the blood-brain barrier (BBB) to serotonin systemically administered in quails. Serotonin injected by a parenteral route (250-1000 microg x kg(-1), sc) elicited a sequence of behavioral events concerned with a sleeping-like state. Sleeping-like behaviors began with feather bristling, rapid oral movements, blinking and finally crouching and closure of the eyes. Previous administration of 5-HT2C antagonist, LY53857 (3 mg x kg(-1), sc) reduced the episodes of feather bristling and rapid oral movements significantly but without altering the frequency of blinking and closure of the eyes. Treatment with the 5-HT2A/2C antagonist, ketanserin (3 mg x kg(-1), sc) did not affect any of the responses evoked by the serotonin. Quipazine (5 mg x kg(-1), sc) a 5-HT2A/2C/3 agonist induced intense hypomotility, long periods of yawning-like and sleeping-like states. Previous ketanserin suppressed gaping responses and reduced hypomotility, rapid oral movements and bristling but was ineffective for remaining responses induced by quipazine. Results showed that unlike mammals, serotonin permeates the BBB and activates hypnogenic mechanisms in quails. Studies using serotoninergic agonist and antagonists have disclosed that among the actions of the serotonin, feather bristling, rapid oral movements and yawning-like state originated from activation of 5-HT2 receptors while blinking and closure of the eyes possibly require other subtypes of receptors.

Transglutaminase-dependent RhoA Activation and Depletion by Serotonin in Vascular Smooth Muscle Cells

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.

Endogenous Serotonin Excites Striatal Cholinergic Interneurons via the Activation of 5-HT 2C, 5-HT6, and 5-HT7 Serotonin Receptors: Implications for Extrapyramidal Side Effects of Serotonin Reuptake Inhibitors

The striatum is richly innervated by serotonergic afferents from the raphe nucleus. We explored the effects of this input on striatal cholinergic interneurons from rat brain slices, by means of both conventional intracellular and whole-cell patch-clamp recordings. Bath-applied serotonin (5-HT, 3-300 microM), induced a dose-dependent membrane depolarization and increased the rate of spiking. This effect was mimicked by the 5-HT reuptake blockers citalopram and fluvoxamine. In voltage-clamped neurons, 5-HT induced an inward current, whose reversal potential was close to the K(+) equilibrium potential. Accordingly, the involvement of K(+) channels was confirmed either by increasing extracellular K(+) concentration and by blockade of K(+) channels with barium. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) profiling demonstrated the presence of 5-HT2C, 5-HT6, and 5-HT7 receptor mRNAs in identified cholinergic interneurons. The depolarization/inward current induced by 5-HT was partially mimicked by the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine and antagonized by both ketanserin and the selective 5-HT2C antagonist RS102221, whereas the selective 5-HT3 and 5-HT4 receptor antagonists tropisetron and RS23597-190 had no effect. The depolarizing response to 5-HT was also reduced by the selective 5-HT6 and 5-HT7 receptor antagonists SB258585 and SB269970, respectively, and mimicked by the 5-HT7 agonist, 5-CT. Accordingly, activation of either 5-HT6 or 5-HT7 receptor induced an inward current. The 5-HT response was attenuated by U73122, blocker of phospholipase C, and by SQ22,536, an inhibitor of adenylyl cyclase. These results suggest that 5-HT released by serotonergic fibers originating in the raphe nuclei has a potent excitatory effect on striatal cholinergic interneurons.

Serotonin Modulates Pallidal Neuronal Activity in the Awake Monkey

Serotonin (5-HT)-containing neurons in the dorsal raphe project to the external and internal segments of the pallidum, which express several 5-HT receptors. Although the involvement of 5-HT in basal ganglia movement control has been suggested, little is known about the physiological action of 5-HT in the pallidum. Previous anatomical studies and in vitro physiological studies in other brain areas have suggested the following possibilities: (1) 5-HT suppresses GABAergic inhibition through presynaptic 5-HT1B receptors; (2) 5-HT decreases the firing of pallidal neurons through postsynaptic 5-HT1A receptors; and (3) 5-HT postsynaptically excites pallidal neurons through activation of 5-HT2C, 5-HT4, or 5-HT7 receptors. To test these possibilities, we examined the effects of locally applied agonists and antagonists of 5-HT on spontaneous neuronal firing and on excitatory and inhibitory responses of pallidal neurons to electrical stimulation of the motor cortex in awake monkeys. Although in vivo experiments could not conclusively determine the receptor types or the active sites involved in the observed effects, the results suggested the following possibilities: (1) 5-HT strongly suppresses GABAergic inhibition probably through 5-HT1B receptors; (2) in the external pallidal segment, the suppression may involve additional receptors or mechanisms; and (3) 5-HT suppresses glutamatergic excitation probably through 5-HT1A (and not 5-HT1B) receptors. The present study did not isolate or identify the existence of strong, direct postsynaptic inhibitory or excitatory effects of 5-HT. Thus, present results imply that 5-HT modulates synaptic inputs of both pallidal segments and exerts a significant role in movement control.

Serotonin-type 3 receptors mediate intestinal lipid-induced satiation and Fos-like immunoreactivity in the dorsal hindbrain

Several gastrointestinal stimuli, including some intestinal nutrients, have been shown to exert their satiating effect via activation of serotonin type-3 (5-HT(3)) receptors. The presence of lipids in the small intestine potently suppresses food intake; however, whether 5-HT(3) receptors play a role in this response has not been directly examined. Therefore, using the selective 5-HT(3) receptor antagonist ondansetron, we tested the hypothesis that duodenal infusion of lipid suppresses intake of both sucrose solution and chow through 5-HT(3) receptor activation. Rats duodenally infused with 72 and 130 mM Intralipid suppressed 1-h 15% sucrose intake by 33 and 67%, respectively. Suppression of sucrose intake by 72 mM Intralipid was significantly attenuated by ondansetron at all doses tested (0.5, 1.0, 2.0, and 5.0 mg/kg ip), whereas the lowest effective dose of ondansetron to attenuate suppression of intake by 130 mM Intralipid was 1.0 mg/kg. Furthermore, infusion of 130 mM Intralipid suppressed 1- and 4-h chow intake by 35 and 20%, respectively. Ondansetron administered as low as 0.5 mg/kg significantly attenuated 1-h Intralipid-induced suppression of chow intake and completely reversed the suppression by 4 h. Administration of ondansetron alone did not alter sucrose or chow intake compared with vehicle injection at any time. Finally, to test whether Intralipid-induced neuronal activation of the dorsal vagal complex is mediated by 5-HT(3) receptors, Fos-like immunoreactivity (Fos-LI) was quantified in ondansetron-pretreated rats following intestinal lipid infusion. Ondansetron (1 mg/kg) significantly attenuated duodenal intralipid-induced Fos-LI in the dorsal hindbrain. These data support the hypothesis that 5-HT(3) receptors mediate both satiation, as well as hindbrain neuronal responses evoked by intestinal lipids.

Electrophysiological and arrhythmogenic effects of 5-hydroxytryptamine on human atrial cells are reduced in atrial fibrillation

5-Hydroxytryptamine (5-HT) is proarrhythmic in atrial cells from patients in sinus rhythm (SR) via activation of 5-HT4 receptors, but its effects in atrial cells from patients with atrial fibrillation (AF) are unknown. The whole-cell perforated patch-clamp technique was used to record L-type Ca2+ current (ICaL), action potential duration (APD) and arrhythmic activity at 37°C in enzymatically isolated atrial cells obtained from patients undergoing cardiac surgery, in SR or with chronic AF. In the AF group, 5-HT (10μM) produced an increase in ICaL of 115±21% above control (n=10 cells, 6 patients) that was significantly smaller than that in the SR group (232±33%; p<0.05; n=27 cells, 12 patients). Subsequent co-application of isoproterenol (1μM) caused a further increase in ICaL in the AF group (by 256±94%) that was greater than that in the SR group (22±6%; p<0.05). The APD at 50% repolarisation (APD50) was prolonged by 14±3ms by 5-HT in the AF group (n=37 cells, 14 patients). This was less than that in the SR group (27±4ms; p<0.05; n=58 cells, 24 patients). Arrhythmic activity in response to 5-HT was observed in 22% of cells in the SR group, but none was observed in the AF group (p<0.05). Atrial fibrillation was associated with reduced effects of 5-HT, but not of isoproterenol, on ICaL in human atrial cells. This reduced effect on ICaL was associated with a reduced APD50 and arrhythmic activity with 5-HT. Thus, the potentially arrhythmogenic influence of 5-HT may be suppressed in AF-remodelled human atrium.

Activation of 5-HT4 receptors inhibits secretion of β-amyloid peptides and increases neuronal survival

Activation of 5-HT4 receptors has been shown to improve memory processes in preclinical cognition models, suggesting potential utility of 5-HT4 agonists for the symptomatic treatment of Alzheimer's disease (AD). Recent studies have shown that 5-HT4 agonists also increase the secretion of the non-amyloidogenic soluble amyloid precursor protein-alpha (sAPPα). In the present study, we demonstrated that a selective 5-HT4 partial agonist, RS67333, inhibited the generation of β-amyloid peptide (Aβ) in primary cortical cultures of Tg2576 transgenic mice expressing human APP K670N/M671L. Furthermore, treatments with RS67333 selectively increased the survival of transgenic neurons in a dose-dependent manner, which was inhibited by 5-HT4 antagonists. These and previous data collectively suggest that the 5-HT4 receptor may be an effective therapeutic target for AD, providing both symptomatic improvements and neuroprotection.

The peristaltic reflex induced by short-chain fatty acids is mediated by sequential release of 5-HT and neuronal CGRP but not BDNF

Short-chain fatty acids (SCFAs) accelerate colonic transit. This study examined whether this action was mediated by activation of the peristaltic reflex. SCFAs (acetate, butyrate, or propionate) were applied to the central compartment of a three-compartment flat-sheet preparation of the rat middle to distal colon. The release of serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and CGRP was measured in all three compartments. Ascending contraction and descending relaxation were measured in the orad and caudad compartments. The addition of SCFAs at physiological to supraphysiological concentrations (0.5-100 mM) to the central compartment elicited concentration-dependent ascending contraction and descending relaxation (EC50 approximately 5 mM). At this concentration, SCFAs induced an 8- to 11-fold increase in 5-HT release and a 2- to 3-fold increase in CGRP release in the central compartment only. They had no effect on BDNF release. CGRP release was inhibited by a 5-HT4 but not a 5-HT3 receptor antagonist. Ascending contraction and descending relaxation were also inhibited by 5-HT4 and by CGRP receptor antagonists added to the central compartment. 5-HT and CGRP release, as well as ascending contraction and descending relaxation induced by mechanical stimulation of the mucosa (2-8 strokes), were significantly augmented by 1 mM acetate. Acetate (1 mM) also doubled propulsive velocity in isolated whole segments of the guinea pig colon. In conclusion, chemical stimulation of the mucosa by SCFAs triggers a peristaltic reflex mediated by the release of 5-HT from mucosal cells and activation of 5-HT4 receptors on sensory CGRP-containing nerve terminals. This SCFA-induced peristaltic pathway augments the peristaltic reflex elicited by mechanical stimulation of the mucosa.

Expression of Serotonin7Receptor and Coupling of Ectopic Receptors to Protein Kinase A and Ionic Currents in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia Causing Cushing’s Syndrome

In ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushing's syndrome, cortisol secretion is controlled by illegitimate membrane receptors. The aim of the present study was to characterize the pharmacological properties and the transduction mechanisms of illegitimate receptors, i.e. receptors for serotonin (5-HT), gastric inhibitory polypeptide (GIP), and LH/human chorionic gonadotropin (hCG), expressed by AIMAH tissues to evaluate the role of ectopic receptors in the physiopathology of Cushing's syndrome. We used in vitro studies on cultured adrenal hyperplasia cells. The setting was a university research laboratory. AIMAH tissues (H1-H3) were removed from three patients previously screened for illegitimate receptors. The main outcome measures were steroidogenic and electrical activities of cultured adrenal hyperplasia cells. In vitro studies showed that the corticotropic effect of 5-HT was mediated by ectopic 5-HT7 receptors in H1 and H2. GIP and hCG stimulated cortisol production via activation of cAMP-dependent protein kinase A in H2. On the contrary, the protein kinase A inhibitor H-89 did not affect hCG-induced cortisol production in H3. Activation of 5-HT7 or GIP receptors enhanced T-type calcium current in H1 or H2 and H3, respectively. In addition, GIP reduced the amplitude of transient and sustained potassium currents in H2. Conversely, hCG did not modify T-type calcium current in H3. These data show that, besides their coupling to the cAMP pathway, illegitimate adrenal receptors can activate additional transduction mechanisms, including modulation of membrane channels.

Investigation of serotonin type 4 receptor expression in human and non-human primate gastrointestinal samples

The serotonin type 4 (5-HT4) receptor has been associated with functions of the gastrointestinal tract such as modulation of the peristaltic reflex, smooth muscle tone, intestinal secretion and visceral sensitivity. The activation of peripheral 5-HT4 receptors with agonists such as tegaserod has been shown to accelerate gastric emptying and improve symptoms of constipation in animals and humans. However, detailed data on the expression profile and on the localization of this receptor subtype are lacking so far. To study the pattern and expression levels of 5-HT4 receptor messenger RNA expression in the gut. Normal tissue samples were collected from the whole gastrointestinal tract of patients undergoing abdominal surgery and, in addition, of monkeys. We performed a comprehensive analysis of 5-HT4 receptor expression by quantitative reverse transcription-polymerase chain reaction, using human and non-human primate tissues from the oesophagus to the rectum. In addition, the brain and heart of non-human primates were analysed. Significantly higher levels of 5-HT4 receptor mRNA were measured in the human stomach, duodenum, jejunum, ileum and caecum and also in the corresponding non-human primate gut segments, ranging from 2- to 12-fold compared with the liver. No differences were found between females and males of both human and non-human primates. These results show 5-HT4 receptor mRNA expression throughout the gastrointestinal tract in humans and primates, and also support the preclinical and clinical findings of 5-HT4 receptors ligands exhibiting multiple effects throughout the gastrointestinal tract.

5‐HT4 receptor activation induces long‐lasting EPSP‐spike potentiation in CA1 pyramidal neurons

Recent studies implicated involvement of the 5-hydroxytryptamine4 (5-HT4) receptor in cognitive and emotional processes. The highest 5-HT4 receptor densities in the brain are found in the limbic system including the hippocampus. Here we used the selective 5-HT4 receptor full agonist, N-pentyl-N'-aminoguanidine carbazimidamide (SDZ-216454) to characterize effects of 5-HT4 receptor activation in whole-cell and field recordings in the area CA1 in hippocampal slices prepared from 3 to 4- and 6 to 9-week-old rats, respectively. Extracellular recordings showed that transient 5-HT4 receptor activation by 10-20 min application of SDZ-216454 induces field excitatory postsynaptic potential (fEPSP)-population spike potentiation (ESP(5-HT4)), which persisted for as long as we held the recordings (> 2 h). ESP(5-HT4) displayed characteristics different from EPSP-spike potentiation that accompanies long-term potentiation; it developed without an associated increase in synaptic transmission, was independent on afferent input, activity of postsynaptic neurons and N-methyl-d-aspartate receptor activation; and was expressed in the presence of GABA receptor antagonists. ESP(5-HT4) was also induced by transient application of the natural neurotransmitter, 5-HT. The increase in the evoked population spike (PS) induced by SDZ-216454 was not prevented by blockers of hyperpolarization-activated cation current (Ih), Cs+ and ZD-7288, but was mimicked and occluded by 150 microm Ba2+. Whole-cell voltage-clamp recordings from pyramidal neurons demonstrated that SDZ-216454 application increases membrane resistance with a concomitant decrease in a Ba2+-sensitive inwardly rectifying K+ current and the Ba2+-insensitive K+ current underlying slow afterhyperpolarization (I(sAHP)). We conclude that 5-HT4 receptor activation may cause a long-lasting excitability increase in CA1 pyramidal neurons by inhibition of a Ba2+-sensitive inwardly rectifying K+ current.

Ligand-Induced 5-HT3 Receptor Internalization in Enteric Neurons in Rat Ileum

Release of 5-hydroxytryptamine (5-HT) from mucosal enterochromaffin cells and activation of 5-HT(3) receptors (5-HT(3)Rs) on neurons in the gut wall is important in the response of the gut to the luminal environment. Intestinal inflammation is associated with increased levels of mucosal 5-HT. The aims of the study were to determine the following: (1) if 5-HT(3)R undergoes ligand-induced internalization in myenteric neurons, and (2) the effect of long-term increase of mucosal 5-HT on 5-HT(3)Rs. Acute effects of exogenous 5-HT or endogenous release of 5-HT by luminal glucose on cellular localization of 5-HT(3)Rs was determined by immunohistochemistry and confocal microscopy. Treatment with the serotonin re-uptake inhibitor, fluoxetine, for 6 days (20 mg/kg daily orally) was used to increase mucosal 5-HT chronically in rats. Net ileal fluid movement was measured in anesthetized rats by the weight change of a 2.5% agarose cylinder. Acute increases in 5-HT induced by exogenous or endogenous 5-HT decreased 5-HT(3)R immunoreactivity at the neuronal cell membrane by 70% and 60%, respectively. Chronic fluoxetine treatment increased mucosal levels of 5-HT and decreased membrane 5-HT(3)R immunoreactivity by 27%. Net fluid absorption was decreased by a 5-HT(3)R agonist or by luminal glucose; this was attenuated 88% and 99%, respectively, by fluoxetine treatment. Long-term increase in 5-HT in the intestinal mucosa results in increased 5-HT(3)R internalization in myenteric neurons. Chronic changes in mucosal 5-HT may alter gastrointestinal secretory and motor function via ongoing loss of receptor from neuronal membrane, causing a mismatch between luminal content and absorption.