Effect of prucalopride on sildenafil-induced inhibition of esophageal peristalsis in healthy adults.

BACKGROUND AND AIM: Prucalopride, a high-affinity 5-hydroxytryptamine 4 receptor agonist, promotes esophageal peristalsis, while phosphodiesterase type 5 inhibitor sildenafil inhibits esophageal peristalsis. The present study was aimed to evaluate whether prucalopride would augment esophageal peristalsis subsequent to the application of sildenafil. METHODS: Seventeen healthy adults underwent high-resolution manometry by a catheter with one injection port located in the mid-esophagus. Secondary peristalsis was assessed by rapid air injections after water swallows. Two sessions were randomly performed including acute administration of sildenafil 50 mg after pretreatment with prucalopride or the placebo. RESULTS: The frequency of primary peristalsis subsequent to the administration of sildenafil was significantly increased by prucalopride (P = 0.02). Prucalopride also significantly increased distal contractile integral of primary peristalsis subsequent to the administration of sildenafil (P = 0.03). No difference in the frequency of secondary peristalsis subsequent to the administration of sildenafil for air injects of 10 mL (P = 0.14) or 20 mL (P = 0.21) was found between prucalopride and placebo. Prucalopride did not change distal contractile integral of secondary peristalsis subsequent to the administration of sildenafil for air injections of 10 mL (P = 0.09) or 20 mL (P = 0.12). CONCLUSIONS: Prucalopride modulates sildenafil-induced inhibition of primary peristalsis by increasing its effectiveness and peristaltic wave amplitude. Our findings suggest that activation of 5-hydroxytryptamine 4 receptors plays a role in mediating sildenafil-induced inhibition of esophageal primary peristalsis rather than secondary peristalsis.

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.

The absence of 5-HT4 receptors modulates depression- and anxiety-like responses and influences the response of fluoxetine in olfactory bulbectomised mice: Adaptive changes in hippocampal neuroplasticity markers and 5-HT1A autoreceptor.

Preclinical studies support a critical role of 5-HT4 receptors (5-HT4Rs) in depression and anxiety, but their influence in depression- and anxiety-like behaviours and the effects of antidepressants remain partly unknown. We evaluated 5-HT4R knockout (KO) mice in different anxiety and depression paradigms and mRNA expression of some neuroplasticity markers (BDNF, trkB and Arc) and the functionality of 5-HT1AR. Moreover, the implication of 5-HT4Rs in the behavioural and molecular effects of chronically administered fluoxetine was assessed in naïve and olfactory bulbectomized mice (OBX) of both genotypes. 5-HT4R KO mice displayed few specific behavioural impairments including reduced central activity in the open-field (anxiety), and decreased sucrose consumption and nesting behaviour (anhedonia). In these mice, we measured increased levels of BDNF and Arc mRNA and reduced levels of trkB mRNA in the hippocampus, and a desensitization of 5-HT1A autoreceptors. Chronic administration of fluoxetine elicited similar behavioural effects in WT and 5-HT4R KO mice on anxiety-and depression-related tests. Following OBX, locomotor hyperactivity and anxiety were similar in both genotypes. Interestingly, chronic fluoxetine failed to reverse this OBX-induced syndrome in 5-HT4R KO mice, a response associated with differential effects in hippocampal neuroplasticity biomarkers. Fluoxetine reduced hippocampal Arc and BDNF mRNA expressions in WT but not 5-HT4R KO mice subjected to OBX. These results demonstrate that the absence of 5-HT4Rs triggers adaptive changes that could maintain emotional states, and that the behavioural and molecular effects of fluoxetine under pathological depression appear to be critically dependent on 5-HT4Rs.

The peptidic antidepressant spadin interacts with prefrontal 5-HT(4) and mGluR(2) receptors in the control of serotonergic function.

This study investigates the mechanism of action of spadin, a putative fast-acting peptidic antidepressant (AD) and a functional blocker of the K(+) TREK-1 channel, in relation with the medial prefrontal cortex (mPFC)-dorsal raphé (DRN) serotonergic (5-HT) neurons connectivity. Spadin increased 5-HT neuron firing rate by 113%, an augmentation abolished after electrolytic lesion of the mPFC. Among the few receptor subtypes known to modulate TREK-1, the stimulation of 5-HT4 receptors and the blockade of mGluR2/3 ones both activated 5-HT impulse flow, effects also suppressed by mPFC lesion. The combination of spadin with the 5-HT4 agonist RS 67333 paradoxically reduced 5-HT firing, an effect reversed by acutely administering the 5-HT1A agonist flesinoxan. It also had a robust synergetic effect on the expression of Zif268 within the DRN. Together, these results strongly suggest that 5-HT neurons underwent a state of depolarization block, and that the mechanisms underlying the influences exerted by spadin and RS 67333 are additive and independent from each other. In contrast, the mGluR2/3 antagonist LY 341495 occluded the effect of spadin, showing that it likely depends on mPFC TREK-1 channels coupled to mGluR2/3 receptors. These in vivo electrophysiological data were confirmed by in vitro Ca(2+) cell imaging performed in cultured cortical neurons. Altogether, our results indicate that spadin, as a natural compound, constitutes a very good candidate to explore the "glutamatergic path" of fast-acting AD research. In addition, they provide the first evidence of 5-HT depolarization block, showing that the combination of 5-HT activators for strategies of AD augmentation should be performed with extreme caution.

Activation of 5-HT4 receptors facilitates neurogenesis of injured enteric neurons at an anastomosis in the lower gut.

Two-photon microscopy (2PM) can enable high-resolution deep imaging of thick tissue by exciting a fluorescent dye and protein at anastomotic sites in the mouse small intestine in vivo. We performed gut surgery and transplanted neural stem cells (NSC) from the embryonic central nervous system after marking them with the fluorescent cell linker, PKH26. We found that neurons differentiated from transplanted NSC (PKH [+]) and newborn enteric neurons differentiated from mobilized (host) NSC (YFP [+]) could be localized within the granulation tissue of anastomoses. A 5-HT4-receptor agonist, mosapride citrate (MOS), significantly increased the number of PKH (+) and YFP (+) neurons by 2.5-fold (P<0.005). The distribution patterns of PKH (+) neurons were similar to those of YFP (+) neurons. On the other hand, the 5-HT4-receptor antagonist, SB-207266 abolished these effects of MOS. These results indicate that neurogenesis from transplanted NSC is facilitated by activation of 5-HT4-receptors. Thus, a combination of drug administration and cell transplantation could be more beneficial than exclusive cell transplantation in treating Hirschsprung's disease and related disorders including post rectal cancer surgery. The underlying mechanisms for its action were explored using immunohistochemistry of the longitudinal mouse ileum and rat rectal preparations including an anastomosis. MOS significantly increased the number of new neurons, but not when co-administered with either of a protein tyrosine kinase receptor, c-RET two inhibitors. The c-RET signaling pathway contributes to enteric neurogenesis facilitated by MOS. In the future, we would perform functional studies of new neurons over the thick granulation tissue at anastomoses, using in vivo imaging with 2PM and double transgenic mice expressing a calcium indicator such as GCaMP6 and channelrhodopsin.

Cardiac PDEs and crosstalk between cAMP and cGMP signalling pathways in the regulation of contractility.

Elucidation of cAMP and cGMP signalling in the heart remains a hot topic, and new regulatory mechanisms continue to appear. Studying the influence of phosphodiesterases on 5-HT4 receptor signalling in porcine atrium, a paper from this issue of the journal expands findings of a crosstalk between cardiac cGMP and cAMP signalling recently discovered in failing rat ventricle to a different species and cardiac region. The overall data suggest that cGMP, produced following stimulation of the NPR-B receptor for C-type natriuretic peptide (CNP), inhibits cAMP degradation by phosphodiesterase 3 and thereby enhances cAMP-mediated signalling from ß-adrenoceptors and 5-HT4 receptors to inotropic effects. In porcine atrium, this effect can be seen both as an increase in inotropic effect and as a reduced fade of the inotropic effect with time. Thus, accumulating evidence brings together several active fields of research, including cardiac phosphodiesterases, compartmentation of cyclic nucleotide signalling and the field of natriuretic peptides. If present in human hearts, this effect of CNP may have clinical implications.

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium.

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT4 receptor agonist prucalopride. Muscles were freeze-clamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently.

5-HT4 receptors constitutively promote the non-amyloidogenic pathway of APP cleavage and interact with ADAM10.

In addition to the amyloidogenic pathway, amyloid precursor protein (APP) can be cleaved by α-secretases, producing soluble and neuroprotective APP alpha (sAPPα) (nonamyloidogenic pathway) and thus preventing the generation of pathogenic amyloid-ß. However, the mechanisms regulating APP cleavage by α-secretases remain poorly understood. Here, we showed that expression of serotonin type 4 receptors (5-HT(4)Rs) constitutively (without agonist stimulation) induced APP cleavage by the α-secretase ADAM10 and the release of neuroprotective sAPPα in HEK-293 cells and cortical neurons. This effect was independent of cAMP production. Interestingly, we demonstrated that 5-HT(4) receptors physically interacted with the mature form of ADAM10. Stimulation of 5-HT(4) receptors by an agonist further increased sAPPα secretion, and this effect was mediated by cAMP/Epac signaling. These findings describe a new mechanism whereby a GPCR constitutively stimulates the cleavage of APP by α-secretase and promotes the nonamyloidogenic pathway of APP processing.

Prostaglandin E1 facilitates inotropic effects of 5-HT4 serotonin receptors and ß-adrenoceptors in failing human heart.

Prostaglandins have displayed both beneficial and detrimental effects in clinical studies in patients with severe heart failure. Prostaglandins are known to increase cardiac output, but the mechanism is not clarified. Here, we tested the hypothesis that prostaglandins can increase contractility in human heart by amplifying cAMP-dependent inotropic responses. Contractility was measured ex vivo in isolated left ventricular strips and phosphodiesterase (PDE) and adenylyl cyclase (AC) activity was measured in homogenates or membranes from failing human left ventricles. PGE(1) (1 µM) alone did not modify contractility, but given prior, amplified maximal serotonin (5-HT)-evoked (10 µM) contractile responses mediated by 5-HT(4) receptors several fold (24 ± 7 % with PGE(1) vs. 3 ± 2 % above basal with 5-HT alone). The 5-HT(4)-mediated inotropic response was amplified by the PDE3 inhibitor cilostamide and further amplified in combination with PGE(1) (26 ± 6 vs. 56 ± 12 % above basal). PGE(1) reduced the time to reach 90 % of both the maximal 5-HT- and isoproterenol-evoked inotropic response compared to 5-HT or isoproterenol alone. PGE(1) did not modify PDE activity in the homogenate, either alone or when given simultaneously with PDE3 and/or PDE4 inhibitors. Neither 5-HT- nor isoproterenol-stimulated AC activity was significantly amplified by PGE(1). Sensitivity of ventricular strips to Ca(2+) was not enhanced in the presence of PGE(1). Our results show that PGE(1) can enhance cAMP-mediated responses in failing human left ventricle, through a mechanism independent of PDE inhibition, amplification of AC activity or increasing sensitivity to calcium. This effect of PGE(1) possibly contributes to the increase of cardiac output, independent of decreased afterload, observed after prostaglandin administration in humans.

Study of the regulation of the inotropic response to 5-HT4 receptor activation via phosphodiesterases and its cross-talk with C-type natriuretic peptide in porcine left atrium.

We studied how 5-HT(4) receptor-mediated inotropic responses are regulated at the level of cAMP in porcine left atrium. We used selective phosphodiesterase (PDE) inhibitors to assess which PDE subtypes are responsible for the fade with time of inotropic responses to 5-HT(4) receptor activation with 5-HT and the 5-HT(4) receptor agonist prucalopride. A possible cross-talk via PDEs between cGMP and 5-HT(4) receptor-induced cAMP signalling was evaluated. Electrically paced left atrial pectinate muscles from young male pigs (15-25 kg) were studied in vitro. Simultaneous inhibition of PDE3 plus PDE4 subtypes was necessary to increase the amplitude and completely prevent the fade of the inotropic response to 5-HT and prucalopride. When responses to 5-HT or prucalopride had faded 1 h after addition, the nonspecific PDE-inhibitor IBMX still fully recovered inotropic responses. Stimulation of particulate guanylyl cyclase, together with PDE2 and PDE4 inhibition, delayed the fade of the response to 5-HT, while stimulation of soluble guanylyl cyclase independently of PDEs accelerated the fade of the response to 5-HT. In conclusion, both PDE3 and PDE4 subtypes are responsible for the suppression and the fade of the inotropic response to 5-HT and prucalopride. Signalling through the 5-HT(4) receptor remains fully active for at least 90 min with PDEs continuously regulating the response. cGMP levels, elevated by activation of particulate guanylyl cyclase under PDE2 inhibition, can indirectly enhance 5-HT(4) receptor-mediated signalling, at least when also PDE4 is inhibited, presumably through inhibition of PDE3. Elevation of cGMP generated by soluble guanylyl cyclase attenuates responses to 5-HT independently of PDEs.

New treatment options for chronic constipation: mechanisms, efficacy and safety.

The present review has several objectives, the first of which is to review the pharmacology and selectivity of serotonergic agents to contrast the older serotonergic agents (which were withdrawn because of cardiac or vascular adverse effects) with the newer generation serotonin receptor subtype 4 agonists. Second, the chloride ion secretagogues that act through the guanylate cyclase C receptor are appraised and their pharmacology is compared with the approved medication, lubiprostone. Third, the efficacy and safety of the application of bile acid modulation to treat constipation are addressed. The long-term studies of surgically induced excess bile acid delivery to the colon are reviewed to ascertain the safety of this therapeutic approach. Finally, the new drugs for opiate-induced constipation are introduced. Assuming these drugs are approved, practitioners will have a choice; however, patient responsiveness will be based on trial and error. Nevertheless, the spectrum of mechanisms and demonstrated efficacy and safety augur well for satisfactory treatment outcomes.

Enteric nervous system neuropathy: repair and restoration.

PURPOSE OF REVIEW: Disordered neurobiology of the enteric nervous system (ENS) underlies a broad assortment of idiopathic, acquired, and congenital pathophysiologies up and down the digestive tract. Progress in two major areas of regenerative medicine related to enteric neuropathy is summarized: new insight into how everyday damage to the ENS might be corrected by indwelling stem cells and prospects for patient-specific replacement of damaged or diseased intestine with one reproduced from pluripotent stem cells derived from embryos or reprogrammed adult cells. RECENT FINDINGS: Germinal centers with undifferentiated stem cells are in position outside ENS ganglia. Messages, which might be released after damage to the ENS or when neurons are lost, direct migration of stem cells into ENS ganglia where they differentiate into one or the other of the specialized classes of interneurons or motor neurons and become 'wired' into the synaptic circuits as neuronal replacements. Action of serotonin and the 5-hydroxytryptamine (HT)4 receptor subtype is a message that initiates the neuronal replacement and circuit restoration process. A reasonable facsimile of a functional intestine can be derived from pluripotent stem cells. SUMMARY: Emerging knowledge of cell and molecular biology of indwelling stem cells in the gut and strategies for application of pluripotential stem cells in patient-specific organ transplantation reflect an emergent revolution in understanding and treating disordered gut function when the underlying cause is ENS neuropathy.

Neuronal stimulation with 5-hydroxytryptamine 4 receptor induces anti-inflammatory actions via α7nACh receptors on muscularis macrophages associated with postoperative ileus.

BACKGROUND: The main symptom of postoperative ileus (POI) is an intestinal motility disorder in which monocytes/macrophages and neutrophils play crucial roles. Prokinetic 5-hydroxytryptamine 4 receptor (5-HT4R) agonists and dopamine receptor antagonists are potential therapeutic agents for directly ameliorating the motility disorder associated with POI. AIM: To determine the effects of the 5-HT4R agonists mosapride citrate (MOS) and CJ-033466 on intestinal smooth muscle contractility relative to immune reactions after POI. METHODS: Intestinal manipulation (IM) was applied to the rat distal ileum. Both MOS (0.3 and 1 mg/kg, s.c.) and CJ-033466 (1 mg/kg, s.c.) were administered to the animals before and after IM. At 24 h after IM, isolated intestinal smooth muscle contractile activity in vitro, gastrointestinal transit in vivo, inflammatory mediator expression and leucocyte infiltration were measured. RESULTS: After IM, ileal circular muscle contractility in vitro and gastrointestinal transit in vivo were reduced and the number of macrophages and neutrophils increased in the inflamed muscle layer, resulting in the induction of inflammatory mediators such as interleukin 1 ß (IL-1ß), IL-6, tumour necrosis factor α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS). Both MOS and CJ-033466 significantly attenuated not only the intestinal motility dysfunction but also the leucocyte infiltration and inflammatory mediator expression after IM. The autonomic ganglionic blocker hexamethonium (1 mg/kg, i.p.) and the α7-nicotinic acetylcholine receptor (α7nAChR) antagonist methyl lycaconitine citrate (0.087 mg/kg, i.p.) blocked MOS-mediated ameliorative actions. Immunohistochemically, α7nAChR is expressed by monocytes/macrophages but not by neutrophils in the inflamed intestine. CONCLUSION: Stimulating the 5-HT4R accelerates acetyl choline (ACh) release from cholinergic myenteric neurons, which subsequently activates α7nAChR on activated monocytes/macrophages to inhibit their inflammatory reactions in the muscle layer. In addition to their gastroprokinetic action, 5-HT4R agonists might serve as novel therapeutic agents for POI characterised by anti-inflammatory potency.

Expression and function of 5-hydroxytryptamine 4 receptors in smooth muscle preparations from the duodenum, ileum, and pelvic flexure of horses without gastrointestinal tract disease.

OBJECTIVE: To evaluate the expression of the 5-hydroxytryptamine 4 (5-HT4) receptor subtype and investigate the modulating function of those receptors on contractility in intestinal tissues obtained from horses without gastrointestinal tract disease. SAMPLE POPULATION: Smooth muscle preparations from the duodenum, ileum, and pelvic flexure collected immediately after slaughter of 24 horses with no history or signs of gastrointestinal tract disease. PROCEDURES: In isometric organ baths, the contractile activities of smooth muscle preparations in response to 5-hydroxytryptamine and electric field stimulation were assessed; the effect of tegaserod alone or in combination with 5-hydroxytryptamine on contractility of intestinal specimens was also investigated. Presence and distribution of 5-HT4 receptors in intestinal tissues and localization on interstitial cells of Cajal were examined by use of an immunofluorescence technique. RESULTS: Widespread 5-HT4 receptor immunoreactivity was observed in all intestinal smooth muscle layers; 5-HT4 receptors were absent from the myenteric plexus and interstitial cells of Cajal. In electrical field-stimulated tissue preparations of duodenum and pelvic flexure, tegaserod increased the amplitude of smooth muscle contractions in a concentration-dependent manner. Preincubation with tegaserod significantly decreased the basal tone of the 5-HT-evoked contractility in small intestine specimens, compared with the effect of 5-HT alone, thereby confirming that tegaserod was acting as a partial agonist. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, 5-HT4 receptors on smooth muscle cells appear to be involved in the contractile response of the intestinal tract to 5-hydroxytryptamine. Results suggest that tegaserod may be useful for treatment of reduced gastrointestinal tract motility in horses.

Safety and efficacy of tegaserod therapy in patients with irritable bowel syndrome or chronic constipation.

BACKGROUND: Tegaserod, a 5-HT4 agonist, is effective for treating irritable bowel syndrome and chronic constipation. However, sales of this drug were recently suspended due to concerns about a higher rate of cardiovascular events in patients receiving tegaserod over placebo in clinical trials. Our aim was to review patients in our practice prescribed tegaserod to determine if any of them had suffered a cardiovascular event or other significant adverse effects while on this therapy. Additionally, we attempted to determine the efficacy of tegaserod in clinical practice. METHODS: Patients with irritable bowel syndrome or chronic constipation in our practice prescribed tegaserod were identified through a search of billing codes and charts reviews. These patients were contacted and questioned about symptoms of cardiovascular events or other adverse events while on tegaserod. The efficacy of this drug was determined by a symptom scale during and after stopping tegaserod. RESULTS: Sufficient data for analysis was retrieved for 51 of 67 patients prescribed tegaserod. Of these, 37 patients (72.5%) experience no adverse events and 14 patients (27.4%) experienced at least one adverse event, including 6 patients (11.8%) with major adverse events (2 patients (3.9%) with atypical chest pain; 4 patients (7.8%) with syncope; and 2 patients (3.9%) who died. One patient died from advanced pancreatic cancer. The other, who had multiple cardiovascular risk factors as well as a previous myocardial infarction, suffered a cardiac arrest 2 days postoperatively following a below knee amputation, and had actually been off tegaserod for 7 days after hospital admission. Patients graded the severity of both abdominal pain and constipation as worse after stopping therapy compared to during therapy (p<0.0002 and p<0.0001, respectively). CONCLUSIONS: The risk of cardiovascular events during tegaserod therapy may be increased in patients with other risk factors. However, this drug is effective for treating irritable bowel syndrome and chronic constipation, and might be used in a select patient population with severe symptoms but without other risk factors for cardiovascular events.

Antiangiogenic effect of a selective 5-HT4 receptor agonist.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) is reported to regulate cell growth in a wide variety of cell types in different carcinomas. 5-HT exerts complex actions on blood vessels, dependent on its interactions with a multiplicity of 5-HT receptors. In the present study, we aimed to investigate the potential antiangiogenic effect of mosapride citrate, a selective 5-HT4 receptor agonist, known to have prokinetic properties on the gastrointestinal tract. For this purpose, cultured human umbilical vein endothelial cells (HUVECs) were used as an in vitro model. MATERIAL AND METHODS: The effect of mosapride citrate on the proliferative activity of HUVECs was assessed by the MTS assay. Then, the apoptosis and the cell cycle detection assays were performed. The effect of mosapride citrate on the ability of HUVECs to adhere and migrate on extracellular matrix proteins (ECMs), as well as their ability to form vascular-like structures on Matrigel was investigated. RESULTS: Mosapride citrate inhibited the proliferative activity of HUVECs, dependent on cell cycle arrest, and not on apoptosis. A dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle in mosapride-treated HUVECs was observed. Mosapride citrate also significantly inhibited the ability of HUVECs to migrate, but not to adhere on ECMs. Additionally, mosapride citrate dose-dependently inhibited the tube-like formation ability of HUVECs on matrigel, an important event in the process of angiogenesis. CONCLUSION: The present results demonstrate the antiangiogenic activity of mosapride citrate in vitro and the possibility of its application as a new anti-cancer agent is suggested.

Ontogenic changes of the control by phosphodiesterase-3 and -4 of 5-HT responses in porcine heart and relevance to human atrial 5-HT(4) receptors.

BACKGROUND AND PURPOSE: Atrial inotropic responses to 5-HT mediated through 5-HT(4) receptors fade, presumably through phosphodiesterase (PDE) activity. We investigated the influence of a selective inhibitor of PDE3 (cilostamide) or of PDE4 (rolipram) on the fade of 5-HT responses in atrial muscle. EXPERIMENTAL APPROACH: 5-HT responses were compared, ex vivo, on sinoatrial beating rate of newborn piglets, porcine atrial and ventricular force, and human atrial force. cAMP levels were assessed in piglet atrium. KEY RESULTS: 5-HT-evoked sinoatrial tachycardia did not fade and was not potentiated by cilostamide (300 nmol.L(-1)) or rolipram (1 micromol.L(-1)). Inotropic responses to 5-HT faded in atria from piglets, adolescent pigs and humans. Cilostamide reduced atrial fade of 5-HT responses in adolescent pigs and humans but not in newborn piglets. Cilostamide disclosed 5-HT ventricular responses in newborn, but not adolescent pigs. Rolipram reduced fade of atrial 5-HT responses in newborn and adolescent pigs but not in humans. Concurrent cilostamide + rolipram abolished fade of 5-HT responses in porcine left atria and facilitated ventricular 5-HT responses, but did not reduce residual fade in human atrium in the presence of cilostamide. 5-HT-evoked increases in cAMP faded; fade was abolished by concurrent cilostamide + rolipram. CONCLUSIONS AND IMPLICATIONS: PDE3-induced control of porcine 5-HT responses differed in atrium and ventricle and changed with age. PDE3 and PDE4 jointly prevented fade of inotropic and cAMP responses to 5-HT in porcine atrium. Unlike porcine atria, only PDE3 induced fade of 5-HT responses in human atria.

Phosphorylation of phospholamban and troponin I through 5-HT4 receptors in the isolated human atrium.

We studied the mRNA expression and function of 5-hydroxytryptamine (5-HT) receptors as well as their signal transduction in right atrial tissue from patients undergoing cardiac surgery and right ventricular tissue from human donor hearts. In isolated, electrically driven strips from human right atrium, 5-HT exerted concentration-dependent positive inotropic effects (EC(50) value = 0.10 +/- 0.01 microM) and hastened relaxation (positive lusitropic effect). The 5-HT(4) receptor antagonists SB203186 or GR125487 antagonised these effects. 5-HT (2 microM) increased the content of cyclic adenosine monophosphate (cAMP) from 6.86 +/- 1.36 to 19.1 +/- 2.45 pmol/mg protein (n = 6, p < 0.05) but did not alter the tissue content of inositol-1,4,5-trisphosphate (IP(3)). With reverse transcription polymerase chain reaction, mRNAs coding for the 5-HT(4) receptor splice variants 5-HT(4(a)), 5-HT(4(b)) and 5-HT(4(c)) were detected in human right atrium and right ventricle. 5-HT(2A) mRNA only was measurable in human atrium. Expression level of total 5-HT(4) receptor mRNA in the right ventricle amounted to 41% (n = 5-8) of that in the right atrium. 5-HT (2 microM) increased the atrial phosphorylation states of phospholamban to 168% at serine-16 and to 150% at threonine-17 (n = 4; p < 0.05) and of the inhibitory subunit of troponin to 150% (n = 6; p < 0.05). In conclusion, the positive inotropic and lusitropic effects of 5-HT in electrically driven human right atria are mediated via 5-HT(4) receptors. These effects are accompanied by and probably due to an increase in cAMP content and the subsequent elevation of the phosphorylation state of Ca(2+) regulatory proteins.

Regulation of aldosterone secretion by several aberrant receptors including for glucose-dependent insulinotropic peptide in a patient with an aldosteronoma.

CONTEXT: Primary adrenal Cushing's syndrome can result from the aberrant adrenal expression of several hormone receptors; this mechanism has not been explored in detail in aldosterone-producing tumors. OBJECTIVE: The objective of the study was to evaluate a 56-yr-old male patient with an aldosteronoma for the regulation of aldosterone secretion by aberrant hormone receptors. RESULTS: Renin-independent stimulation of aldosterone secretion was observed in vivo after a mixed meal, oral glucose, or administration of glucose-dependent insulinotropic peptide (GIP), vasopressin, and tegaserod. The mixed meal-mediated stimulation of aldosterone was not present in five other cases of aldosteronoma. A smaller response of aldosterone after GIP infusion was observed in a normal subject. Aldosterone secretion was stimulated by GIP in primary cultures of this patient's aldosteronoma. Increased expression of GIP receptor was found in this aldosteronoma by real-time RT-PCR and immunohistochemistry. The GIP receptor protein was also found at lower levels in zona glomerulosa cells of the normal adjacent adrenal gland. Increased expression of serotonin 4 and ACTH receptors was also present in this aldosteronoma. CONCLUSIONS: This case report provides new evidence of the implication of aberrant hormone receptors in the regulation of this aldosteronoma and suggests that further detailed studies of the role of aberrant hormone receptors in this frequent pathology should be undertaken.

Increased particulate phosphodiesterase 4 in the prefrontal cortex supports 5-HT4 receptor-induced improvement of object recognition memory in the rat.

RATIONALE: Serotonin receptors (5-HT4Rs) are critical to both short-term and long-term memory processes. These receptors mainly trigger the cyclic adenosine monophosphate (cAMP)/protein kinase A signaling pathway, which is regulated by cAMP phosphodiesterases (PDEs). OBJECTIVES: We investigated the mechanisms underlying the effect of the selective activation of 5-HT4R on information acquisition in an object recognition memory task and the putative regulation of PDE. MATERIALS AND METHODS: The effect of RS 67333 (1 mg/kg, intraperitoneally [i.p.], injected 30 min before the sample phase) was examined at different delay intervals in an object recognition task in Sprague-Dawley rats. After the testing trial, PDE activity of brain regions implicated in this task was assayed. RESULTS: RS 67333-treated rats spent more time exploring the novel object after a 15-min (P < 0.001) or 4-h delay (P < 0.01) but not after a 24-h delay, whereas control animals showed no preference for the novel object for delays greater than 15 min. We characterized the specific patterns and kinetic properties of PDE in the prefrontal and perirhinal cortices as well as in the hippocampus. We demonstrated that particulate PDE activities increase in both the prefrontal cortex and hippocampus following 5-HT4R stimulation. In the prefrontal cortex, PDE4 activities support the RS 67333-induced modification of PDE activities, whereas in the hippocampus, all cAMP-PDE activities varied. In contrast, particulate PDE variation in the hippocampus was not found to support improvement of recognition memory after a 4-h delay. CONCLUSIONS: We provide evidence that the increase in particulate PDE4 activity in the prefrontal cortex supports the 5-HT4R-induced increase in information acquisition.