Effect of selective 5HT3 antagonist (GR 38032F) on small intestinal transit and release of gastrointestinal peptides

Antagonists of 5-hydroxytryptamine type 3 (5HT₃) receptors reduce the nausea induced by cisplatinum, but the effects of these agents on 5HT₃ receptors in the human gut remain to be defined. We examined the actions of one of these drugs (Glaxo GR 38032F) on small intestinal transit and mouth-to-cecum transit times in healthy man. We also quantified its effects on the release of peptide YY (PYY), neurotensin, human pancreatic polypeptide, gastrin-cholecystokinin, and motilin. Ten healthy volunteers were enrolled in a randomized, double-blind, placebo-controlled crossover study. Following a single intravenous dose of GR 38032F (0.15 mg/kg), we measured the time to appearance in plasma of sulfapyridine after injection of salicylazosulfapyridine into the duodenum. This was used as a measure of duodenocecal transit. The appearance of hydrogen in breath after ingestion of a meal containing lactulose was also correspondingly used to quantify the mouth-to-cecum transit of the head of the meal. Gastrointestinal hormones were assayed in plasma by specific RIAs; samples were drawn fasting (10 min after injection) and after breakfast (358 calories: 15.7 g protein, 55.4 g carbohydrate, 8.1 g fat). The postprandial integrated response and peak release of PYY was decreased by GR 38032F. There was also a trend for the peak release of neurotensin to be reduced. GR 38032F did not significantly alter small intestinal transit times or mouth-to-cecum transit times. We conclude that GR 38032F does not have a major effect on small intestinal transit in health.Supported in part by grants from Glaxo Inc., USA, and from the National Institutes of Health (RR585, DK34988, and DK32121).     

Effects of a serotonin 5-HT(4) receptor antagonist SB-207266 on gastrointestinal motor and sensory function in humans

BACKGROUND: Serotonin 5-HT(4) receptors are located on enteric cholinergic neurones and may regulate peristalsis. 5-HT(4) receptors on primary afferent neurones have been postulated to modulate visceral sensation. While 5-HT(4) agonists are used as prokinetic agents, the physiological role of 5-HT(4) receptors in the human gut is unknown. AIMS: Our aim was to characterise the role of 5-HT(4) receptors in regulating gastrointestinal motor and sensory function in healthy subjects under baseline and stimulated conditions with a 5-HT(4) receptor antagonist. METHODS: Part A compared the effects of placebo to four doses of a 5-HT(4) receptor antagonist (SB-207266) on the cisapride mediated increase in plasma aldosterone (a 5-HT(4) mediated response) and orocaecal transit in 18 subjects. In part B, 52 healthy subjects received placebo, or 0.05, 0.5, or 5 mg of SB-207266 for 10-12 days; gastric, small bowel, and colonic transit were measured by scintigraphy on days 7-9, and fasting and postprandial colonic motor function, compliance, and sensation during distensions were assessed on day 12. RESULTS: Part A: 0.5, 5, and 20 mg doses of SB-207266 had significant and quantitatively similar effects, antagonising the cisapride mediated increase in plasma aldosterone and acceleration of orocaecal transit. Part B: SB-207266 tended to delay colonic transit (geometric centre of isotope at 24 (p=0.06) and 48 hours (p=0.08)), but did not have dose related effects on transit, fasting or postprandial colonic motor activity, compliance, or sensation. CONCLUSION: 5-HT(4) receptors are involved in the regulation of cisapride stimulated orocaecal transit; SB 207266 tends to modulate colonic transit but not sensory functions or compliance in healthy human subjects.     

The 5-HT(3) receptor antagonist alosetron inhibits the colorectal distention induced depressor response and spinal c-fos expression in the anaesthetised rat

BACKGROUND: Noxious intestinal distention elicits a reflex depressor response in the sodium pentobarbitone anaesthetised rat, which can be used as an index of visceral nociception. 5-HT(3) receptor antagonists inhibit this reflex. Repeated colorectal distention (CRD) induces Fos like immunoreactivity (Fos-LI) in the rat spinal cord. AIMS: To examine the effect of the 5-HT(3) receptor antagonist alosetron on the depressor response to CRD, and on Fos expression in the lumbosacral spinal cord. METHODS: Male rats were anaesthetised with sodium pentobarbitone, and mean arterial blood pressure monitored during repeated colorectal balloon inflation before and after treatment with alosetron or saline. Rats anaesthetised with urethane and treated with alosetron or saline underwent a repeated CRD paradigm, after which the lumbosacral spinal cord was removed and processed for visualisation of Fos-LI. RESULTS: CRD elicited reproducible, volume dependent falls in arterial blood pressure, and repeated distention-effect curves were constructed. Alosetron (1-100 microg/kg intravenously) inhibited the depressor response to CRD in a dose related manner, with an ID(50) value of 3.0 microg/kg. Following repeated CRD, numbers of Fos-LI neurones were significantly increased to 1246 (total in 12 sections at 120 microm intervals from L6 to S1) compared with 49 in sham distended animals. Pretreatment with alosetron (100 microg/kg) significantly reduced numbers of Fos-LI neurones to 479.8. CONCLUSION: The 5-HT(3) receptor antagonist alosetron inhibits the depressor response to CRD in a potent and dose dependent manner. It also inhibits CRD induced Fos-LI in the spinal cord. These results suggest that 5-HT(3) receptors are involved in visceral nociceptive transmission, perhaps located on primary afferent or spinal neurones.     

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E₂ (PGE₂) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3?µM) used alone or in combination with the P2Y₁₂ antagonist cangrelor (1?µM), both without and with aspirin (100?µM). Platelet aggregation and P-selectin expression were measured in whole blood (n?=?10) following stimulation with the thromboxane A₂ (TXA₂) mimetic U46619 (0.3 or 1?µM) in combination with either the EP3 agonist sulprostone (0.1?µM), or PGE₂ (1?µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n?=?42) were randomly assigned to receive no background treatment, clopidogrel (300?mg loading dose plus 75?mg daily) or clopidogrel and aspirin (75?mg daily) for 10 days alongside DG-041 (200?mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y₁₂ antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y₁₂ blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.     

A randomized,double-blind,placebo-controlled clinical trial of the effectiveness of the novel serotonin type 3 receptor antagonist ramosetron in both male and female Japanese patients with diarrhea-predominant irritable bowel syndrome

Objective. Irritable bowel syndrome is characterized by abdominal discomfort and/or pain associated with altered bowel habits. The neurotransmitter serotonin and serotonin type 3 receptors that are extensively distributed on enteric neurons in the human gastrointestinal tract play a role in increasing the sensation of pain and affecting bowel habits in patients with irritable bowel syndrome. The aim of this study was to evaluate the efficacy and safety of the serotonin type 3 receptor antagonist ramosetron hydrochloride in Japanese patients with diarrhea-predominant irritable bowel syndrome. Material and methods. In a double-blind, placebo-controlled, parallel group-comparative study with a 1-week run-in period, 539 patients with diarrhea-predominant irritable bowel syndrome meeting the Rome II diagnostic criteria received either 5 µg ramosetron hydrochloride (n=270) or placebo (n=269) once daily for 12 weeks. Results. Forty-seven percent of ramosetron hydrochloride-treated patients were monthly responders in the primary end-point, “Patient-reported global assessment of relief of irritable bowel syndrome symptoms”, compared with 27% for placebos (p<0.001). The most frequently reported adverse event in the ramosetron hydrochloride-treated group compared with the placebo group was hard stool. Conclusions. Ramosetron hydrochloride 5 µg once daily is effective and well tolerated in the treatment of abdominal pain, discomfort and bowel habits in patients with diarrhea-predominant irritable bowel syndrome.     

Stimulation by nizatidine, a histamine H(2)-receptor antagonist, of duodenal HCO(3)(-)secretion in rats:relation to anti-cholinesterase activity

AIM:To examine whether nizatidine stimulates duodenal HCO(3)(-) secretion in rats by inhibiting AChE activity.METHODS:Under pentobarbital anesthesia, a proximal duodenal loop was perfused with saline, and the HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 10mM HCl. Nizatidine, neostigmine, carbachol or famotidine was administered i.v. as a single injection.RESULTS:Intravenous administration of nizatidine (3-30mg/kg) dose-dependently increased duodenal HCO(3)(-) secretion, and the effect at 10mg/kg was equivalent to that obtained by carbachol at 0.01mg/kg. This nizatidine action was observed at the same dose range that inhibited acid secretion and enhanced gastric motility, mimicked by i.v. injection of neostigmine(0.03mg/kg), and significantly attenuated by bilateral vagotomy and prior s.c. administration of atropine but not by indomethacin, a cyclooxygenase inhibitor, or NG-nitro-L-arginine methyl ester, a NO synthase inhibitor. The HCO(3)(-) secretory response to acetylcholine (0.001mg/kg) was significantly potentiated by the concurrent administration of nizatidine (3mg/kg,i.v.). The IC(50) of nizatidine for AChE of rat erythrocytes was 1.4·10(-6) M, about 12 times higher than that of neostigmine. Neither famotidine (> 10(-3) M, 30mg/kg, i.v.) nor cisapride (> 10(-3) M,3mg/kg, i.v.) had any influence on AChE activity or duodenal HCO(3)(-) secretion. Duodenal damage induced by acid perfusion (100mM HCl for 4h) in the presence of indomethacin was significantly prevented by nizatidine and neostigmine, at the doses that increased the HCO(3)(-)secretion.CONCLUSION:Nizatidine stimulates duodenal HCO(3)(-) secretion, in both vagal dependent and atropine sensitive manners, and the action is associated with the anti-AChE activity of this agent.     

High-frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT3AB receptor

The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB(Y129S) receptor are 20- and 10-fold slower, respectively, than those of the WT receptor. Single-channel measurements reveal a 7-fold-increased mean open time of 5-HT3AB(Y129S) receptors compared with WT receptors. The augmented signaling displayed by 5-HT3AB(Y129S) receptors may confer protection against the development of depression. The variant also may influence the development and/or treatment of nausea and other disorders involving 5-HT3 receptors. Thus, the impact of the high-frequency variant 5-HT3B(Y129S) on 5-HT3AB receptor signaling calls for a search for additional phenotypes, and the variant may thus aid in establishing the role of the 5-HT3AB receptor in pathophysiology.     

Preferential accumulation in vivo of 24R,25-dihydroxyvitamin D(3) in growth plate cartilage of rats

Vitamin D₃ is metabolized in vivo through 25-(OH)D₃ (25D) to both 1α,25-(OH)₂D₃ (1,25D) and 24R,25-(OH)₂D₃ (24,25D). Whereas it is assumed that this metabolism occurs primarily in the kidney, recent studies show that there are extrarenal 1α-and 24R-hydroxylase activities as well, and in chondrocytes, these enzymes are regulated by hormones and growth factors. Furthermore, chondrocytes from the resting zone of growth plate cartilage are a target cell population for 24,25D action, suggesting that this vitamin D metabolite may be targeted to this tissue in vivo. To test this hypothesis, 30 normal male Sprague Dawley rats (120 ±20 g) were divided into three groups of eight animals each, and a control group of six animals, and fed ad libitum for 2 wk, a standard rat chow (Teklad LM-485), which contained 3 IU vitamin D₃/g. The rats were then injected im daily at 9:00am, for 4 consecutive d, with 0.1 mL of either [³H]-25D, [³H]-1,25D or [³H]-24,25D. Each dose contained 13 pmol of hormone (0.36 μCi/dose). The distribution of these metabolites was assessed in tibial bone (B) following ablation of the bone marrow, articular cartilage from the tibia (AC), costochondral growth plate cartilage (GC), serum (S), small intestine (I), and kidney (K). The use of high specific activity tritiated vitamin D metabolites facilitated determining tissue localization and further metabolism without perturbation of the body pools of each major metabolite. Accumulation of [³H]-1,25D or [³H]-24,25D in each tissue was compared to circulating serum levels. In rats dosed with [³H]-25D, the tissue:serum ratios for 1,25D were 4.1 (AC), 35.4 (GC), 1.3 (B), 0.7 (K), and 3.0 (I); and tissue:serum ratios for 24,25D were 1.6 (AC), 9.9 (GC), 0.04 (B), 0.2 (K), and 0.4 (I). In rats dosed with [³H]-24,25D alone, GC was the only tissue to accumulate the administered metabolite at a concentration significantly higher than that of serum. Similarly, in rats dosed with [³H]-1,25D alone, GC was the only tissue to accumulate 1,25D at a concentration higher than that of serum. These results demonstrate, for the first time, that under in vivo conditions, GC specifically accumulates 24,25D and 1,25D. This suggests that growth plate may be a target organ for these two hormones.     

Effect of the 5-HT3 receptor antagonist, ondansetron, on gastric size in dyspeptic patients with impaired gastric accommodation

BACKGROUND: A reduction of gastric accommodation after a meal has been documented in patients with idiopathic dyspepsia. In these patients the administration of a 5-HT3 receptor antagonist may reduce some of the dyspeptic symptoms; it is not clear however, whether these drugs influence gastric adaptation to distension as well. AIM: To evaluate the effects of the 5-HT3 receptor antagonist, ondansetron, on gastric distension after a liquid meal in dyspeptic patients with reduced gastric accommodation. METHODS: Before and after a 500 ml water load, gastric accommodation (area of the proximal and distal stomach) was evaluated using real-time ultrasonography in 21 idiopathic dyspepsia patients and 26 healthy controls. In dyspeptic patients, the test was repeated twice: after the administration of placebo and after ondansetron 8 mg i.v. (in both cases, 15 min prior to the water load). Secondary outcomes were epigastric pain, fullness and nausea as assessed by a visual analogue scale at basal and after ondansetron. RESULTS: Fasting gastric size was similar in dyspeptic and controls. Compared with controls, dyspeptic patients showed a statistically significant smaller area of the proximal stomach (14.7+/-1.2 cm(2) vs. 18.6+/-1.4 cm(2), respectively; p=0.0247). In dyspeptic patients, gastric proximal and distal size did not change significantly following placebo, whereas after the administration of ondansetron the mean area of the proximal and distal stomach significantly increased (proximal stomach: 14.6+/-1.6 cm(2) placebo, 20.4+/-1.9 cm(2) ondansetron, p=0.0095; distal stomach: 8.9+/-0.9 placebo, 11.4+/-1.2cm(2) ondansetron, p=0.0409). Of the symptoms, only nausea was significantly reduced after ondansetron. CONCLUSION: In dyspeptic patients with impaired gastric accommodation, ondansetron reverts gastric accommodation to within the range of controls.     

Characterization of Ionic Transport in Li2O-(Mn:Fe)2O3-P2O5 Glasses for Li Batteries

We present a systematic study of the lithium-ion transport upon the mixed manganese-iron oxide phosphate glasses 3Li₂O-xMn₂O₃-(2-x)Fe₂O₃-3P₂O₅(LM_xF_2−xPO; 0≤ x ≤2.0) proposed for the use in a cathode for lithium secondary batteries. The glasses have been fabricated using a solid reaction process. The electrical characteristics of the glass samples have been characterized by electrical impedance in the frequency range from 100 Hz to 30 MHz and temperature from 30 °C to 240 °C. Differential thermal analysis and X-ray diffraction were used to determine the thermal and structural properties. It has been observed that the dc conductivity decreases, but the activation energies of dc and ac and the glass-forming ability increase with the increasing Mn₂O₃ content in LM_xF_2−xPO glasses. The process of the ionic conduction and the relaxation in LM_xF_2−xPO glasses are determined by using power–law, Cole–Cole, and modulus methods. The Li⁺ ions migrate via the conduction pathway of the non-bridging oxygen formed by the depolymerization of the mixed iron–manganese–phosphate network structure. The mixed iron–manganese content in the LM_xF_2−xPO glasses constructs the sites with different depths of the potential well, leading to low ionic conductivity.     

Central Modulation of Rectal Distension-Induced Blood Pressure Changes by Alosetron, A 5-HT3 Receptor Antagonist

Irritable bowel syndrome (IBS) represents one ofthe most common gastrointestinal-related diagnoses.Although the precise etiologic basis of IBS is notknown, a common presenting symptom is abdominal pain or discomfort that is thought to develop, atleast in part, from a heightened awareness of visceralnociceptive input. Agents capable of reducing thisheightened visceral nociception would, therefore, have utility in the treatment of IBS. In this studywe evaluated the effects of intravenous andintracerebroventricular administration of a5-HT₃ receptor antagonist, alosetron, onblood pressure changes associated with rectal distension in anesthetized andawake dogs. This vasoactive reflex serves as a model forvisceral nociception. For intracerebroventricularstudies, the cerebroventricular guides were placed over the lateral ventricle. In anesthetized studies,blood pressure was measured by femoral arterycannulation. In awake studies, blood pressure wasmonitored by noninvasive measurement. A rectal balloonwas placed in the rectum of each dog and maintainedthroughout the experiments. Each dose of alosetron wasgiven to the dogs as an intravenous orintracerebroventricular bolus, and every 30 min therectal balloon was inflated and blood pressure responsesobserved. In both anesthetized and awake dogs alosetronproduced a significant inhibition of the vasoactivereflex. In particular, alosetron showed high potency when administered intracerebroventricularly.Alosetron, administered either centrally orperipherally, appears to modulate the visceralnociceptive effect of rectal distension indogs.     

Thermo-Exfoliated Graphite Containing CuO/Cu2(OH)3NO3:(Co2+/Fe3+) Composites: Preparation,Characterization and Catalytic Performance in CO Conversion

Thermo-exfoliated graphite (TEG)/CuO/Cu₂(OH)₃NO₃:(Co²⁺/Fe³⁺) composites were prepared using a wet impregnation method and subsequent thermal treatment. The physicochemical characterization of the composites was carried out by powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) and Ar temperature-desorption techniques. The catalytic efficiency toward CO conversion to CO₂ was examined under atmospheric pressure. Characterization of species adsorbed over the composites taken after the activity tests were performed by means of temperature programmed desorption mass-spectrometry (TPD MS). (TEG)/CuO/Cu₂(OH)₃NO₃:(Co²⁺/Fe³⁺) composites show superior performance results if lower temperatures and extra treatment with H₂SO₄ or HNO₃ are used at the preparation stages. The catalytic properties enhancements can be related to the Cu₂(OH)₃NO₃ phase providing reaction centers for the CO conversion. It has been found that prevalence of low-temperature states of desorbed CO₂ over high-temperature ones in the TPD MS spectra is characteristic of the most active composite catalysts.     

Ins(1,4,5)P(3) regulates phospholipase Cbeta1 expression in cardiomyocytes

The functional significance of the Ca²⁺-releasing second messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃, IP₃) in the heart has been controversial. Ins(1,4,5)P₃ is generated from the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP₂) along with sn-1,2-diacylglycerol, and both of these are important cardiac effectors. Therefore, to evaluate the functional importance of Ins(1,4,5)P₃ in cardiomyocytes (NRVM), we overexpressed IP₃ 5-phosphatase to increase degradation. Overexpression of IP₃ 5-phosphatase reduced Ins(1,4,5)P₃ responses to α₁-adrenergic receptor agonists acutely, but with longer stimulation, caused an overall increase in phospholipase C (PLC) activity, associated with a selective increase in expression of PLCβ1, that served to normalise Ins(1,4,5)P₃ content. Similar increases in PLC activity and PLCβ1 expression were observed when Ins(1,4,5)P₃ was sequestered onto the PH domain of PLCδ1, a high affinity selective Ins(1,4,5)P₃-binding motif. These findings suggested that the available level of Ins(1,4,5)P₃ selectively regulates the expression of PLCβ1. Cardiac responses to Ins(1,4,5)P₃ are mediated by type 2 IP₃-receptors. Hearts from IP₃-receptor (type 2) knock-out mice showed heightened PLCβ1 expression. We conclude that Ins(1,4,5)P₃ and IP₃-receptor (type 2) regulate PLCβ1 and thereby maintain levels of Ins(1,4,5)P₃. This implies some functional significance for Ins(1,4,5)P₃ in the heart.     

Polymorphisms in the novel serotonin receptor subunit gene HTR3C show different risks for acute chemotherapy-induced vomiting after anthracycline chemotherapy

The aim of this study was to correlate chemotherapy-induced nausea and vomiting (CINV) with commonly occurring single nucleotide polymorphisms (SNP) in the 5-hydroxytryptamine receptor 3 genes (HTR3). Women with breast cancer without previous chemotherapy were eligible for this prospective study. All patients received epirubicin, with or without cyclophosphamide, and preventive medication with ondansetron and dexamethasone. The patients documented every vomiting event on an hourly basis. Real-time polymerase chain reaction (PCR) analysis was performed for the following nonsynonymous SNPs: p.Y129S (HTR3B), p.K163N (HTR3C) and p.A405G (HTR3C). The overall proportion of patients (total n = 110) who reported vomiting in the first 24 h after chemotherapy was 31.8%. The variant genotype of K163N (HTR3C) was associated with vomiting, which occurred in 50.0% (P = 0.009). Polymorphisms in the HTR3C gene could serve as a predictive factor for CINV in patients undergoing moderately emetogenic chemotherapy.     

From the Cover: PNAS Plus: Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence

Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P₂ and probably PI(3,4,5)P₃ as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and PI(3,4,5)P₃ in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P₃ is 55-fold slower than 5-phosphatase activity against PI(4,5)P₂; thus, PI(4,5)P₂ generated more slowly from dephosphorylating PI(3,4,5)P₃ might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P₂ in parallel to PI(3,4,5)P₃ dephosphorylation, emphasizing that VSPs can cleave the 3-phosphate of PI(3,4,5)P₃.This paper concerns the substrate specificity and voltage dependence of a unique voltage-sensitive phosphoinositide (PI) phosphatase in intact live cells. Bioelectricity, caused by ion channels and differences in ion concentrations between the inside and outside of a cell, regulates essential biological activities like generation, propagation, and processing of neuronal signals; muscle contraction; and secretion of hormones. Voltage-gated ion channels were the first protein family identified that possessed bioelectric voltage-sensing domains (VSDs) and participated in these signaling activities. Recently, a quite unanticipated voltage-sensing enzyme with a VSD was cloned from the sea squirt Ciona intestinalis (1). Biochemical and electrophysiological examination revealed a voltage-dependent phosphatase activity toward polyphospho-PIs that was given the name C. intestinalis voltage-sensing phosphatase (Ci-VSP). Since then, homologs have been discovered in other vertebrates, for example, Danio rerio (zebrafish; Dr-VSP), African frog (Xi-VSP and Xt-VSP), chicken (Gg-VSP), and salamander (Hn-VSP and Cp-VSP) (2–5). In addition, two mammalian homologs have been discovered in human Hs-transmembrane phosphatase with tensin homology (TPTE) and mouse (Mm-VSP) tissues (6, 7). Although knowledge about the physiological function of these two proteins is still lacking, a recent study showed that mouse VSP seems to be localized to intracellular membranes of neuronal cells, suggesting a different role for mammalian VSPs than for plasma membrane-localized Ci-VSP or Dr-VSP (7). As in voltage-gated ion channels, the VSD of VSPs consists of four transmembrane segments, S1–S4, with the charged voltage-sensing S4 segment being moved by the intense electric fields across the plasma membrane upon depolarization (8). However, VSPs are monomeric and have a cytosolic catalytic domain instead of the pore-forming domain (S5–S6) of ion channels. This enzyme domain is homologous to tumor suppressor phosphatase and tensin homolog (PTEN), a phosphoinositide 3-phosphatase that dephosphorylates both phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (1). Three distinctive regions of PTEN, an N-terminal phospholipid-binding motif that anchors the protein at the plasma membrane, a phosphatase domain that has the enzymatic site, and a C-terminal lipid-interacting C2 domain (9, 10), are well conserved in sequence and structure in the VSPs (11–14).Propagation of the depolarization-induced conformational changes of the VSD to the cytosolic catalytic domain activates the unique voltage-activated phosphoinositide phosphatase activity (12, 14, 15). Unlike its analog PTEN, which has only 3-phosphatase activity (9, 16), Ci-VSP was seen initially to cleave the 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and PI(3,4,5)P₃ in response to membrane depolarization, generating phosphatidylinositol 4-phosphate [PI(4)P] and PI(3,4)P₂, respectively (1, 17–19). Subsequently, VSPs were reported to cleave 3-phosphate from PI(3,4)P₂, generating PI(4)P upon larger depolarization (20), and, very recently, Ci-VSP was indicated to cleave 3-phosphate from PI(3,4,5)P₃, generating PI(4,5)P₂ (21, 22). Because different substrate reactions are best seen at different voltages, several authors have suggested that changing electric fields can drive VSPs successively through several catalytically active states that favor one set of substrates or reactions over another (20–22). The active site of VSPs is well conserved among species (12) and shows only a single amino acid difference from the active site of PTEN (1). When this residue in Ci-VSP was mutated to the corresponding amino acid of the PTEN active site, the mutated Ci-VSP still showed both 5- and 3-phosphatase activities toward polyphosphoinositides (14). Therefore, the observed substrate specificity and the voltage-dependent dual phosphatase activity of VSPs might be determined by the environment surrounding the active site rather than only by the active site itself (14). When the entire enzymatic domain of Ci-VSP was replaced by PTEN, the resulting chimera, called VSPTEN, had the enzymatic properties of a voltage-dependent pure 3-phosphatase (23).VSPs are found in remarkably diverse tissues, including the testis and brain of mice (7, 24, 25); testis, brain, and stomach of humans (6); testis and neuronal complex of the ascidian (1); and testis, ovary, kidney, and liver of the African frog (26). Surprisingly, the physiological functions of this widespread enzyme remain a puzzle. Recent work suggests that VSPs might have roles in egg fertilization (26) and in neuronal signaling in the brain (7). To understand its physiological enzymology more completely, we screened Dr-VSP–induced phosphoinositide changes in living cells by engineered Förster resonance energy transfer (FRET) probes that specifically report the cellular dynamics of PI(4)P, PI(3,4)P₂, PI(4,5)P₂, and PI(3,4,5)P₃. Our results show that when exogenous expression of PI(4)P 5-kinase type Iγ (PIPKIγ) was used selectively to counteract the 5-phosphatase activity of Dr-VSP, PI(4,5)P₂ accumulated, revealing an intrinsic 3-phosphatase activity of VSP toward PI(3,4,5)P₃. The voltage dependence and substrate specificity of 3- and 5-phosphatase subreactions of Dr-VSP were then extracted quantitatively by a comprehensive kinetic systems analysis. Together, our data demonstrate that Dr-VSP possesses both 3- and 5-phosphatase activities toward PI(3,4,5)P₃, with the same voltage dependence but with quite different absolute catalytic rates. These results should help clarify the roles of VSPs in fertilization, neural computations, and other signaling events that involve voltage changes.     

Demonstration of a high affinity receptor for 1,25-dihydroxyvitamin D3 in rat pancreas

The existence of a high affinity receptor for 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in rat pancreas was biochemically demonstrated in this study. In order to study the properties of this putative receptor, we took advantage of the analysis of low ionic strength chromatin-localized 1,25(OH)₂D₃ receptor. Using this method, the susceptibility of receptor protein to enzymatic degradation was so decreased, and the contamination by plasma vitamin D binding protein (DBP) component was so efficiently eliminated that a specific, saturable binding for 1,25(OH)₂D₃ could be demonstrated in the saturation analysis and the peak for the receptor was consistently apparent in the sucrose density gradient analysis. The equilibrium dissociation constant (Scatchard K_d) was found to be 3.7 ± 1.5 × 10^-10 (M), and the concentration of specific binding sites was calculated to be 1.22 ± 0.40 (fmol/mg protein). The number of specific binding sites in the rat pancreas was only 0.44% of that present in rat intestine (277 ± 19 (fmol/mg protein)) and 6.7% of that in rat kidney (18.1 ± 1.0 (fmol/mg)). However, when a correction is made for the 1,25(OH)₂D₃ receptor distribution in the tissues and expressed as the receptor concentration per receptor-containing cells, the rat pancreatic receptor level was calculated to be about 30% of the rat intestine. Sucrose density gradient sedimentation of this receptor yielded a value of 3.2 ± 0.1 (S) for the sedimentation coefficient and this peak was displaceable by a 100-fold excess of nonradioactive 1,25(OH)₂D₃. These data provide evidence for the presence of a specific 1,25(OH)₂D₃ receptor in mammalian pancreas, and we suggest, in conjunction with the facts that vitamin D₃ and its active metabolites play a physiological role on insulin secretion from rat pancreatic β-cells, that this receptor might be involved in the mechanisms of action of vitamin D₃ on insulin secretion from rat pancreas via a genomic effect.     

Effects of a novel 5-HT1A receptor agonist,E4424, on gastric adherent mucus levels following restraint stress in rats

Several novel arylpiperazine serotonin 1_A receptor agonists, developed as anxiolytics, have antisecretory and gastroprotective effects in rats. E4424 (2-}4-[4-(4-chloropyrazol-1-yl)butyl]-1-piperazinyl{pyrimidine; Lesopitron dihydrochloride), has potent anti-gastric secretory and antiulcer effects. Preliminary data indicated an enhancing effect of E4424 on gastric mucus that may underlie its gastroprotective actions. We therefore tested the effects of acute and chronic administration of E4424 and of a reference 5-HT_1A receptor agonist, 8-OHDPAT [8-hydroxy-2-(di-n-propylamino)tetralin], on gastric mucus levels in rats subjected to cold-restraint stress, a procedure associated with depletion of gastric mucus and the development of mucosal injury. Acute oral administration of E4424 increased adherent mucus levels by 12%, 11%, and 13%, relative to controls. Chronic E4424 significantly increased gastric mucus relative to controls (69% increase). Acute oral treatment with 8-OHDPAT did not affect gastric mucus level. Acute intraperitoneal 8-OHDPAT slightly increased mucus levels. Chronic twice per day 8-OHDPAT did not affect mucus levels; however, chronic once per day treatment with 8-OHDPAT significantly elevated gastric mucus levels at the highest doses used. For E4424, there is a strong correlation between reduction of gastric mucosal injury and increase in gastric mucus level, suggesting that the action of E4424 on glandular mucus levels is an important mechanism underlying its gastroprotective effects.     

Role of the 5-HT2AReceptor and α1-adrenoceptor in the Contractile Response of Rat Pulmonary Artery to 5-HT in the Presence and Absence of Nitric Oxide

This study investigated the role of 5-HT_2Areceptors andα₁ -adrenoceptors in the contractile response to 5-HT in the first branch pulmonary artery of the rat and their interaction with endogenous nitric oxide. 5-HT and phenylephrine induced concentration-dependent contractions. Theα₁ -adrenoceptor antagonists prazosin, HV723 and phentolamine produced concentration-dependent rightward shifts of the 5-HT concentration-response curves (CRC) consistent with an action at α₁-adrenoceptors. The 5-HT₂receptor antagonists ritanserin, ketanserin and methysergide produced rightward shifts that were less than would have been predicted for an action solely at 5-HT_2Areceptors. 5-HT and phenylephrine CRCs were shifted to the left by -NAME. Endothelium denudation also increased the tissue sensitivity to 5-HT. In the presence of -NAME, ketanserin produced greater antagonism of the 5-HT CRC but not the phenylephrine CRC. Ketanserin also produced greater antagonism of the 5-HT CRC in endothelium denuded rings compared with endothelium intact rings. These findings indicate (a) that both theα₁ -adrenoceptor class and the 5-HT_2Areceptor is involved in the contractile response to 5-HT; (b) in the presence of endogenous nitric oxide the contractile response to 5-HT is mediated predominently byα₁ -adrenoceptors; (c) inhibition of endogenous nitric oxide potentiates the 5-HT_2Areceptor-mediated component of the contraction.     

Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor

Defining key differences between agonist and antagonist binding to hormone receptors is important and will aid rational drug design. Glu(1.35) in transmembrane helix 1 (TM1) of the human oxytocin receptor (OTR) is absolutely conserved in all OTRs cloned to date. We establish that Glu(1.35) is critical for high affinity binding of agonists (full and partial) but is not required for antagonist binding (peptide or non-peptide). Consequently, the mutant receptor [E1.35A]OTR exhibited markedly decreased OT affinity (>1200-fold) and disrupted second messenger generation. Substitutions of Glu(1.35) by Asp, Gln or Arg were incapable of supporting wild-type OTR agonist binding or signaling. Molecular modeling revealed that Glu(1.35) projects into the receptor's central binding crevice and provides agonist-specific contacts not utilized by antagonists. This study explains why Glu is absolutely conserved at residue-1.35 in all receptors binding OT and related peptides, and provides molecular insight into key differences between agonist-receptor and antagonist-receptor binding modes.     

Safety, tolerability and efficacy of indacaterol, a novel once-daily beta(2)-agonist, in patients with COPD: a 28-day randomised, placebo controlled clinical trial

In patients with chronic obstructive pulmonary disease (COPD) classified as moderate onwards, Global Initiative for Chronic Obstructive Lung Disease (GOLD) Guidelines recommend regular treatment with one or more long-acting bronchodilators, such as beta(2)-agonists or anticholinergics. In contrast to currently available long-acting beta(2)-agonists, which have a duration of action of 12 h, indacaterol has demonstrated effective 24-h bronchodilation on once-daily dosing. A double-blind, randomised, placebo-controlled study was conducted to compare the safety, tolerability and efficacy of indacaterol with that of placebo, over a 28-day period, in patients with moderate COPD (as defined by GOLD 2001 criteria; equivalent to moderate-to-severe COPD in the GOLD 2005 criteria). Patients were randomised 2:2:1 to receive indacaterol 400 microg or 800 microg or placebo once-daily (between 07:00 and 11:00 h) via a single-dose dry-powder inhaler for 28 days. Assessments included monitoring of adverse events (AEs), blood chemistry (including serum potassium and blood glucose), vital signs (blood pressure and heart rate), electrocardiograms and spirometry. One hundred and sixty-three patients were randomised, with 155 (95%) completing the study. There were no statistically significant differences between treatment groups in the overall incidence of AEs, with AEs reported by 35%, 51% and 25% of patients in the indacaterol 400 microg, 800 microg and placebo groups, respectively. The majority of AEs were mild or moderate in severity, and there were no study-drug related serious AEs. There were no statistically significant differences between indacaterol groups and placebo in mean pulse rate and QTc interval, and isolated statistically significant (p<0.05) treatment-placebo differences in mean blood pressure, blood glucose and serum potassium. There was a statistically significant improvement in FEV(1) vs placebo at all post-baseline timepoints for both indacaterol treatment groups; 30 min post-dose, adjusted mean+/-SE FEV(1) indacaterol-placebo differences were: Day 1, 220+/-36 ml and 210+/-36 ml; Day 14, 320+/-50 ml and 270+/-50 ml; Day 28, 260+/-61 ml and 200+/-61 ml for 400 and 800 microg, respectively (all p<0.01 vs placebo). Bronchodilation was still apparent after 24h, with pre-dose (i.e. trough) adjusted mean+/-SE FEV(1) indacaterol-placebo differences of: Day 14, 230+/-44 ml and 210+/-44 ml; Day 28, 220+/-49 ml and 210+/-49 ml for indacaterol 400 and 800 microg, respectively (all p<0.0001 vs placebo). Once-daily indacaterol was well tolerated at doses up to 800 microg with a good overall safety profile. There was no statistical difference at any dose between the safety of indacaterol and placebo. Furthermore, this study supports the previously demonstrated 24-h bronchodilator efficacy of indacaterol.