Early diagnosis of bladder cancer through the detection of urinary tyrosine-phosphorylated proteins.

BACKGROUND: A noninvasive, highly sensitive and specific urine test is needed for bladder cancer (BC) diagnosis and surveillance in addition to the invasive cystoscopy. We previously described the diagnostic effectiveness of urinary tyrosine-phosphorylated proteins (UPY) and a new assay (UPY-A) for their measurement in a pilot study. The aim of this work was to evaluate the performances of the UPY-A using an independent cohort of 262 subjects. METHODS: Urinary tyrosine-phosphorylated proteins were measured by UPY-A test. The area under ROC curve, cutoff, sensitivity, specificity and predictive values of UPY-A were determined. The association of UPY levels with tumour staging, grading, recurrence and progression risk was analysed by Kruskal-Wallis and Wilcoxon's test. To test the probability to be a case if positive at the UPY-A, a logistic test adjusted for possible confounding factor was used. RESULTS: Results showed a significant difference of UPY levels between patients with BC vs healthy controls. For the best cutoff value, 261.26 Standard Units (SU), the sensitivity of the assay was 80.43% and the specificity was 78.82%. A statistically significant difference was found in the levels of UPY at different BC stages and grades between Ta and T1 and with different risk of recurrence and progression. A statistically significant increased risk for BC at UPY-A ⩾261.26 SU was observed. CONCLUSIONS: The present study supplies important information on the diagnostic characteristics of UPY-A revealing remarkable performances for early stages and allowing its potential use for different applications encompassing the screening of high-risk subjects, primary diagnosis and posttreatment surveillance.

A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients.

BACKGROUND: Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine. METHODS: The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated. RESULTS: Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform. CONCLUSIONS: A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker. GENERAL SIGNIFICANCE: The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine.

Use of porcine luteinizing hormone at oestrous onset in a protocol for fixed-time artificial insemination in gilts.

The aim of this study was to evaluate the effect of porcine luteinizing hormone (pLH) given at oestrous onset in gilts, by different routes and doses, on the interval between onset of oestrus and ovulation (IOEO) and reproductive performance using a single fixed-time artificial insemination (FTAI). A total of 153 gilts were submitted to oestrous detection at 8-h intervals and assigned to three groups: control - without hormone application and inseminated at 0, 24 and 48 h after oestrous onset; VS2.5FTAI - 2.5 mg pLH by the vulvar submucosal route at oestrous onset and a single FTAI 16 h later; IM5FTAI - 5 mg pLH by the intramuscular route at oestrous onset and a single FTAI 16 h later. More VS2.5FTAI gilts (47.1%; p < 0.05) ovulated within 24 h after oestrous onset than control gilts (25.5%) whereas IM5FTAI gilts had an intermediate percentage (31.4%; p > 0.05). The IOEO tended to be shorter (p = 0.06) in VS2.5FTAI (30.2 ± 1.4 h) than in control (34.7 ± 1.4 h) gilts, but there was no difference (p > 0.05) between control and IM5FTAI (32.8 ± 1.4 h) gilts. Farrowing rate was not different (p > 0.05) among treatments. Total born piglets (TB) was lower (p < 0.05) in VS2.5FTAI (12.3 ± 0.4) than in control gilts (14.1 ± 0.4), whereas intermediate TB was observed in IM5FTAI gilts (13.3 ± 0.4). Due to the advancement of ovulation, reduction of the hormonal dose and the ease of application, the vulvar submucosal route would be the best option for FTAI protocols, but their negative impact on litter size remains to be elucidated. Taking into account the good fertility results obtained in IM5FTAI gilts whose ovulation was not advanced, the possibility of a single FTAI without any hormonal treatment should be further investigated, to establish reliable FTAI protocols for gilts.

The post-cervical insemination does not impair the reproductive performance of primiparous sows.

The study evaluated the reproductive performance of primiparous sows submitted to post-cervical insemination (PCAI) compared with cervical artificial insemination (CAI). Difficulty with catheter introduction, the occurrence of bleeding or semen backflow during insemination, and volume and sperm cell backflow up to 60 min after insemination were also evaluated. Sows were homogenously distributed, according to body weight loss in lactation, lactation length, weaned piglets, weaning-to-oestrus interval and total born in previous farrowing, in two treatments: PCAI (n = 165) with 1.5 × 10(9) sperm cells in 45 ml (2.4 ± 0.04 doses per sow) and CAI (n = 165) with 3 × 10(9) sperm cells in 90 ml (2.5 ± 0.04 doses per sow). During PCAI, sows were inseminated in the absence of boars. Transabdominal real-time ultrasonography was performed at oestrus onset, immediately before the first insemination and at 24 h after last insemination. There was no difference (P > 0.05) between treatments in farrowing rate (91.5% × 89.1%) and litter size (12.5 × 11.9 piglets born, respectively for PCAI and CAI sows). Successful passage of the intrauterine catheter in all the inseminations was possible in 86.8% (165/190) of sows initially allocated to PCAI treatment. Difficulty of introducing the catheter in at least one insemination did not affect the reproductive performance of PCAI sows (P > 0.05). Bleeding during insemination did not affect (P > 0.05) the farrowing rate in both treatments, but litter size was reduced in CAI and PCAI sows (P ≤ 0.06). Percentage of spermatozoa present in backflow within 1 h after insemination was greater in CAI than PCAI sows (P < 0.01). More than 85% of primiparous sows can be successfully post-cervical inseminated with doses containing 1.5 × 10(9) sperm cells in the absence of the boar during insemination without impairing the reproductive performance.

Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.

Proposal of an improved prognostic classification for pT3 renal cell carcinoma.

PURPOSE: The prognostic accuracy of the current TNM 2002 staging system for locally advanced renal cell carcinoma has been questioned. To contribute to the development of a more accurate classification for this stage of disease we assessed the correlation between patterns of invasion in the pT3 category and outcomes in a large multi-institutional series. MATERIALS AND METHODS: Pathological data and clinical followup on 513 pT3 renal cell carcinoma cases treated with radical nephrectomy between 1983 and 2005 at 3 Italian academic centers were retrospectively reviewed. Cause specific survival rates were calculated with the Kaplan-Meier method and multivariate analysis was performed using the Cox proportional hazards regression model. RESULTS: Estimated overall 5-year cause specific survival was 50.1% at a median followup of 61.5 months in survivors. The current TNM classification was not a significant outcome prognosticator. Patients with a tumor invading only the perirenal or sinus fat were at lowest risk for death from the disease. Patients at intermediate risk had tumors with invasion of the venous system alone. Simultaneous perirenal fat and sinus fat invasion or perirenal fat and vascular invasion as well as adrenal gland involvement characterized high risk tumors. Low risk tumors could be further divided into 2 groups with different outcomes based on a size cutoff of 7 cm. Our classification was a significant predictor of survival on multivariate analysis as well as M stage, N stage, Fuhrman grade and tumor size. CONCLUSIONS: We confirm that the prognostic usefulness of the current 2002 TNM system for pT3 renal cell carcinoma is limited. We have identified 4 groups of tumors with distinct patterns of invasion and significantly different survival probabilities in this category. Large prospective series are needed to validate these findings.

Insulin modulation of an endothelial nitric oxide component present in the alpha2- and beta-adrenergic responses in human forearm.

We explored in 51 normal subjects, distributed in various series of experiments, whether endothelium nitric oxide may play a role in insulin modulation of alpha2- and beta-adrenergic- evoked vascular responses. In particular, we examined the forearm blood flow response (FBF, ml.min-1.dl-1) to intrabrachial infusion of BHT-933 (0.5, 1, and 2 microg.min-1.dl-1) or isoproterenol (1, 3, and 6 ng. min-1.dl-1) in control conditions, during intrabrachial infusion of insulin alone (0.05 mU.kg-1.min-1) and associated with l-N-monomethylarginine (L-NMMA) (0.05 microg.min-1.dl-1), a nitric oxide synthase inhibitor. In control conditions both BHT-933 and isoproterenol induced a dose-dependent vascular response. Local hyperinsulinemia (deep venous plasma insulin 68.5+/-4 microU/ml) did not change basal FBF whereas attenuated BHT-933 vasoconstriction and enhanced isoproterenol vasodilation. L-NMMA reduced basal FBF and abolished the insulin effect on BHT-933 and isoproterenol response. To clarify whether a nitric oxide component is included in alpha2- and beta-adrenergic response and may be responsible for insulin vascular effect, we further examined BHT-933 and isoproterenol responses during nitric oxide inhibition. Interestingly, L-NMMA potentiated the BHT-933 vasoconstriction and attenuated the isoproterenol vasodilation and, in these conditions, insulin was no more able to exhibit its vascular effects. Finally, to rule out the possibility that the conteracting effect of L-NMMA may not be specifically related to insulin action, dose-response curves to phenylephrine (0.5, 1, and 2 microg.min-1.dl-1) or sodium nitroprusside (1, 2, and 4 microg.min-1.dl-1) were also performed. Both insulin and L-NMMA were unable to alter the phenylephrine-induced vasoconstriction and the sodium nitroprusside vasodilation. In conclusion, our data demonstrate an endothelial nitric oxide component in the alpha2- and beta-adrenergic vascular responses which is the target of the insulin vascular action.

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.

Extralobar pulmonary sequestration showing high CA 19-19 levels.

We report a case of 40 year-old woman with left thoracic pain who was diagnosed as having a cystic mass located posteriorly at the basis of the left pleural cavity. The preoperative serum CA 19-9 level was increased (2 900 IU/mL). Further investigations did not find neoplastic lesions in the gastrointestinal tract. The intraoperative finding of an anomalous systemic arterial supply to the mass suggested the diagnosis of extralobar sequestration, confirmed at the histopathological examination. The association between pulmonary sequestration and increased tumor markers levels is overlooked in western literature, but it is often reported by many Japanese authors. This case report would underline the practical usefulness for preoperative diagnosis of pulmonary sequestration, when the CT-scan does not demonstrated an anomalous systemic vessel. The common embryogenic origin of both respiratory and digestive apparatus can explain the increased levels of tumor markers such as CA 19-9 and carcinoembryonic antigen in bronchogenic cyst, intestinal duplication and pulmonary sequestration.

Single fixed-time artificial insemination in gilts and weaned sows using pLH at estrus onset administered through vulvar submucosal route.

This study evaluated the use of a single fixed-time artificial insemination (FTAI) in gilts and weaned sows using 2.5 mg of porcine luteinizing hormone (pLH) administered through vulvar submucosal route, at the onset of estrus. In experiment 1 (Exp.1), 318 pubertal gilts were assigned to two groups: control-G-no hormonal application and artificial inseminations (AIs) at 12, 36, and 60 hours after the onset of estrus if they were still in standing estrus; and FTAI-G-use of pLH at the onset of estrus and a single FTAI 12 hours later. In experiment 2 (Exp. 2), 309 weaned sows were assigned to three groups: Control-S-no hormone application and AIs at 0, 24, and 48 hours after the onset of estrus if they were still in standing estrus; FTAI-NH-no hormone application and a single FTAI at 24 hours after the onset of estrus, and FTAI-pLH-use of pLH at the onset of estrus and a single FTAI 24 hours later. Transabdominal real time B-mode ultrasonography was performed to determine whether the insemination had been performed within 24 hours before ovulation, considered as the optimal interval. In Exp. 1, ultrasound evaluation (12-hour intervals) was carried out to determine the interval between the onset of estrus and ovulation. In both experiments, 2 × 10(9) sperm cells in 80 mL were used to perform cervical and postcervical deposition of semen in gilts and sows, respectively. Compared with control-G, FTAI-G gilts had shorter (P < 0.05) duration of estrus (57.7 vs. 61.2 hours) and interval between the onset of estrus and ovulation (36.3 vs. 42.3 hours). The adjusted farrowing rate (AFR) was lower (P < 0.05) in FTAI-G (86.0%) compared with control-G (93.5%), but total piglets born (TPB) did not differ between these groups (12.3 vs. 12.5 piglets). Within the FTAI-G group, the AFR was lower (P < 0.05) in the presence (50.0%) than in the absence (94.9%) of semen backflow during AI. Also in the FTAI-G group, the insemination outside the optimal interval reduced (P < 0.05) the TPB (10.5 vs. 12.9 piglets) in comparison with gilts inseminated within the optimal interval. In Exp. 2, there were no differences in the AFR (Control-S: 94.1%; FTAI-NH: 86.1%; FTAI-pLH: 88.0%) and TPB (Control-S: 12.8; FTAI-NH: 12.7, and FTAI-pLH: 12.0 piglets) among treatments. The presence of semen backflow reduced (P < 0.05) the TPB in FTAI-pLH and FTAI-NH sows. In the FTAI-pLH, a single insemination performed too late relative to ovulation reduced the AFR (P < 0.05) compared with sows inseminated within the optimal interval. In conclusion, 2.5-mg pLH applied at the vulvar submucosa at the onset of estrus advances the ovulation in gilts, but a single FTAI performed 12 hours later reduces the farrowing rate. A single FTAI performed at 24 hours after the onset of estrus in weaned sows does not affect their reproductive performance, regardless of pLH application.

Noradrenergic vascular hyper-responsiveness in human hypertension is dependent on oxygen free radical impairment of nitric oxide activity.

BACKGROUND: Noradrenergic vascular hyper-responsiveness is a hallmark of essential hypertension. To evaluate whether nitric oxide plays a role in the enhanced vascular response to norepinephrine in hypertension, we examined 32 hypertensives and 28 normotensives who were distributed in 3 experimental series. METHODS AND RESULTS: In the first series, we measured the forearm blood flow (FBF) response to a norepinephrine infusion under control conditions and during the infusion of L-N-monomethylarginine (L-NMMA). Norepinephrine evoked dose-dependent vasoconstriction that was greater in hypertensives than in normotensives (maximum FBF, -61+/-1 versus -51+/-1%; P<0.01). During L-NMMA infusion, norepinephrine vasoconstriction was not modified in hypertensives; however, it was potentiated in normotensives (maximum FBF, -64+/-2%; P<0.01). In the second series, we tested whether norepinephrine vasoconstriction could be affected by an antioxidant such as ascorbic acid. Norepinephrine vasoconstriction was blunted by ascorbic acid administration only in hypertensives (maximum FBF, -49+/-3 versus -63+/-2%; P<0.01); the vasoconstriction became similar to that observed in normotensives. During ascorbic acid plus L-NMMA administration, the vascular response to norepinephrine increased to a similar extent in both study groups. To rule out the possibility that the effect of ascorbic acid on norepinephrine vasoconstriction could depend on adrenergic receptor-induced nitric oxide release, in the last series we inhibited endogenous nitric oxide and replaced it with an exogenous nitric oxide donor (sodium nitroprusside). Even in these conditions, ascorbic acid attenuated norepinephrine vasoconstriction only in hypertensives (maximum FBF, -50+/-2 versus -62+/-1%; P<0.01). CONCLUSIONS: Our data demonstrate that noradrenergic vascular hyper-responsiveness in hypertension is dependent on an impairment of nitric oxide activity that is realized through norepinephrine-induced oxygen free radical production.

Fluid dynamics modeling of particulate deposition in the lungs.

Detailed knowledge of the transport of air and particles in the human lungs is needed for two reasons: the selection of the right dosage of aerosol drugs used in respiratory therapy and the analysis of the maximum allowable concentration for particulate in air. This work is the first step of a more complex study, purpose of which is to provide some predictive relationships in order to evaluate the depth reached by the particles in the lungs as a function of their size using numerical modeling. In this phase we validated our numerical method, comparing the obtained results with those found in the literature. The Computational Fluid Dynamics code FLUENT 6 with the Eulerian-Lagrangian approach was used to simulate particle trajectories. A model of double bifurcation,based on the morphometric studies by Weibel and Hammersley and Olson, was adopted in order to represent the whole central part of the respiratory system with the same geometry,appropriately scaled down. A method to create a realistic velocity profile at the inlet of the domain was developed, in order to obtain data about particle deposition also reliable about the first bifurcation, unlike previous works.

Characterization and leaching of NiCd and NiMH spent batteries for the recovery of metals.

Since NiMH and NiCd batteries are still used in the electronic devices market, a treatment and recycling plant has many advantages both from the environmental and the economic points of view. Unfortunately, there is no relationship between shape, size and chemical composition of spent batteries, consequently the characterization and the leaching method of the starting material becomes an important step of the overall treatment process in choosing the best conditions for the selective separation of the metals by hydrometallurgy. Leaching at 20 degrees C with H(2)SO(4) 2M for about 2h seems to be a good solution in terms of cost and efficiency for both battery types. The hydroxide compounds can be readily leached while Ni present as metallic form requires more aggressive conditions due to kinetic constraints. In this paper, the characterization of NiMH and NiCd spent batteries and the results of leaching tests in different conditions are reported.

Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK-Positive Anaplastic Large Cell Lymphoma.

UNLABELLED: ALK is a tyrosine kinase receptor involved in a broad range of solid and hematologic tumors. Among 70% to 80% of ALK(+) anaplastic large cell lymphomas (ALCL) are caused by the aberrant oncogenic fusion protein NPM-ALK. Crizotinib was the first clinically relevant ALK inhibitor, now approved for the treatment of late-stage and metastatic cases of lung cancer. However, patients frequently develop drug resistance to Crizotinib, mainly due to the appearance of point mutations located in the ALK kinase domain. Fortunately, other inhibitors are available and in clinical trial, suggesting the potential for second-line therapies to overcome Crizotinib resistance. This study focuses on the ongoing phase I/II trial small-molecule tyrosine kinase inhibitor (TKI) AP26113, by Ariad Pharmaceuticals, which targets both ALK and EGFR. Two NPM-ALK(+) human cell lines, KARPAS-299 and SUP-M2, were grown in the presence of increasing concentrations of AP26113, and eight lines were selected that demonstrated resistance. All lines show IC50 values higher (130 to 1,000-fold) than the parental line. Mechanistically, KARPAS-299 populations resistant to AP26113 show NPM-ALK overexpression, whereas SUP-M2-resistant cells harbor several point mutations spanning the entire ALK kinase domain. In particular, amino acid substitutions: L1196M, S1206C, the double F1174V+L1198F and L1122V+L1196M mutations were identified. The knowledge of the possible appearance of new clinically relevant mechanisms of drug resistance is a useful tool for the management of new TKI-resistant cases. IMPLICATIONS: This work defines reliable ALCL model systems of AP26113 resistance and provides a valuable tool in the management of all cases of relapse upon NPM-ALK-targeted therapy.

Factors that influence peak bone mass formation: a study of calcium balance and the inheritance of bone mass in adolescent females.

We suggested that calcium may be an important determinant of peak bone mass. For further elucidation, calcium balances in adolescent females with different calcium intakes (270-1637 mg/d), and a 2-y intervention study of calcium supplementation were performed. Hereditary influences on bone status were also evaluated by comparing subjects' and parents' bone mass. The main determinant of calcium balance was calcium intake; net calcium absorption increased with intake and urinary calcium did not change. Adolescent females retained 200-500 mg Ca/d, suggesting that inadequate calcium intake may translate into inadequate calcium retention and a reduction in peak bone mass. There was a more pronounced increase in bone mass over time in the calcium-supplemented group (1640 mg Ca/d) than in the control group (750 mg Ca/d), but the differences between bone mass measurements were not statistically significant, possibly because of a type II error. By the age of 16 y daughters had accumulated 90-97% of the bone mass of their premenopausal mothers.

The effects of novel, selective 5-hydroxytryptamine (5-HT)4 receptor ligands in rat spatial navigation.

Activation of central 5-hydroxytryptamine (5-HT4) receptors may enhance cognitive performance. In the present study, the effects of two novel, potent and selective 5-HT4 receptor agonists, RS 67333 (1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-n-burtl-4-piperidinyl)- 1-propanone) and RS 67506 (1-(4-amino- 5-chloro-2-methoxyphenyl)-3-[1-[2-[(methylsulfonyl)amino]ethyl]-4- piperidinyl]-1-propanone), were studied in a rat model of spatial learning and memory; the Morris water maze. RS 67333 (0.1, 10 and 1000 micrograms/kg, intraperitoneally (i.p.)), a highly potent, selective and hydrophobic 5-HT4 receptor agonist, reversed the decrements in cognitive performance induced by atropine (30 mg/kg, i.p.). By contrast, no effect was seen to RS 67506 (0.1, 10 and 1000 micrograms/kg, i.p.), a hydrophilic 5-HT4 receptor agonist, of equivalent potency and selectivity to RS 67333. This differential effect may reflect the enhanced ability of RS 67333 to enter the CNS, with respect to RS 67506. The ameliorative actions of RS 67333 on cognitive dysfunction were abolished by prior treatment with a selective 5-HT4 receptor antagonist, RS 67532 [1-(4-amino-5-chloro-2-(3, 5-dimethoxy benzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone; 1 mg/kg, i.p.]. When given alone, or in naive rats, RS 67532 (0.1, 10 and 1000 micrograms/kg, i.p.), was without effect. None of the compounds tested affected the swim speed at any of the doses used. In separate locomotor studies, RS 67532 reduced activity at 1 and 10 mg/kg, i.p., although no effect was seen with RS 67333 or RS 67506 (0.01-10 mg/kg, i.p.). These data suggest that RS 67333 reversed the cognitive deficit induced by atropine and support a role of 5-HT4 receptors in rat spatial learning and memory.

Stereoselective effects of (R)- and (S)-zacopride on cognitive performance in a spatial navigation task in rats.

In the present studies we investigated the actions of (R)- and (S)-zacopride, potent 5-HT3 receptor antagonists with 5-HT4 receptor agonists properties, on performance in a spatial learning and memory task in rats, the Morris water maze. A significant cognitive/performance deficit, as indicated by the increased escape latency across several trials, was produced by systemic administration of the muscarinic receptor antagonist atropine (30 mg/kg, IP). (R)-zacopride (0.001-1 microgram/kg, but not 10 or 100 micrograms/kg) significantly reduced escape latency in atropine-treated animals. (S)-Zacopride was inactive over the entire dose range examined (0.001-100 micrograms/kg, i.p.). Moreover, pretreatment with (S)-zacopride (1 or 100 micrograms/kg) did not alter the procognitive effects of (R)-zacopride (1 microgram/kg). These data demonstrate that the cognition enhancing properties of zacopride in this model of cholinergic hypofunction are exclusive to its (R)-enantiomer and imply that this action is unrelated to 5-HT, receptor antagonism or 5-HT4 receptor agonism. The possibility that the procognitive effects of (R)-zacopride may be related to actions at the novel "(R)-zacopride site" is discussed.

Comparison of potencies of 5-HT3 receptor antagonists at inhibiting aversive behavior to illumination and the von Bezold-Jarisch reflex in the mouse.

The inhibitory effects of ondansetron and R and S zacopride on aversive behavior to light and the von Bezold-Jarisch reflex have been compared in mouse. The potencies (ID50, microgram/kg i.v.) of compounds at inhibiting the von Bezold-Jarisch reflex, elicited by 2-methyl-5-HT (mouse 100 micrograms/kg, i.v.; rat 10-80 micrograms/kg i.v.) were: S zacopride (0.02), granisetron (0.17), R zacopride (0.30) ondansetron (3.16). A similar rank order of ID50 values was observed in rat, i.e. S zacopride (0.02), granisetron (0.36), R zacopride (0.25) and ondansetron (2.65). These data suggest that the activity of compounds at 5-HT3 receptors mediating this effect was similar in both mouse and rat. In mouse behavioral studies, ondansetron and R and S zacopride potently inhibited aversive behavior to light (0.0003-30 micrograms/kg, p.o.), when the amount of time spent in the dark and locomotor activity were measured. Thus, at 0.3 ng/kg, the mean percentage time spent in the dark significantly decreased from 84 to 72, for both R and S zacopride, respectively. The maximal effects of these compounds were modest in comparison to the 'anxiolytic' effects of diazepam (0.3-1.4 mg/kg, i.p.; at 0.3 mg/kg the mean percentage time spent in the dark significantly decreased from 84 to 36) or chlordiazepoxide (3-40 mg/kg, i.p.; at 3 mg/kg, the mean percentage time spent in the dark significantly decreased from 85 to 40). The doses of the 5-HT3 antagonists were approx 1000-fold lower than those effective inhibitory doses determined in the von Bezold-Jarisch reflex studies, in either mouse or rat.(ABSTRACT TRUNCATED AT 250 WORDS)