Amperometry approach curve profiling to understand the regulatory mechanisms governing the concentration of intestinal extracellular serotonin.

Enterochromaffin (EC) cells located within the intestinal mucosal epithelium release serotonin (5-HT) to regulate motility tones, barrier function and the immune system. Electroanalytical methodologies have been able to monitor steady state basal extracellular 5-HT levels but are unable to provide insight into how these levels are influenced by key regulatory processes such as release and uptake. We established a new measurement approach, amperometry approach curve profiling, which monitors the extracellular 5-HT level at different electrode-tissue (E-T) distances. Analysis of the current profile can provide information on contributions of regulatory components on the observed extracellular 5-HT level. Measurements were conducted from ex vivo murine ileum and colon using a boron-doped diamond (BDD) microelectrode. Amperometry approach curve profiling coupled with classical pharmacology demonstrated that extracellular 5-HT levels were significantly lower in the colon when compared to the ileum. This difference was due to a greater degree of activity of the 5-HT transporter (SERT) and a reduced amount of 5-HT released from colonic EC cells. The presence of an inhibitory 5-HT4 autoreceptor was observed in the colon, where a 40% increase in extracellular 5-HT was the half maximal inhibitory concentration for activation of the autoreceptor. This novel electroanalytical approach allows estimates of release and re-uptake and their contribution to 5-HT extracellular concentration from intestinal tissue be obtained from a single series of measurements.

Age-Related Changes in Central Nervous System 5-Hydroxytryptamine Signalling and Its Potential Effects on the Regulation of Lifespan.

Serotonin or 5-hydroxytryptamine (5-HT) is an important neurotransmitter in the central nervous system and the periphery. Most 5-HT (~99%) is found in the periphery where it regulates the function of the gastrointestinal (GI) tract and is an important regulator of platelet aggregation. However, the remaining 1% that is found in the central nervous system (CNS) can regulate a range of physiological processes such as learning and memory formation, mood, food intake, sleep, temperature and pain perception. More recent work on the CNS of invertebrate model systems has shown that 5-HT can directly regulate lifespan.This chapter will focus on detailing how CNS 5-HT signalling is altered with increasing age and the potential consequences this has on its ability to regulate lifespan.

Decreased behavioural and neurochemical effects of angiotensin IV following prenatal alcohol exposure in the mouse.

Angiotensin IV (ang IV) is known to improve learning and memory in animal models but the mechanism is unclear. We have previously demonstrated sex differences in the pro-cognitive effects of ang IV, and that prenatal alcohol exposure (PAE) abolishes these effects. This study aimed to explore a possible mechanism underlying the sex differences and the effects of PAE in male mice. Mouse breeding harems received 5% ethanol in drinking water throughout pregnancy and lactation in a two-bottle schedule. The effects of ang IV were assessed in offspring at 4 months of age using the open field test, novel object recognition test and elevated plus maze. Aminopeptidase activity of brain insulin-regulated aminopeptidase (IRAP), a putative target of ang IV, was determined. As seen in a previous similar study, ang IV administered immediately after the second training trial significantly improved novel object recognition 24 h later in male mice but not female. PAE abolished this pro-cognitive effect in males. PAE also increased anxiety-like behaviour in male but not female offspring. Ang IV decreased the aminopeptidase activity of brain IRAP in control male, but not female, mice; PAE abolished this inhibitory effect. Ang IV improved memory consolidation in male but not female mice and PAE abolished this effect in the males. While the effects of PAE may be related to increased anxiety; ang IV decreased the aminopeptidase activity in male but not female mice and PAE abolished this inhibitory effect. The results therefore suggest that improvements in learning and memory induced by peripheral administration of ang IV correlate with a reduction of the enzyme activity of IRAP. This is the first demonstration that ang IV administered peripherally can induce long-term (24 h) changes in IRAP function which are probably not simple competitive inhibition and the first demonstration that PAE alters IRAP activity.

Valoración de la elección de tratamiento conservador en la enfermedad renal crónica / Assessment of the choice of conservative treatment in chronic kidney disease

Introducción: El tratamiento conservador es una opción de tratamiento en la enfermedad renal crónica. Esta elección es decisión del paciente y/o familia. Objetivo: Analizar el proceso de elección de tratamiento conservador, identificar el perfil de paciente que lo elige, su supervivencia y quien comunica la elección. Material y Método: Estudio observacional, retrospectivo. Se incluyeron pacientes que eligieron tratamiento conservador entre 2010-2017. Los datos se obtuvieron de los registros de enfermería en historia clínica. Se recogieron variables demográficas, supervivencia, Índice de Comorbilidad de Charlson, índice de Barthel y valores de los pacientes utilizando la herramienta Tarjetas de valores. Resultados: Se estudiaron 95 pacientes: 41,05% hombres, edad media 82,36±9 años, 27,37% institucionalizados. La familia comunicó la elección en el 62,11% de los casos. La media del filtrado glomerular al inicio de la información fue 11,53±2,73 ml/min, mediana del Charlson 8 (13-3), Barthel 55 (100-0) puntos. En el proceso de evidenciar valores, la tarjeta más elegida fue "personal sanitario responsable del tratamiento". La supervivencia media fue 496,19 días±553,8. Viven menos los hombres y los institucionalizados, sin diferencia significativa. El riesgo de muerte es mayor, al aumentar el Charlson y disminuir el filtrado glomerular (p=0,01). La familia comunicó la elección de seguir tratamiento conservador en el 62,11% de los casos. Conclusiones: El paciente que opta por tratamiento conservador es, una persona anciana, dependiente, con comorbilidades, supervivencia media en torno a 18 meses y en más de la mitad de los casos es la familia quien comunica la decisión de optar por ese tratamiento

Introduction: Conservative treatment is a treatment option in chronic kidney disease. The election of this treatment is determined by patient and / or the family. Objective: The objective of the study was to analyze the process of choosing conservative treatment, identify the patient profile, the patient' survival and who communicate the election. Material and Method: Observational, retrospective study. Patients who chose conservative treatment between 2010-2017 were included. The data were obtained from nursing records in clinical history. Demographic variables, survival, Charlson Comorbidity Index, Barthel Index and patient values were collected using the Values Cards tool. Resultados: The sample was 95 patients, 41.05% men, mean age 82.36±9 years, 27.37% institutionalized. The family reported the choice in 62.11% of the cases. The mean glomerular filtration rate at the beginning of the information was 11.53±2.73 ml/min, the median of Charlson index: 8 (13-3), median of Barthel index: 55 (100-0) points. In the process of contrasting values, the most chosen card was "health personnel responsible for treatment". The median survival was 496.19±553.8 days. Men and the institutionalized live less, without significant difference. The risk of death is greater, as the Charlson index increases and the glomerular filtration rate decreases (p=0.01). The family announced the election to continue conservative treatment in 62.11% of cases. Conclusions: Patients who opts for conservative treatment are elderly persons, dependents, with comorbidities, average survival of 18 months, and in more than half of the cases, the family is the one that communicates the decision to opt for that treatment

Prenatal alcohol exposure reduces 5-HT concentration in mouse intestinal muscle and mucosa.

The influence of prenatal alcohol exposure on the serotoninergic system in the brain has been well studied, however its influence on the serotoninergic system in the gastrointestinal system remains unknown. The objective of the study was to use a mouse model of prenatal alcohol exposure to investigate the effects on serotonin and its metabolites and precursors in colonic tissue. This study used treatment of mouse breeding harems with 5% ethanol with saccharin via drinking water throughout pregnancy and compared the results with a saccharin control group. Tryptophan, serotonin (5-HT) and 5- hydroxyindoleacetic acid (5-HIAA) concentrations were measured in the longitudinal muscle myenteric plexus (LMMP) and mucosa of intestinal tissue by high-performance liquid chromatography (HPLC). Decreased 5-HT concentrations in mucosa and LMMP (females only) were observed in prenatally exposed mice compared to controls. Increases in mucosal and LMMP tryptophan concentration were only observed in prenatally exposed female mice. In conclusion, prenatal alcohol exposure causes a decrease in conversion of tryptophan to 5-HT in both muscle and mucosa although the effect is more pronounced in females. The observed sex difference may be related to changes associated with the estrous cycle.

Low-dose chronic prenatal alcohol exposure abolishes the pro-cognitive effects of angiotensin IV.

Prenatal ethanol exposure (PAE) in humans results in a spectrum of disorders including deficits in learning and memory. Animal models to date have typically used high ethanol doses but have not identified the biochemical changes underlying the cognitive deficit. This study used treatment of mouse breeding harems with 5% ethanol via drinking water throughout pregnancy and lactation and explored the behavioural consequences in the progeny at 3-6 months of age using the open field test, novel object recognition test and elevated plus maze to measure anxiety and memory consolidation. The effects of angiotensin IV on behaviour of the progeny were also determined. The results indicated that PAE increased anxiety-like behaviour as determined in the open field test in male but not female progeny. In control animals, angiotensin IV enhanced memory consolidation in males, but this effect was abolished by PAE. The abolition of the pro-cognitive effect of angiotensin IV was not a consequence of increased anxiety, and there was some evidence of a long-lasting anxiolytic effect of angiotensin IV in the male PAE progeny. These results suggest that PAE may act via alteration of the actions of the brain renin-angiotensin system to impair memory consolidation, but these effects may be partially sex-dependent.

The TNF-α antagonist etanercept reverses age-related decreases in colonic SERT expression and faecal output in mice.

Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age-related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24-month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg-1). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to "anti-TNFα" drugs as potential treatments for age-related chronic constipation.

Changes of renin-angiotensin system-related aminopeptidases in early stage Alzheimer's disease.

Activities of aminopeptidases A, B, and N (ApA, ApB & ApN) and insulin-regulated aminopeptidase (IRAP) have been seen to be decreased amongst patients with Alzheimer's disease (AD). All of these enzymes are involved with the brain renin-angiotensin system which is believed to be involved with learning and memory. This study aimed to explore the time course and the mechanisms underlying these changes. Serum samples were collected from 45 AD patients at the start of the study, and again 13months later (n=37). The control group was 22 healthy, older, adults. Enzyme activity was determined at two substrate concentrations to allow Michaelis-Menten analysis of the enzyme activity. The results indicated that there was decreased activity of ApA, ApB and ApN amongst AD patients but no difference in serum IRAP activity. There were no associations between enzyme activity and age, gender nor scores on psychomotor tests. Consideration of the data for the two time points for AD patients showed that the changes in ApB occurred at an early stage of the disease and persisted, whilst those of ApA and ApN only became apparent at later stages of the disease. Although differences in Michaelis-Menten parameters were not statistically significant, consideration of the values suggested that the decrease in ApB activity may be a result of changes in enzyme protein conformation, whilst that of ApN may be a consequence of decreased enzyme expression. Importantly, the different time courses of the effects and the differential changes in enzyme affinity and expression indicated that the observed changes with progression of AD were not a 'class effect' for serum aminopeptidases but were idiosyncratic for the individual enzymes.

Acute paraquat exposure impairs colonic motility by selectively attenuating nitrergic signalling in the mouse.

Paraquat, a common herbicide, is responsible for large numbers of deaths worldwide through both deliberate and accidental ingestion. Previous studies have eluded that the bioavailability of paraquat increases substantially with increasing dose and that these changes may in part be due to the effects that these high concentrations have on the gastrointestinal tract (GI tract). To date, the actions of acute, high concentrations (20mM for 60 min) of paraquat on the GI tract, particularly the colon which is a major site of paraquat absorption, are unknown. This study examined the effects of acute paraquat administration on colonic motility in the C57BL/6 mouse. Acute paraquat exposure decreased colonic motility and the amplitude of colonic migrating motor complexes (CMMCs), which are major motor patterns involved in faecal pellet propulsion. In isolated segments of distal colon, paraquat increased resting tension and markedly attenuated electrical field stimulation-evoked relaxations. Pharmacological dissection of paraquat's mechanism of action on both the CMMCs and field stimulated tissue using the nitric oxide synthase inhibitor NG-nitro-L-arginine and direct measurement of NO release from the myenteric plexus, demonstrated that paraquat selectively attenuates nitrergic signalling pathways. These changes did not appear to be due to alterations in colonic oxidative stress, inflammation or complex 1 activity, but were most likely caused by paraquat's ability to act as a redox couple. In summary, these data demonstrate that acute paraquat exposure attenuates colonic transit. These changes may facilitate the absorption of paraquat into the circulation and so facilitate its toxicity.

Impaired colonic motility and reduction in tachykinin signalling in the aged mouse.

Ageing is associated with an increased incidence of constipation in humans. The contribution that the ageing process makes to this condition is unclear. The aim of this study was to determine the effects of age on faecal output and colonic motility in male C57BL/6J mice and to determine the role that altered tachykinin signalling plays in this process. Total faecal output recorded over a 24h period decreased with age due to a reduction in the number of pellets produced and their water content. These changes occurred in the absence of any significant change in food and water intake. There was an increase in the amount of faecal matter stored in the isolated colon with age which caused a proportional increase in colonic length. Analysis of colonic motility using an artificial pellet demonstrated that pellets moved in a stepwise fashion through the colon. There was an age-related increase in pellet transit time due to decreases in the step distance, velocity, and frequency of stepwise movements. These changes were reversed using the neurokinin 2 (NK2) receptor agonist neurokinin A. Addition of the NK2receptor antagonist GR159897 significantly increased transit time in the young animals by decreasing step distance, velocity and frequency, but was without effect in the aged colon. In summary, the ageing C57BL/6J mouse shows an impaired motility phenotype. These effects appear, at least in part, to be due to an attenuation of tachykinin signalling via NK2 receptors.

Age-related changes in melatonin release in the murine distal colon.

Constipation and fecal impaction are conditions of the bowel whose prevalence increases with age. Limited information is known about how these conditions manifest; however, functional deficits are likely to be due to changes in signaling within the bowel. This study investigated the effects of age on colonic mucosal melatonin (MEL) release and the consequences this had on colonic motility. Electrochemical measurements of MEL overflow demonstrated that both basal and mechanically stimulated MEL release decreased with age. The MEL/serotonin also decreased with increasing age, and the trend was similar to that of MEL overflow, suggestive that age-related changes were primarily due to a reduction in MEL levels. Levels of N-acetylserotonin and the N-acetylserotonin/serotonin ratio were reduced with age, providing an explanation for the reduction in MEL release. Decreases in colonic motility were observed in animals between 3 and 24 months old. Exogenous application of MEL could reverse this deficit in aged colon. In summary, we propose that the age-related decline in MEL release may be due to either decreases or alterations in mechanosensory channels and/or a loss in levels/activity of the N-acetyltransferase enzyme responsible for the synthesis of N-acetylserotonin. Decreases in MEL release may explain the decreases in colonic motility observed in 24 month old animals and could offer a new potential therapeutic treatment for age-related constipation.

Serotonin: from top to bottom.

Serotonin is a monoamine neurotransmitter, which is phylogenetically conserved in a wide range of species from nematodes to humans. In mammals, age-related changes in serotonin systems are known risk factors of age-related diseases, such as diabetes, faecal incontinence and cardiovascular diseases. A decline in serotonin function with aging would be consistent with observations of age-related changes in behaviours, such as sleep, sexual behaviour and mood all of which are linked to serotonergic function. Despite this little is known about serotonin in relation to aging. This review aims to give a comprehensive analysis of the distribution, function and interactions of serotonin in the brain; gastrointestinal tract; skeletal; vascular and immune systems. It also aims to demonstrate how the function of serotonin is linked to aging and disease pathology in these systems. The regulation of serotonin via microRNAs is also discussed, as are possible applications of serotonergic drugs in aging research and age-related diseases. Furthermore, this review demonstrates that serotonin is potentially involved in whole organism aging through its links with multiple organs, the immune system and microRNA regulation. Methods to investigate these links are discussed.

Chromatographic analysis of age-related changes in mucosal serotonin transmission in the murine distal ileum.

BACKGROUND: In the upper bowel, alterations in motility and absorption of key nutrients have been observed as part of the normal ageing process. Serotonin (5-HT) is a key signalling molecule in the gastrointestinal tract and is known to influence motility, however little is known of how the ageing process alters 5-HT signalling processes in the bowel. RESULTS: An isocratic chromatographic method was able to detect all 5-HT precursors and metabolites. Using extracellular and intracellular sampling approaches, we were able to monitor all key parameters associated with the transmission process. There was no alteration in the levels of tryptophan and 5-HTP between 3 and 18 month old animals. There was a significant increase in the ratio of 5-HT:5-HTP and an increase in intracellular 5-HT between 3 and 18 month old animals suggesting an increase in 5-HT synthesis. There was also a significant increase in extracellular 5-HT with age, suggesting increased 5-HT release. There was an age-related decrease in the ratio of intracellular 5-HIAA:extracellular 5-HT, whilst the amount of 5-HIAA did not change with age. In the presence of an increase in extracellular 5-HT, the lack of an age-related change in 5-HIAA is suggestive of a decrease in re-uptake via the serotonin transporter (SERT). CONCLUSIONS: We have used intracellular and extracellular sampling to provide more insight into alterations in the neurotransmission process of 5-HT during normal ageing. We observed elevated 5-HT synthesis and release and a possible decrease in the activity of SERT. Taken together these changes lead to increased 5-HT availability and may alter motility function and could lead to the changes in adsorption observed in the elderly.

Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.

Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca(2+). As an "adhesion G-protein-coupled receptor," LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, we used LPH1's extracellular domain to purify its endogenous ligand. A single protein of ∼275 kDa was isolated from rat brain and termed Lasso. Peptide sequencing and molecular cloning have shown that Lasso is a splice variant of teneurin-2, a brain-specific orphan cell surface receptor with a function in neuronal pathfinding and synaptogenesis. We show that LPH1 and Lasso interact strongly and specifically. They are always copurified from rat brain extracts. Coculturing cells expressing LPH1 with cells expressing Lasso leads to their mutual attraction and formation of multiple junctions to which both proteins are recruited. Cells expressing LPH1 form chimerical synapses with hippocampal neurons in cocultures; LPH1 and postsynaptic neuronal protein PSD-95 accumulate on opposite sides of these structures. Immunoblotting and immunoelectron microscopy of purified synapses and immunostaining of cultured hippocampal neurons show that LPH1 and Lasso are enriched in synapses; in both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. A C-terminal fragment of Lasso interacts with LPH1 and induces Ca(2+) signals in presynaptic boutons of hippocampal neurons and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can form transsynaptic complexes capable of inducing presynaptic Ca(2+) signals, which might affect synaptic functions.

Alternatively spliced protein variants as potential therapeutic targets for male infertility and contraception.

Mammalian sperm were previously shown to express the PP1gamma2 isoform of protein phosphatase 1 (PP1) as well as its regulatory proteins inhibitor 2 and glycogen synthase kinase 3. Furthermore, the development of sperm motility during transit through the epididymis correlates with changes in PP1 activity. Thus, since PP1 cellular activity is determined by the partners it binds, we embarked on a study aimed at defining the specific interactomes of PP1gamma1 and PP1gamma2 (the two known alternatively spliced variants of PP1gamma). To this end, exhaustive screens were performed on a human testis cDNA library using the yeast two-hybrid method. Among the various proteins detected, the most abundant interactors with PP1gamma2 were Nek2A and R15B. Closer sequence analysis revealed novel alternatively spliced variants of Nek2A and NIPP1, which we designated Nek2A-T and NIPP1-T, respectively. They were shown to be highly expressed in rat and human testis by Northern analysis and to result from alternative splicing events by RT-PCR. Thus, both the previously known Nek2A isoform and the novel Nek2A-T and NIPP1-T variants appear to bind PP1gamma2 in vitro (blot overlays) and in vivo by coexpression in yeast. The usefulness of testis-specific alternatively spliced proteins as targets for the development of novel therapeutic strategies for male infertility and contraception is discussed. PP1gamma2, Nek2A-T, and NIPP1-T are currently being investigated as alternatively spliced targets for signal transduction therapeutics.