On the function of proton-gated ion channels ASICs and TRPV1 : a patch-clamp study

AbstractAcidosis is encountered during tissue inflammation and triggers pain in humans. H+-gated ion channels are expressed at high levels in sensory neurons of the peripheral nervous system. Ion channels from two different families present the required pH sensitivity to detect the acidosis associated with peripheral inflammation: Acid-Sensing Ion Channels (ASICs) and the Transient Receptor Potential Vanilloid-1 (TRPV1) channel.ASICs are members of the Degenerin/Epithelial Na+ Channel family of ion channels. Six ASIC subunits have been identified in mammals (ASICla, -lb, -2a, -2b, -3 and -4). ASICs form In-activated voltage-insensitive homo- or heterotrimeric Na+ channels. TRPV1 is a member of the TRP family of ion channels and forms non-selective cation channels that mediate a sustained current. TRPV1 is activated by H+, heat (T>43°C), lipids, capsaicin, voltage and other stimuli. A stimulus can increase TRPV1 response to a different stimulus. For example H+ can shift the capsaicin concentration dependence of TRPV1 to lower values. ASICs and TRPV1 have been shown to be involved in inflammatory pain. Using the patch-clamp technique, we studied different aspects of the function of ASICs and TRPV1 in the physiological context of pain.In the first part of this thesis, we characterize the effect of a temperature increase from 25 to 35°C on the function of ASICs and TRPV1 in transfected CHO cells and primary cultures of rat DRG sensory neurons. ASICs give rise to transient currents while TRPV1 mediates a sustained current. In addition, ASICs and TRPV1 respond to H+ with distinct pH dependences. We assess the relative contribution of ASICs and TRPV1 to H+-evoked electrical signaling in rat DRG neurons and we conclude that ASICs are the most important pH sensors in the pH range 7.4 to 6.0 at 35°C in sensory neurons.ASICs and TRPV1 are expressed in the epithelium lining the lumen of the bladder (urothelium). The Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) is a painful condition associated with a dysfunction of the urothelial barrier and with inflammation. In the second part of this thesis, we show that human urothelial cells -the cell line TEU2 and primary cultures of human bladder urothelium- express functional ASICs but no functional TRPV1 channels. In addition, we show that the levels of ASIC2 and ASIC3 mRNA are increased in the urothelium of patients suffering from BPS/IC. These data suggest that ASICs are involved in the pathology of BPS/IC.Finally, we demonstrate that APETx2 inhibits the sensory neuron specific voltage-dependent Na+ channel Nav1.8. APETx2 was previously shown to inhibit homo- or heterotrimeric ASIC3- containing channels with IC5o from 0.08 to 1 μΜ. We show that APETx2 also inhibits Nav1.8 with an ICsoof «2.6 μΜ. APETx2 reduces the maximal conductance and induces a depolarizing shift in the voltage dependence of activation of Nav1.8. In current-clamp experiments, APETx2 reduces the number of action potentials (APs) evoked by a current ramp. Nav1.8 mediates most of the current during the AP upstroke and has been shown to be an important mediator of inflammatory pain. The fact that APETx2 inhibits two ion channels involved in inflammatory pain suggests that APETx2 or derivatives may represent novel analgesic compounds.RésuméL'acidose tissulaire est observée durant l'inflammation et entraine la douleur chez l'humain. Des canaux ioniques activés par les protons (H+) sont fortement exprimés dans les neurones sensoriels du système nerveux périphérique. De ceux-ci, les Acid-Sensing Ion Channels [ASICs) et Transient Receptor Potential Vanilloid-1 (TRPV1) présentent une sensibilité adéquate à l'acidité pour servir de détecteurs d'acidose.Les ASICs sont membres de la famille Degenerin/Epithelial Na* Channel. Six sous-unités ASIC ont été identifiées chez les mammifères (ASICla, -lb, -2a, -2b, -3 et -4). Les ASICs forment des canaux sélectifs au Na\ insensibles au voltage et activés par les H+. Les canaux fonctionnels sont des homo- ou hétérotrimères de sous-unités ASIC. TRPV1 est un membre de la famille TRP de canaux ioniques. Les canaux TRPV1 sont activés par les H+, la chaleur (T>43°Ç), les lipides, la capsaicine, le voltage et d'autres stimulus. L'activation de TRPV1 entraine un courant soutenu non-sélectif. Un stimulus peut augmenter la réponse de TRPV1 à un autre stimulus. Les H+ peuvent, par exemple, induire un décalage vers des valeurs plus faibles de la courbe de dépendance à la concentration de TRPV1 pour la capsaicine. Il a été démontré que les ASICs et TRPV1 sont impliqués dans la douleur inflammatoire. En utilisant la technique du patch-clamp, nous avons étudié différents aspects de la fonction des ASICs et de TRPV1 dans des contextes associés à la douleur.Dans la première partie de cette thèse, nous caractérisons l'effet d'une augmentation de température de 25 à 35°C sur la fonction des canaux ASICs et TRPV1, dans des cellules CHO transfectées et dans des cultures primaires de neurones sensoriels (DRG) de rat. L'activation des ASICs entraine l'apparition d'un courant transitoire tandis que l'activation de TRPV1 entraine un courant soutenu. De plus, les ASICs et TRPV1 possèdent des dépendances au pH différentes. Nous évaluons la contribution relative des ASICs et de TRPV1 au signalement électrique induit par les H+ et nous concluons que les ASICs sont les senseurs d'acidité les plus importants dans les neurones sensoriels, dans le domaine de pH de 7.4 à 6.0, à température corporelle.Les ASICs et TRPV1 sont exprimés dans l'épithélium recouvrant l'intérieur de la vessie (l'urothélium). Le Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) est une condition médicale douloureuse associée à une dysfonction de la barrière urothéliale et à une inflammation. Dans la seconde partie de cette thèse, nous démontrons que des cellules urothéliales (de la lignée cellulaire TEU2) et des cellules provenant de cultures primaires d'épithéliums de vessies humaines expriment des canaux ASIC fonctionnels mais pas de TRPV1 fonctionnels. De plus, nous montrons que le niveau d'expression de ASIC2 et -3 est augmenté dans l'urothélium de la vessie de patients souffrant de BPS/IC. Ces données suggèrent que les ASICs sont impliqués dans la pathologie BPS/IC.Pour finir, nous démontrons que la toxine APETx2 inhibe le canal spécifique aux neurones sensoriels Nav1.8, un membre de la famille des canaux sodiques dépendants du potentiel. Il a été démontré précédemment que la toxine APETx2 inhibe les canaux contenant une ou plusieurs sous-unités ASIC3 avec un ICso entre 0.08 et 1 μΜ. Nous montrons que la toxine APETx2 inhibe Nav1.8 avec un IC50 de «2.6 μΜ. La toxine APETx2 réduit la conductance maximale et induit un décalage de la dépendance au potentiel de Nav1.8 vers des valeurs plus positives. Dans des expériences de courant imposé sur des neurones sensoriels, la toxine APETx2 réduit le nombre de potentiels d'action induits par une rampe de courant. Nav1.8 est responsable de la majeure partie du courant durant la phase ascendante du potentiel d'action et a été démontré comme étant un médiateur important de la douleur inflammatoire. L'inhibition de deux types de canaux, impliqués dans la douleurs inflammatoire, par la toxine APETx2, suggère que cette dernière ou ses dérivés représentent des composés analgésiques prometteurs.

Heritability of renal function in hypertensive families of African descent in the Seychelles (Indian Ocean).

BACKGROUND: We estimated the heritability of three measures of glomerular filtration rate (GFR) in hypertensive families of African descent in the Seychelles (Indian Ocean). METHODS: Families with at least two hypertensive siblings and an average of two normotensive siblings were identified through a national hypertension register. Using the ASSOC program in SAGE (Statistical Analysis in Genetic Epidemiology), the age- and gender-adjusted narrow sense heritability of GFR was estimated by maximum likelihood assuming multivariate normality after power transformation. ASSOC can calculate the additive polygenic component of the variance of a trait from pedigree data in the presence of other familial correlations. The effects of body mass index (BMI), blood pressure, natriuresis, along with sodium to potassium ratio in urine and diabetes, were also tested as covariates. RESULTS: Inulin clearance, 24-hour creatinine clearance, and GFR based on the Cockcroft-Gault formula were available for 348 persons from 66 pedigrees. The age- and gender-adjusted correlations (+/- SE) were 0.51 (+/- 0.04) between inulin clearance and creatinine clearance, 0.53 (+/- 0.04) between inulin clearance and Cockcroft-Gault formula and 0.66 (+/- 0.03) between creatinine clearance and Cockcroft-Gault formula. The age- and gender-adjusted heritabilities (+/- SE) of GFR were 0.41 (+/- 0.10) for inulin clearance, 0.52 (+/- 0.13) for creatinine clearance, and 0.82 (+/- 0.09) for Cockcroft-Gault formula. Adjustment for BMI slightly lowered the correlations and heritabilities for all measurements whereas adjustment for blood pressure had virtually no effect. CONCLUSION: The significant heritability estimates of GFR in our sample of families of African descent confirm the familial aggregation of this trait and justify further analyses aimed at discovering genetic determinants of GFR.

Closeness in relationships as a mediator between sexual abuse in childhood or adolescence and psychopathological outcome in adulthood.

The risk of adverse psychological outcomes in adult victims of childhood and adolescent sexual abuse (CSA) has been documented; however, research on possible mediating variables is still required, namely with a clinical perspective. The attachment literature suggests that secure interpersonal relationships may represent such a variable. Twenty-eight women who had experienced episodes of CSA, and 16 control women, were interviewed using Bremner's Early Trauma Inventory and the DSM-IV Global Assessment of Functioning; they also responded to Collins' Relationship Scales Questionnaire, evaluating adult attachment representations in terms of Closeness, Dependence and Anxiety. Subjects with an experience of severe abuse reported significantly more interpersonal distance in relationships (low index of Closeness) than other subjects. The index of psychopathological functioning was correlated with both the severity of abuse and attachment (low index of Closeness). Regression analysis on the sample of abused women revealed that attachment predicted psychopathology when abuse was controlled for, whereas abuse did not predict psychopathology when attachment was controlled for. Therefore, preserving a capacity for closeness with attachment figures in adulthood appears to mediate the consequences of CSA on subsequent psychopathological outcome.

Sexual Health Promotion Strategy and Action Plan

A five year sexual health strategy including addendum 2008-2013

Tackling Sexual Violence at Home

Tackling Violence at Home Action Plans

Tackling Sexual Violence and Abuse 09-10 Action Plan

Tackling Sexual Violence and Abuse

Estimation of glomerular filtration rate in hospitalised patients: are we overestimating renal function?

QUESTIONS UNDER STUDY AND PRINCIPLES: Estimating glomerular filtration rate (GFR) in hospitalised patients with chronic kidney disease (CKD) is important for drug prescription but it remains a difficult task. The purpose of this study was to investigate the reliability of selected algorithms based on serum creatinine, cystatin C and beta-trace protein to estimate GFR and the potential added advantage of measuring muscle mass by bioimpedance. In a prospective unselected group of patients hospitalised in a general internal medicine ward with CKD, GFR was evaluated using inulin clearance as the gold standard and the algorithms of Cockcroft, MDRD, Larsson (cystatin C), White (beta-trace) and MacDonald (creatinine and muscle mass by bioimpedance). 69 patients were included in the study. Median age (interquartile range) was 80 years (73-83); weight 74.7 kg (67.0-85.6), appendicular lean mass 19.1 kg (14.9-22.3), serum creatinine 126 μmol/l (100-149), cystatin C 1.45 mg/l (1.19-1.90), beta-trace protein 1.17 mg/l (0.99-1.53) and GFR measured by inulin 30.9 ml/min (22.0-43.3). The errors in the estimation of GFR and the area under the ROC curves (95% confidence interval) relative to inulin were respectively: Cockcroft 14.3 ml/min (5.55-23.2) and 0.68 (0.55-0.81), MDRD 16.3 ml/min (6.4-27.5) and 0.76 (0.64-0.87), Larsson 12.8 ml/min (4.50-25.3) and 0.82 (0.72-0.92), White 17.6 ml/min (11.5-31.5) and 0.75 (0.63-0.87), MacDonald 32.2 ml/min (13.9-45.4) and 0.65 (0.52-0.78). Currently used algorithms overestimate GFR in hospitalised patients with CKD. As a consequence eGFR targeted prescriptions of renal-cleared drugs, might expose patients to overdosing. The best results were obtained with the Larsson algorithm. The determination of muscle mass by bioimpedance did not provide significant contributions.

Ant brood function as life preservers during floods.

Social organisms can surmount many ecological challenges by working collectively. An impressive example of such collective behavior occurs when ants physically link together into floating 'rafts' to escape from flooded habitat. However, raft formation may represent a social dilemma, with some positions posing greater individual risks than others. Here, we investigate the position and function of different colony members, and the costs and benefits of this functional geometry in rafts of the floodplain-dwelling ant Formica selysi. By causing groups of ants to raft in the laboratory, we observe that workers are distributed throughout the raft, queens are always in the center, and 100% of brood items are placed on the base. Through a series of experiments, we show that workers and brood are extremely resistant to submersion. Both workers and brood exhibit high survival rates after they have rafted, suggesting that occupying the base of the raft is not as costly as expected. The placement of all brood on the base of one cohesive raft confers several benefits: it preserves colony integrity, takes advantage of brood buoyancy, and increases the proportion of workers that immediately recover after rafting.

Tackling Sexual Violence and Abuse - A regional Strategy 2008-2013 (PDF 20.2 MB)

Tackling Sexual Violence and Abuse - A regional Strategy 2008-2013

Natural Arabidopsis brx loss-of-function alleles confer root adaptation to acidic soil.

Soil acidification is a major agricultural problem that negatively affects crop yield. Root systems counteract detrimental passive proton influx from acidic soil through increased proton pumping into the apoplast, which is presumably also required for cell elongation and stimulated by auxin. Here, we found an unexpected impact of extracellular pH on auxin activity and cell proliferation rate in the root meristem of two Arabidopsis mutants with impaired auxin perception, axr3 and brx. Surprisingly, neutral to slightly alkaline media rescued their severely reduced root (meristem) growth by stimulating auxin signaling, independent of auxin uptake. The finding that proton pumps are hyperactive in brx roots could explain this phenomenon and is consistent with more robust growth and increased fitness of brx mutants on overly acidic media or soil. Interestingly, the original brx allele was isolated from a natural stock center accession collected from acidic soil. Our discovery of a novel brx allele in accessions recently collected from another acidic sampling site demonstrates the existence of independently maintained brx loss-of-function alleles in nature and supports the notion that they are advantageous in acidic soil pH conditions, a finding that might be exploited for crop breeding.

Public Health Function Review

Public Health Function Review

Cirp function and characterisation in RNA and microRNAs

In order to search for novel genes involved in cell proliferation, the hypothesis was that by infecting primary cells with a cDNA library of immortal cells would render immortalizing genes. Consequently it has been discovered CIRP (Cold inducible RNA-binding protein). Mammalian cells exposed to mild hypothermia show a general inhibition of protein synthesis and a concomitant increase in the expression of a small number of cold-shock mRNAs and proteins. Rbm3, another RNA binding protein belonging to the same family, has been postulated to facilitate protein synthesis at mild cold shock. To investigate if the same occurs for CIRP, CIRP was overexpressed in primary cells and protein sintesis was measured. Interestingly, CIRP increased protein synthesis, however, such increase did not involve an increase in the polysome fraction or affected the ribosome profile. In addition, the effect caused by CIRP inhibition or knockdown was also analyzed. Different siRNAs against CIRP were tested. Once checked their efficiency by decreasing CIRP at mRNA and protein levels, proliferation was tested by BrdU, cell number (DAPI) and proliferation curves were performed. Interestingly, CIRP provoke a decreased proliferation in primary cells: MEFs, HMEC; and cancer cells: TERA2 and HeLa. In conclusion, we describe for the first time that CIRP bypasses replicative senescence when over-expressed at physiological temperature (37ºC) by increasing a general protein synthesis. This effect is achieved through ERK1/2 activation in MEFs.The decrease in growth rate found in mammalian cells treated with mild cold stress is not entirely attributable to arrested metabolism. This decrease may also involve an active process in which CIRP and other stress-responsive proteins play a fundamental role in stimulating proliferation. Although most cell proteins are down-regulated or inhibited with cold stress, CIRP is activated to maintain cells in an active proliferative status and its overexpression at 37°C might be potentially oncogenic.

Cefalea tusígena, de esfuerzo y relacionada con la actividad sexual:descripción de una serie de 50 casos

Las cefaleas tusígena, de esfuerzo y sexual son entidades infrecuentes y poco descritas. Hemos realizado un estudio retrospectivo de pacientes que consultaron durante 5 años por dicha patología en nuestro centro. De una muestra de 50 pacientes, 25 consultaron por cefalea tusigena, 15 por esfuerzo y 10 relacionadas con actividad sexual (74%, 80% y 80% de formas respectivamente). Las tres entidades podrían tratarse del espectro de una misma patología. Los principales elementos diferenciadores respecto a las formas secundarias fueron la asociación en estas de sintomatología neurológica, exploración patológica y la edad de debut más temprana. Ambas pueden responder a Indometacina