Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Control of a power supply with cycling current using different controllers

Fast Field Cycling (FFC) Nuclear Magnetic Resonance (NMR) relaxometers require controlled current sources in order to get accurate flux density with respect to its magnet. The main elements of the proposed solution are a power semiconductor, a DC voltage source and the magnet. The power semiconductor is commanded in order to get a linear control of the flux density. To implement the flux density control, a Hall Effect sensor is used. Furthermore, the dynamic behavior of the current source is analyzed and compared when using a PI controller and a PD2I controller.

Control of a power supply with cycling current using different controllers

Fast Field Cycling (FFC) Nuclear Magnetic Resonance (NMR) relaxometers require controlled current sources in order to get accurate flux density with respect to its magnet. The main elements of the proposed solution are a power semiconductor, a DC voltage source and the magnet. The power semiconductor is commanded in order to get a linear control of the flux density. To implement the flux density control, a Hall Effect sensor is used. Furthermore, the dynamic behavior of the current source is analyzed and compared when using a PI controller and a PD2I controller.

Reversible current power supply for fast-field cycling nuclear magnetic resonance equipment

The Fast Field-Cycling Nuclear Magnetic Resonance (FFC-NMR) is a technique used to study the molecular dynamics of different types of materials. The main elements of this equipment are a magnet and its power supply. The magnet used as reference in this work is basically a ferromagnetic core with two sets of coils and an air-gap where the materials' sample is placed. The power supply should supply the magnet being the magnet current controlled in order to perform cycles. One of the technical issues of this type of solution is the compensation of the non-linearities associated to the magnetic characteristic of the magnet and to parasitic magnetic fields. To overcome this problem, this paper describes and discusses a solution for the FFC-NMR power supply based on a four quadrant DC/DC converter.

Correlation between the mechanical cycling behavior and microstructure in laser welded joints using NiTi memory shape alloys

Disserta����o para obten����o do Grau de Mestre em Engenharia de Materiais

Comparing standard and low-cost tools for gradient evaluation along potential cycling paths

Promoting the use of non-motorized modes of transport, such as cycling, is an important contribution to the improvement of mobility, accessibility and equity in cities. Cycling offers a fast and cheap transportation option for short distances, helping to lower pollutant emissions and contributing to a healthier way of life. In order to make the cycling mode more competitive in relation to motorized traffic, it is necessary to evaluate the potential of alternatives from the perspective of the physical effort. One way to do so consists of assessing the suitability of locations for implementing cycling infrastructures. In this work, four tools to determine the gradient along potential cycling paths are compared. Furthermore, an evaluation of the reliability of some low-cost tools to measure this parameter was conducted, by comparison with standard measurements using cartographic plans, on a field case study applied to the city of Braga, Portugal. These tools revealed a good level of accuracy for the planning stage, but proved to be less reliable for use in design.

Importancia del Ca2+ en la fisiolog��a esperm��tica: modulaci��n de su actividad por progesterona y prote��nas del plasma seminal. Importance of calcium in sperm physiology: its regulation by progesterone and proteins from the seminal plasma.

El objetivo general del proyecto es estudiar el efecto de la progesterona y de algunas prote��nas del plasma seminal sobre la actividad del Ca2+ en diferentes procesos fisiol��gicos que ocurren en el espermatozoide, los cuales est��n estrechamente relacionados con la capacidad fertilizante de esta c��lula. La progesterona, principal esteroide secretado por las c��lulas del cumulus oophorus, ejerce su efecto a trav��s de un receptor no-gen��mico provocando aumento en el calcio intracelular de los espermatozoides y, consecuentemente, promoviendo la capacitaci��n, la respuesta quimiot��ctica y la exocitosis acrosomal. Pese a estas observaciones, los mecanismos a trav��s de los cuales la progesterona estimula fen��menos tan diversos en el espermatozoide son a��n desconocidos. Tampoco se conoce con exactitud el papel funcional y los mecanismos de acci��n de algunas prote��nas del plasma seminal que interaccionan y se unen a los espermatozoides, con alta especificidad, durante la eyaculaci��n. Por lo tanto, resulta altamente interesante profundizar los estudios sobre las propiedades funcionales de las prote��nas caltrin (calcium transport inhibitor) y ��-microseminoprotein (MSP) del plasma seminal de mam��feros, las cuales responden a las caracter��sticas mencionadas. Los estudios hasta ahora realizados han dado cuenta de que caltrin inhibe la incorporaci��n de Ca2+ extracelular, previene la exocitosis acrosomal espont��nea y promueve la uni��n espermatozoide-zona pel��cida. Tambi��n hay datos preliminares que sugieren un efecto inhibitorio sobre la movilidad hiperactivada de los espermatozoides. Respecto a MSP, s��lo se sabe que inhibe la exocitosis acrosomal espont��nea y que su contenido, en el plasma seminal, guarda una relaci��n inversa con la fertilidad. Por todo lo expuesto, se propone estudiar los mecanismos de acci��n de la progesterona y las prote��nas caltrin y MSP sobre los procesos fisiol��gicos antes indicados. Para ello, se estudiar��n las variaciones de Ca2+ intracelular en espermatozoides individuales sometidos a diferentes tratamientos (gradientes de progesterona, capacitaci��n en presencia y ausencia de caltrin y/o MSP, etc.), usando video microscop��a de fluorescencia y an��lisis computarizado de im��genes. Tambi��n se examinar�� la influencia de estas mol��culas sobre la interacci��n de gametas y la fertilizaci��n.

Mecanismo de interacci��n, calcio dependiente, entre Calcineurina y PERK, involucrado en el Estr��s de Ret��culo Endopl��smico. Ca2+ -dependent mechanism of interaction between Calcineurin and PERK involved in Endoplasmic Reticulum Stress.

El Estr��s de Ret��culo Endopl��smico (RE) es inducido por la acumulaci��n de prote��nas sin plegar en el lumen de la organela. Esto se puede observar en diversas situaciones fisio-patol��gicas como durante una infecci��n viral o en proceso isqu��mico. Adem��s, contribuye a la base molecular de numerosas enfermedades ya sea ��ndole metab��lico (Fibrosis qu��stica o Diabetes Miellitus) o neurodegenerativas como mal de Alzheimer o Parkinson (Mutat Res, 2005, 569). Para restablecer la homeostasis en la organela se activa una se��al de transducci��n (UPR), cuya respuesta inmediata es la atenuaci��n de la s��ntesis de prote��na debido a la fosforilaci��n de subunidad alpha del factor eucari��tico de iniciaci��n de translaci��n (eIF2��) v��a PERK. Esta es una prote��na de membrana de RE que detecta estr��s. Bajo condiciones normales, PERK est�� inactiva debido a la asociaci��n de su dominio luminar con la chaperona BIP (Nat Cell Biol, 2000, 2: 326). Frente a una situaci��n de estr��s, la chaperona se disocia causando desinhibici��n. Recientemente, (Plos One 5: e11925) se observ��, bajo condiciones de estr��s, un aumento de Ca2+ citos��lico y un r��pido incremento de la expresi��n de calcineurina (CN), una fosfatasa citos��lica dependiente de calcio, heterodim��rica formada por una subunidad catal��tica (CN-A) y una regulatoria (CN-B). Adem��s, CN interacciona, sin intermediarios, con el dominio citos��lico de PERK favoreciendo su trans-autofosforilaci��n. Resultados preliminares indican que, astrocitos CNA��-/- exhibieron, en condiciones basales, un mayor n��mero de c��lulas muertas y de niveles de eIF2�� fosforilado que los astrocitos CNA��-/-. Hip��tesis: CNA��/B interacciona con PERK cuando el Ca2+ citos��lico esta incrementado luego de haberse inducido Estr��s de RE, lo cual promueve dimerizaci��n y auto-fosforilaci��n de la quinasa, acentu��ndose as�� la fosforilaci��n de eIF2�� e inhibici��n de la s��ntesis de prote��nas. Esta activaci��n citos��lica de PERK colaborar��a con la ya descrita, desinhibici��n luminal llevada cabo por BIP. Cuando el Ca2+ citos��lico retorna a los niveles basales, PERK fosforila a CN, reduciendo su afinidad de uni��n y disoci��ndose el complejo CN/PERK. Objetivo general: Definir las condiciones por las cuales CN interacciona con PERK y regula la fosforilaci��n de eIF2�� e inhibici��n de la s��ntesis de prote��na. Objetivos espec��ficos: I-Estudiar la diferencia de afinidades y dependencia de Ca2+, de las dos isoformas de CN (�� y ��) en su asociaci��n con PERK. Adem��s verificar la posible participaci��n de la subunidad B de CN en esta interacci��n. II-Determinar si la auto-fosforilaci��n de PERK es diferencialmente regulada por las dos isoformas de CN. III-Discernir la relaci��n del estado de fosforilaci��n de CN con su uni��n a PERK. IV-Determinar efectos fisiol��gicos de la interacci��n de CN-PERK durante la respuesta de Estr��s de RE. Para llevar a cabo este proyecto se realizar��n experimentos de biolog��a molecular, interacci��n prote��na-prote��na, ensayos de fosforilaci��n in vitro y un perfil de polisoma con astrocitos CNA��-/- , CNA���-/- y astrocitos controles. Se espera encontrar una mayor afinidad de uni��n a PERK de la isoforma �� de CN y en condiciones donde la concentraci��n de Ca2+ sea del orden micromolar e imite niveles del i��n durante un estr��s. Con respecto al estado de fosforilaci��n de CN, debido a los resultados preliminares, donde solo se la encontr�� fosforilada en condiciones basales, se piensa que CN podr��a interactuar con mayor afinidad con PERK cuando CN se encuentre desfosforilada. Por ��ltimo, se espera encontrar un aumento de eIF2�� fosforilado y una acentuaci��n de la atenuaci��n de la s��ntesis de prote��na como consecuencia de la mayor activaci��n de PERK por su asociaci��n con la isoforma �� de CN en astrocitos donde el Estr��s de RE se indujo por privaci��n de oxigeno y glucosa. Estos experimentos permitir��n avanzar en el estudio de una nueva funci��n citoprotectora de CN recientemente descrita por nuestro grupo de trabajo y sus implicancias en un modelo de isquemia. The accumulation of unfolded proteins into the Endoplasmic Reticulum (ER) activates a signal transduction cascade called Unfolding Protein Response (UPR), which attempts to restore homeostasis in the organelle. (PKR)-like-ER kinase (PERK) is an early stress response transmembrane protein that is generally inactive due to its association with the chaperone BIP. During ER stress, BIP is tritrated by the unfolded protein, leading PERK activation and phosphorylation of eukaryotic initiation factor-2 alpha (eIF2alpha), which attenuates protein s��ntesis. If ER damage is too great and homeostasis is not restored within a certain period of time, an apoptotic response is elicited. We recently demonstrated a cytosolic Ca2+ increase in Xenopus oocytes after induce ER stress. Moreover, calcineurin A/B, a an heterotrimeric Ca2+ dependent phosphatases (CN-A/B), associates with PERK increasing its auto-phosphorylation and significantly enhancing cell viability. Preliminary results suggest that, CN-A��-/- knockout astrocytes exhibit a significant higher eIF2�� phosphorylated level compared to CN-A��-/- astrocytes. Our working hypothesis establishes that: CN binds to PERK when cytosolic Ca2+ is initially increased by ER stress, promoting dimerization and autophosphorylation, which leads to phosphorylation of elF2�� and subsequently attenuation of protein translation. When cytosolic Ca2+ returns to resting levels, PERK phosphorylates CN, reducing its binding affinity so that the CN/PERK complex dissociates. The goal of this project is to determine the conditions by which CN binding to PERK attenuates protein translation during the ER stress response and subsequently, to determine how the interaction of CN with PERK is terminated when stress is removed. To perform this project is planed to do molecular biology experiments, pull down assays, in vitro phosphorylations and assess overall mRNA translation efficiency doing a polisome profile.

Exerc��cio e restri����o alimentar aumentam o RNAm de prote��nas do tr��nsito de Ca2+ mioc��rdico em ratos

FUNDAMENTO: Treinamento f��sico (TF) aumenta a sensibilidade dos horm��nios tireoidianos (HT) e a express��o g��nica de estruturas moleculares envolvidas no movimento intracelular de c��lcio do mioc��rdio, enquanto a restri����o alimentar (RIA) promove efeitos contr��rios ao TF. OBJETIVO: Avaliar os efeitos da associa����o TF e RIA sobre os n��veis plasm��ticos dos HT e a produ����o de mRNA dos receptores HT e estruturas moleculares do movimento de c��lcio do mioc��rdio de ratos. M��TODOS: Utilizaram-se ratos Wistar Kyoto divididos em: controle (C, n = 7), RIA (R50, n = 7), exerc��cio f��sico (EX, n = 7) e exerc��cio f��sico + RIA (EX50, n = 7). A RIA foi de 50% e o TF foi nata����o (1 hora/dia, cinco sess��es/semana, 12 semanas consecutivas). Avaliaram-se as concentra����es s��ricas de triiodotironina (T3), tiroxina (T4) e horm��nio tireotr��fico (TSH). O mRNA da bomba de c��lcio do ret��culo sarcoplasm��tico (SERCA2a), fosfolamban (PLB), trocador Na+/Ca+2 (NCX), canal lento de c��lcio (canal-L), rianodina (RYR), calsequestrina (CQS) e receptor de HT (TRα1 e TRβ1) do mioc��rdio foram avaliados por rea����o em cadeia da polimerase (PCR) em tempo real. RESULTADOS: RIA reduziu o T4, TSH e mRNA do TRα1 e aumentou a express��o da PLB, NCX e canal-L. TF aumentou a express��o do TRβ1, canal-L e NCX. A associa����o TF e RIA reduziu T4 e TSH e aumentou o mRNA do TRβ1, SERCA2a, NCX, PLB e correla����o do TRβ1 com a CQS e NCX. CONCLUS��O: Associa����o TF e RIA aumentou o mRNA das estruturas moleculares c��lcio transiente, por��m o eixo HT-receptor n��o parece participar da transcri����o g��nica dessas estruturas.

Chronic Stress Improves NO- and Ca2+ Flux-Dependent Vascular Function: A Pharmacological Study

Background: Stress is associated with cardiovascular diseases. Objective: This study aimed at assessing whether chronic stress induces vascular alterations, and whether these modulations are nitric oxide (NO) and Ca2+ dependent. Methods: Wistar rats, 30 days of age, were separated into 2 groups: control (C) and Stress (St). Chronic stress consisted of immobilization for 1 hour/day, 5 days/week, 15 weeks. Systolic blood pressure was assessed. Vascular studies on aortic rings were performed. Concentration-effect curves were built for noradrenaline, in the presence of L-NAME or prazosin, acetylcholine, sodium nitroprusside and KCl. In addition, Ca2+ flux was also evaluated. Results: Chronic stress induced hypertension, decreased the vascular response to KCl and to noradrenaline, and increased the vascular response to acetylcholine. L-NAME blunted the difference observed in noradrenaline curves. Furthermore, contractile response to Ca2+ was decreased in the aorta of stressed rats. Conclusion: Our data suggest that the vascular response to chronic stress is an adaptation to its deleterious effects, such as hypertension. In addition, this adaptation is NO- and Ca2+-dependent. These data help to clarify the contribution of stress to cardiovascular abnormalities. However, further studies are necessary to better elucidate the mechanisms involved in the cardiovascular dysfunction associated with stressors. (Arq Bras Cardiol. 2014; [online].ahead print, PP.0-0)

Obesity Resistance Promotes Mild Contractile Dysfunction Associated with Intracellular Ca2+ Handling

Abstract Background: Diet-induced obesity is frequently used to demonstrate cardiac dysfunction. However, some rats, like humans, are susceptible to developing an obesity phenotype, whereas others are resistant to that. Objective: To evaluate the association between obesity resistance and cardiac function, and the impact of obesity resistance on calcium handling. Methods: Thirty-day-old male Wistar rats were distributed into two groups, each with 54 animals: control (C; standard diet) and obese (four palatable high-fat diets) for 15 weeks. After the experimental protocol, rats consuming the high-fat diets were classified according to the adiposity index and subdivided into obesity-prone (OP) and obesity-resistant (OR). Nutritional profile, comorbidities, and cardiac remodeling were evaluated. Cardiac function was assessed by papillary muscle evaluation at baseline and after inotropic maneuvers. Results: The high-fat diets promoted increase in body fat and adiposity index in OP rats compared with C and OR rats. Glucose, lipid, and blood pressure profiles remained unchanged in OR rats. In addition, the total heart weight and the weight of the left and right ventricles in OR rats were lower than those in OP rats, but similar to those in C rats. Baseline cardiac muscle data were similar in all rats, but myocardial responsiveness to a post-rest contraction stimulus was compromised in OP and OR rats compared with C rats. Conclusion: Obesity resistance promoted specific changes in the contraction phase without changes in the relaxation phase. This mild abnormality may be related to intracellular Ca2+ handling.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Neural Wiskott-Aldrich syndrome protein modulates Wnt signaling and is required for hair follicle cycling in mice.

The Rho family GTPases Cdc42 and Rac1 are critical regulators of the actin cytoskeleton and are essential for skin and hair function. Wiskott-Aldrich syndrome family proteins act downstream of these GTPases, controlling actin assembly and cytoskeletal reorganization, but their role in epithelial cells has not been characterized in vivo. Here, we used a conditional knockout approach to assess the role of neural Wiskott-Aldrich syndrome protein (N-WASP), the ubiquitously expressed Wiskott-Aldrich syndrome-like (WASL) protein, in mouse skin. We found that N-WASP deficiency in mouse skin led to severe alopecia, epidermal hyperproliferation, and ulceration, without obvious effects on epidermal differentiation and wound healing. Further analysis revealed that the observed alopecia was likely the result of a progressive and ultimately nearly complete block in hair follicle (HF) cycling by 5 months of age. N-WASP deficiency also led to abnormal proliferation of skin progenitor cells, resulting in their depletion over time. Furthermore, N-WASP deficiency in vitro and in vivo correlated with decreased GSK-3beta phosphorylation, decreased nuclear localization of beta-catenin in follicular keratinocytes, and decreased Wnt-dependent transcription. Our results indicate a critical role for N-WASP in skin function and HF cycling and identify a link between N-WASP and Wnt signaling. We therefore propose that N-WASP acts as a positive regulator of beta-catenin-dependent transcription, modulating differentiation of HF progenitor cells.

Physiological differences between cycling and running: lessons from triathletes

The purpose of this review was to provide a synopsis of the literature concerning the physiological differences between cycling and running. By comparing physiological variables such as maximal oxygen consumption (V O(2max)), anaerobic threshold (AT), heart rate, economy or delta efficiency measured in cycling and running in triathletes, runners or cyclists, this review aims to identify the effects of exercise modality on the underlying mechanisms (ventilatory responses, blood flow, muscle oxidative capacity, peripheral innervation and neuromuscular fatigue) of adaptation. The majority of studies indicate that runners achieve a higher V O(2max) on treadmill whereas cyclists can achieve a V O(2max) value in cycle ergometry similar to that in treadmill running. Hence, V O(2max) is specific to the exercise modality. In addition, the muscles adapt specifically to a given exercise task over a period of time, resulting in an improvement in submaximal physiological variables such as the ventilatory threshold, in some cases without a change in V O(2max). However, this effect is probably larger in cycling than in running. At the same time, skill influencing motor unit recruitment patterns is an important influence on the anaerobic threshold in cycling. Furthermore, it is likely that there is more physiological training transfer from running to cycling than vice versa. In triathletes, there is generally no difference in V O(2max) measured in cycle ergometry and treadmill running. The data concerning the anaerobic threshold in cycling and running in triathletes are conflicting. This is likely to be due to a combination of actual training load and prior training history in each discipline. The mechanisms surrounding the differences in the AT together with V O(2max) in cycling and running are not largely understood but are probably due to the relative adaptation of cardiac output influencing V O(2max) and also the recruitment of muscle mass in combination with the oxidative capacity of this mass influencing the AT. Several other physiological differences between cycling and running are addressed: heart rate is different between the two activities both for maximal and submaximal intensities. The delta efficiency is higher in running. Ventilation is more impaired in cycling than in running. It has also been shown that pedalling cadence affects the metabolic responses during cycling but also during a subsequent running bout. However, the optimal cadence is still debated. Central fatigue and decrease in maximal strength are more important after prolonged exercise in running than in cycling.