Integrated Approach to Human Functions: Understanding Biological Transports

1. 27/05/2015 BiosystemMAN Prof. PavelKučera FBMI Kladno Winter 2013 Integrated approach to human functions with accent on BIOLOGICAL TRANSPORTS Obecné cíle: -pochopit • funkční organizaci živých organizmů • základní koncepty systémového přístupu k lidskému organismu • experimentální a vyšetřovací metody užívané ve fyziologii a medicíně • integrované funkce systémů skýtajících uplatnění pro biomedicínské techniky a inženýry - být schopen - definovat zajímavé problémy a navrhnout jejich řešení - využít získaných znalostí: - v oblasti biomedicinského inženýrství - v oblasti biotechnologie koncepce → vývoj → validace → uplatnění nových technologií Plán: ÚVOD OD BUŇKY KE TKÁNI A ORGÁNU DÝCHACÍ A OBĚHOVÝ SYSTÉM VYLUČOVACÍ SYSTÉM TRÁVICÍ SYSTÉM NERVOVÝ A POHYBOVÝSYSTÉM 1

2. 27/05/2015 1 :ÚVOD Na konci této kapitoly bych měl být schopen: definovat živý systém v termodynamickémpojetí definovat buňku a její hlavnífunkce vysvětlit koncept homeostásy na úrovni jednobuněčného a mnohobuněčnéhoorganismu popsat složení a objemy základních elektrolytických oddílů lidskéhotěla definovat a klasifikovat (spřažené) transporty charakterizující živéorganismy vysvětlit, že základem buněčných funkcí jsou konformační změnyproteinů vysvětlit jak živočisné buňky generují a využívajíenergii Oblast vědy dovolující splnit tyto cíle jeFYZIOLOGIE 2 , , What is PHYSIOLOGY ( ) ? 2 1 Science that • studies the normal functionsof livingsystems 3 • aims to understand mechanismsof • uses experimental approach4 adaptation • is interdisciplinary ( biology, morphology,chemistry, • physics, psychology...) • Physiology represents the foundations of Systemsbiology • ? How do organisms function from molecular level to whole body coordinate behavior? 3

3. 27/05/2015 1 SYSTEM = a portion of universedelimited for consideration° Characteristics: • 1EXCHANGES • noexchange: • energyonly: isolated closed UNIVERSE - energy & matter: open • 2 STATE • parameters do not vary: • means of para- stable barrier(membrane) meters do not vary:stationary periodic 3EVOLUTION Initial state (U1) → final state(U2) U2 - U1 = dU = Q +W* dU = TdS + PdV + µdn + edq +… SYSTEM n V T° P q … internal energyU SURROUNDINGS (environment) 4STRUCTURE of forces =0; net fluxes absent: equilibrium of forces ≠0 netfluxespresent: dissipative 4 * work done ON thesystem ° existing or arbitrarily defined by ourthought Structure equilibrium: distribution of variables the most likely the most homogeneous the most stable sum of forces = 0; absence of net fluxes coldplate dissipative: distribution of variables unlikely inhomogeneous sum of forces ≠ 0 presence of net fluxes sum of fluxes = 0 hotplate Benard-Marangoni instability at the free surface: state far from equilibrium maintained by the heat transfer temperature gradient → surface tension gradient → ejection of fluid from the hot to the cold region. To conserve mass, hot fluid ascends from the lower plane. transfer of heat = generator of losses → CHAOS transfer of heat = generator of ORDER ClassicalT°dynamics: Non-equilibriumT°dynamics: 5

4. 27/05/2015 What is life? C. E. Folsome, The Origin of Life. The little warm pond (1979): Life is that property of matter that results from cycling of bioelements in aqueous solution, ultimately driven by radiant energy to attain maximal complexity. organic organic organic Organic molecules: proteins carbohydrates lipids nucleic acids phosphorylated compounds … NITROGEN CARBON inorganic inorganic PHOSPHORUS Simplebioelements: “CHNOPS” organic organic inorganic HYDROGEN SULPHUR organic inorganic inorganic OXYGEN . inorganic • Ecology: • Such a cycling takes place at planet scale and cannot exist without • - primary producers • users • scavengers Radiant energy drives the phosphorus cycle to which the other cycles are chemically coupled according to their specific turn ratios:. Coupled bioelemental cycles (L. Onsager, Nobel in chemistry1968) 6 Summary Living organisms(systems): : are opensystems external energy source selective barriers exchanges of matter & energy are in non equilibrium state internal energy higher than that of the same elementsseparated and at the same low temperatue & pressure:instability are dissipativestructure maintaining their order (their stable structure), in the face of a constant flux of matter and energy passing through them, by dissipating entropy to their environment show irreversible evolution birth differentiation stationary state ageing death t i m e show auto-organisation autogenesis, autodelimitation, autoorganization, autoreplication Life appears as the supreme expression of self-generating and self-ordering processes we know. I.Prigogine (Nobel in Chemistry1977), Order out of chaos (1984) 7

5. 27/05/2015 How is a living organism (HUMAN) organised? ORGANISM(man) organ assembly: "SYSTEM" (e.g.digestive) ORGAN STIMULI TISSUE CELL BEHAVIOUR INTERNALMILIEU ORGANELLE RESPONSES MOLECULAR COMPLEX Units* that may be classified according to their levels of functionalhierarchy. *Integrons (F. Jacob, Nobel price, "La logique duvivant”) CELL: the simplest system able to survive in a non-living but convenientenvironment 8 CELL: the simplest system capable to survive in a non-living but convenient environment Structure of an animal cell 14 5-15 " … omne vivum e cellula …" 9

6. 27/05/2015 2 Normal functions (system = animalcell) Growth +Division 1 Movement Form Masse Volume Energy metabolism: ATPproduction +heat 2 Transports(fluxes): ion pumps & channels: electricalpotential, acid-basebalance Macromolecules 4 5 8 Force wastes cyto- squelet 3 Anabolism (syntheses: renewal,reserves) 3+7 precursors 6 4 Catabolism & rejection ofwastes H+ Na+ Ca+ ADP+Pi Glucose +O2 5 Contractility 1 2 ATP + 6 Internal clock (cellularoscillators) 7 Memory K+ H2O + CO2 +heat 8 Signal reception andemission = Everything flows!10 ≡ exchange ≡ transport ≡ flux ≡current 3 ADAPTATION: How to remain stable when everything flows ? 3a) case of an unicellular organism: external milieu (volume∞) Transports across the membrane: Bextracell Cintracell Bintracell Cextracell membrane (selectivity) A M C M:Metabolism (chemicaltransformations): B C B Volume <1mm3 of fluxes=0 parameters (T, P, V, composition, pH etc.) remain stable : steady state. Hence, although the cell is constantly traversed by matter and energy, its state and structure do not vary: HOMEOSTASIS (ό= similar & = tostand) Homeostatic mechanisms in unicellular organisms areoperating: 1 at the membrane level (control of transports) 2 3 at the cytosol level at the nuclear level (control of the speed of chemical reactions, turnover) (control of gene expression) 11

7. 27/05/2015 3 b) case of a multicellular organism (with internalised extracellular milieu): S S P Man: Volume ~ 7.10 7 mm3 • Problem: • The volume of extracellular space is small so that : • substrates (S) quickly consumed • product (P) quickly accumulated S T I M U L I Respiration Nervous system S Skin P Blood Immune system • Task: • Homeostasis then consists of: • maintaining of cell composition • maintaining of internal milieu (blood + extracellular fluid) composition internalmilieu circulation Hormonal system • R E S P O • N Solution: • S exchanges beween • E • S - organism and surroundings • blood and extracellular fluid • extracellular fluid and cells • adaptive behaviour Digestion Locomotor system Kidney P P of fluxes (S,P) =0 stable parameters (T, P, V, composition, pHetc.) Homeostatic mechanisms in an multicellular organism are calledREGULATIONS Regulations are based on cooperation of all exchanging systems and they are controlled by nervous, endocrine and immune systems 12 4 Experimental approach inphysiology 1 Initial hypothesis based on a comprehensive representation of the reality : modeling 6 Verification 2 Controlled perturbation of thesystem (stimulus:electrical,mechanical,chemical,thermal...) 5 New representation modeling 3 Study of the system responses (e.g., experimental recording of electrical potential, muscular force, urine composition, temperature, behavior....) 4 Analysis of the stimulus - response relationship (transfer function, limits...) † o temperature int C 40 man: homeotherm 36.8 37.8 30 † Transferfunction example: warming of asystem inert matter, poikilotherm 20 10 0 oC 13 temperatureext 10 20 30 40

8. 27/05/2015 Model ofthermoregulation neuralcode centralT° CNS: detected T° compared to a reference Metabolism: BODY HEAT ENVIRONEMENT A regulation consists of: receptor(s) sensory message reference value error detector motor command effector(s) neuralcode skinT° Two types ofmodels: • models for teaching that help to transmit knowledge • (even though often rather caricatured representations of reality, they are very useful) • models for research that tend to simulate and permit predictions ! A good model is an approximation, it is a TOOL : it allows to imagine future experiments ! 14 Realworld System Insight System reduce organism organ tissue cell organelle network transcript gene molecule Parts integrate Modelling ? - is a simplified representation of thereality - allows exploration of(sub)systems Parts -EXPERIMENTAL → data on structure - function - timerelationships + reintegrate these data = Understanding of reality • CONCEPTUAL(thought) • FORMAL(computation) Systems biology - provides a framework for targeted analysis and application of modelling for bio-medical research and development - combines reductionist and integrative tools andtechniques 15

9. 27/05/2015 HUMAN PHYSIOME PROJECT Hunter et al. Pflügers Arch2002 SYSTEM molecular events diffusion motility mitosis EVENTS protein specific turnover functions behaviour GENE NETWORKS PATHWAYS STOCHASTIC DIFFERENTIALEQUATIONS MODELS SYSTEMS 10-6 10-3 100103106 timescale 109 seconds 16 • BIOLOGICAL TRANSPORTS • are basis of life • are exchanges of matter, energy and information • between living systems and their surroundings • within living systems • are processes • highly organised in space and in time • intimately inter-related with all the ecosystem • underlie all functions • are targets of controlling mechanisms • enable the systems to maintain their organisation • 17

10. 27/05/2015 WHAT IS TRANSPORTED IN LIVING SYSTEMS Mass (gases, water, neutral solutes, charged solutes (ions, proteins..)), Energy, Signals WHAT CAUSES THE TRANSPORTS Transports are generated by their conjugated driving forces by means of coupling coefficients. According to Lars Onsager : → → Ji = Lii x Fi (transport i = conjugated force i x coupling coefficient ii ( L from “Leitfähigkeit” ) Examples of coupling coefficients: Examples of driving forces: Across small x: (~ pressure difference) (~ concentration difference) (~ potential difference) (~ temperature difference) • mobility • diffusioncoefficient • electricalconductance • thermalconductivity mechanical gradient: chemical gradient: electrical gradient: dP/dx dC/dx dV/dx temperature gradient: dT/dx UNITS (transports are vectors: value &direction!) Mass / time Volume / time Energy / time Charge/ time Information / time g/day, mmol/min … litre/day, ml/min… J/s (W), kcal/day ... Cb/s (A) bit/s (as concentration = mass/vol → mass/time = (Vol./time) xconc. As biological transports often take place across bi-dimensional barriers (membranes) they are also expressed with respect to surface area (e.g.: mol.s-1.cm-2). 18 COUPLEDTRANSPORTS → → If a transport Ji is also dependent on an additional force Fjthen: → → → → Ji = Lij x Fj and Jj = Lji x Fi where Lij =Lji coupling Example: In simultaneous presence of fluxes of heat and of matter, the heat flow per unit of pressure difference and the density (matter) flow per unit of temperature difference are equal (reciprocity relations of Onsager). CHEMICAL COUPLING OF FLUXES: N.B. Most biological transports are coupled! 1) Reactions with high free energy (G) drive reactions with lower free energy 2) Both reactions need a commonintermediate CH3 H H I I I H2N─ C─C─ N─aa─ C─N – aan Example: protein synthesis = formation of peptide bonds I II H O II O 2 LeucylGlycine G: +4.5Kcal/mole Leu +Gly t-RNA ATP AMP+PP G: - 8 Kcal/mole Recall: for a chemical reaction A+B → C+D at equilibrium the corresponding G = -RTln [C].[D] / [A].[B] 19

11. 27/05/2015 “ Microtransports” inside the internalspace “Macrotransports” deduced from humanbehaviour HelloJohn! transcellular paracellular AIR WATER + NUTRIENTS HelloMary! HEAT trans intra cellular WORK membrane Metabolism WASTES • diapedesis • transcytosis • cytoplasmic flux ( macromolecularmovements) • filtration • -diffusion -simple • -facilitated • pumping • respiration • eating &drinking • excretion - work - body posture &movements • heat exchange -sweating • communication - perception &expression ! Homeostatic BALANCE: what enters + what leaves = 0 - molecular & atomicoscillation 20 • Diffusion is the transport of particlesthat • takes place in absence of convection (movement ofmilieu), • is a consequence of constant thermal motion ofparticles, • is due to an inhomogeneity in themilieu, • proceeds in the sense of decreasingconcentration, • results in uniformdistribution, • is an irreversiblephenomenon. a JSout→in 1cm2 transport along the gradient [S]i [S] out in 0 [S]o -[S]i The flux of molecule S (JS) across a membrane of thickness dx is proportional to the concentration gradient across the membrane d [S] /dx which gives : Fick's First Law of Diffusion: JS ~ d[S] /dx or JS= - DS.d[S] /dx a in ∫ JSdx = -DS∫d[S] Rearranginggives: o out Integration with x varying between 0 and a (thickness of the membrane), and [A] from [A]0 (out) to [A]i (in) gives: JS = -DS {[S]i - [S]o} = DS {[S]o - [S]i} or JS = (DS/x) {[S]o - [S]i} or JS = PS {[S]o - [S]i} A plot of JSversus {[S]0 - [S]i} is linear, with a slope of PS . JS: initial flux (mol.cm-2.s-1, measured for a short time (concentrations of S on both sides of the membrane do not change significantly. DS : diffusion coefficient (cm-2s-1), negative sign as concentration increases in the opposite direction of net flux. PS : permeability coefficient (cms-1). 21

12. 27/05/2015 Transport of ions generates membrane electricalpotential Conditions: 1) inhomogeneous distribution of ions across the membrane → gradients ofconcentration 2) selective ionic permeabilty of the membrane → flux of ions → electricalgradients C1 > C2 membrane is fully permeable for the particle: If the substance bears a charge (e.g. +): - + - + ++ V2 + V1 - - + + + + + + + + C2 + + C 2 C1 C 1 + 1 2 + + 1 2 The diffusion creates a loss of + charges in 1: V =V2-V1. The free energy (work required to transfer 1 mole of substance from 1 to 2 is: The free energy (work required to transfer 1 mole of ion against this potential difference) is: G = z F V C2 C1 G = RTln when concentration and electrical gradients are balanced the potential difference across the membrane is called equilibrium (or Nernst ) potential of this ion: where RT C 2-1 E = -ln 2 ion C1 zF 22 C: ionic concentration; z: ionic valence; F: Faraday’s constant; R: gas constant; T: absolute temperature (see units in tables!) Kinetics: Nature of transmembranemicrotransports A B Membrane FluxA→B gas,lipids dissolution water, ions channels Simplediffusion transport along the gradient GradientA-B uniports Facilitateddiffusion FluxA→B many compounds saturation symports transport along the gradient Gradient A-B antiports symports many compounds Secundary active transport antiports FluxB→A saturation transport against the gradient (needs energy) H+ ATP Ca++ Primary active transport (pumps = ATPases) ATP H+ GradientA-B 3Na+ ATP 23 2K+

13. 27/05/2015 Structure of the cellmembrane Water channel(aquaporine) • Fluidmosaic: • double layer of phospholipids(fluidity) • inserted proteins (functionalunits) Na-K-ATPase (sodium-potassiumpump) Membraneproteins: • channels(pores) • carriers • “pumps” • receptors TRANSPORTS depend on PROTEIN SPATIAL CONFORMATION 24 CELL ENERGETICS: ATP as energy quantum of the livingsystem Adenine CELLFUNCTIONS Ribose 3 phosphoricacids energy transduction: conformation Adenosine molecularphosphorylation Adenosine-tri-phosphate(ATP) ATPases ATP4- +H2O ADP3- + HPO42- + H+ + 31kJ ATP synthase heat 31kJ 32-36x cytosol SUBSTRATE CATABOLISM,e.g.: Glucose → 2pyruvates 2 pyruvates + 6O2 → 6CO2 +6H2O mitochondrion 2700 kJ =-G ATP provides phosphoryl groups at high group-transfer potential in order to drive thermodynamically unfavorable reactions 25

14. 27/05/2015 The main components of the mitochondrial ATPproduction GLYCOLYSIS LIPIDCATABOLISM innermembrane pyruvate fattyacid matrix H+ + acetylCoA H+ ~0.22V citricacid - Krebscycle CO2 NADH2(FADH2) NAD+(FAD) H2O F1F0 e- H+ ATP H+ ADP +Pi ½O2 E T S Chemiosmotichypothesis Mitchell, Nobel Prize1978 H+ H+ H+ O2 26 ETS: electron transport system F1F0 : ATPsynthase Structure of F1F0 ATP synthase and binding change mechanism for ATP synthesis John Walker (structure) Nobel Prize1997 Paul Boyer (function) Step 1 matrix Step 2 Step3 • The subunits have 3 interacting and conformationally distinct active sites: • the open (O) conformation is inactive and has a low affinity for ligands; • the L conformation (with “loose” affinity for ligands) is also inactive; • the tight (T) conformation is active and has a high affinity for ligands. Synthesis of ATP: step 1: binding of ADP and Pi to an L site; • step 2: an energy-driven conformational change converts: the L site to a T conformation • and T to O and O to L; step 3: ATP is synthesized at the T site and released from the O site. 27

15. 27/05/2015 Speaking about composition: What are we made of? molecules: Proteins Carbohydrates Lipids Nucleic acids Minerals elements: C P H S O Ca SOLIDS 28kg 40 % of body weight Fe … &others N EAU 60 % LIQUIDS 42kg Male 25 years 70 kg Na+ K+ CATIONS Ca++ Mg++ Cl - IONS HCO- ANIONS 3 HPO3- PROTEINS 4 RCOO- 28 Volumes & compositions of body electrolytic compartments TO REMEMBER: endothelium cell membrane Intracellular fluid principal cation K + principal anions phosphates proteins Posm () ~ 300 mOsm / ℓ Extracellular fluid principal cation Na + principal anion Cl - Posm ) ~ 300 mOsm / ℓ mEq /ℓ 150 Na+ 100 K + Na+ K+, Ca++, Mg++ 50 Mg++ 0 Cl- HCO3 - Plasma: Hematocrit: volume ERY volume total -50 PO43- Cl- Proteins 70 g/ℓ -100 Protn- P oncotic () ~ 25 mmHg HCO3- Cell membrane permeable: H 2 O, Na+ K ,+ Cl - gradients <-> fluxes electrical potential: cell interior negative impermeable: proteins -150 1.5 ℓ 3.5ℓ 10.5ℓ ERY PLASMA INTERSTITIAL FLUID 28 ℓ INTRACELLULAR FLUID VOLUMES 29