mac gregor paracellular la 03192007

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  • An Introduction to the Shunt or Intercellular Pathway

  • Requests for Paracellular Presentation

  • MacGregor, Just go over the basics. No-one wants to hear your theories on paracellular permeability, keep them to yourself.-Anonymous RSMRequests for Paracellular Presentation

  • MacGregor, Just go over the basics. No-one wants to hear your theories on paracellular permeability, keep them to yourself.-Anonymous RSMMacGregor, pure basics would be good, definitions, (how to measure TER, etc), not so much detail so that were all going to want to set up our own electrophysiology labs in our basements or anything.-Anonymous RSMRequests for Paracellular Presentation

  • An epithelia is a finely regulated barrier between compartments with different composition that is able to withstand steep chemical and electrical gradients.This requires a seal, otherwise gradients would dissipate through intercellular space.Seal expected to be located at limit between lumen and intercellular spaceIntroduction to the Epithelial Physiology

  • The Intercellular PathwayIntercellular PathwayIntracellular Pathway

  • Since the epithelia of choice for many years was frog skin, with negligible fluxes through intercellular space.Frog skin RT~40,000 cm2Seal was expected to be impermeable, hence for almost 100 years called, Schlussleisten, bandelettes de fermeture, tight junctions, gaskets, belts.Introduction to the Epithelial Physiology

  • Diamond, JM and Bossert, WH, J Cell Biol. 1968. 37:694-702. Jared DiamondUltrastructural Geometry in backwards fluid transporting epitheliaMany fluid transporting epithelia posses dead-end long and narrow channels

  • Jared DiamondPulitzer Prize Winning Author

  • Epithelia like the small intestine or gallbladder that exhibit little or no ISC and have a very low RTThese were thought to be too fragile and had been damaged while mounting in Ussing chamberHowever, leaky epithelia did show a robust ability to transport water and solutesIntroduction to the Epithelial Physiology

  • Electrical resistance across leaky epithelia is much lower ~ 4 - 80 cm2Eventually concluded that leakiness was not due to damage and was property of large intercellular permeation (shunt) route (~1970)Introduction to the Epithelial Physiology

  • Electrical Resistances of Various EpitheliaPowell, DW. 1981.Am. J. Physiol. 4: G275-G288.

  • In contrast to relative impermeability of frog skin Ussing and Windhager (1964)The first demonstrations of intercellular ion permeation was obtained by electrophysiological studies in amphibian proximal tubule and gallbladder by:-Windhager, Boulpaep and Giebisch (1967)Fromter (1972)Fromter and Diamond (1972)Introduction to Leaky Epithelia

  • From this work came the concepts of tight and leaky epithelia.

    Basically tight epithelia cm2, and leaky epithelia, cm2.

    Introduction to Leaky Epithelia

  • Introduction to Leaky Epithelia1982

  • Introduction to Leaky Epithelia

  • Introduction to Leaky Epithelia

  • Boulpaep revised definitions of tight and leaky epithelia.

    Basically tight epithelia cm2, and leaky epithelia, cm2.

    Revised concepts of Tight and Leaky Epithelia

  • Intracellular and Intercellular routesPowell, DW. 1981.Am. J. Physiol. 4: G275-G288.

  • Research in intercellular movement surfaced 1962-1965.It did not gain recognition until early 1970sDuring 1970-80 most studies on shunt pathway were concerned with ultraleaky epithelia where shunt accounts for over 90% of conductance.Introduction to Leaky Epithelia

  • This prompted studies with small hydrophilic solutes to determine an upper size of solutes permitted to traverse this junction.Further experiments, although controversial, suggested that H2O traverses the junction.Introduction to Leaky Epithelia

  • Due to large permeability of solutes and ions and since this may account for up to 90% of total flux, the name tight junction is misleading.Introduction to Leaky Epithelia

  • Intercellular pathways across leaky epithelia can be viewed as nondiscriminating, water-filled imperfections between cells.These properties are ideally suited for epithelia whose physiological function is to absorb or secrete fluid isotonically and which are not normally faced with large transepithelial differences in ionic concentrations or total osmolarityIntroduction to Leaky Epithelia

  • In fact, these shunts virtually ensure the absence of significant transepithelial asymmetries.

  • 1970The Paracellular Pathway

  • The Paracellular Pathway1970

  • The Paracellular Pathway1970Emile Boulpaep

  • Advances that shaped the field

  • Studying the Tight Junction

  • When TJ is characterized by electrical resistance the measurement usually reflects conductance for Na+ and Cl-.It has recently been discovered that the TJ can discriminate among different ions.This data has led to speculation that TJs are aqueous protein-lined channels of defined size.Introduction to the Tight Junction

  • Studies of junctional permeability involve measurement of fluxes (ions, water, non-electrolytes).Electrical conductance.Transepithelial voltage changes upon exposure to solutions of different composition.Studying the Tight Junction

  • Due to ease, accuracy and time resolution, electrical measurements are generally preferred when studying junctional permeability (especially leaky epithelia).Studying the Tight Junction

  • Studying the Tight Junction

  • Paracellular fluxVmNa+Cl-+-

  • Na+Cl-+0Simplified Ussing Chamber TechniqueMeasure I

  • Devor et al., J Gen. Physiol. 1999. 113:743-760. Methods for measuring RTShort-circuit current Isc measurements with voltage deflections of 2mV.

  • MacVinish et al., Br. J. Pharmacol. 2007. e-pub ahead of printMethods for measuring RTShort-circuit current Isc measurements with a voltage deflection of 1mV.

  • Grahammer et al., J Biol. Chem. 2001. 276:42268-42275. Methods for measuring RTOpen-circuit VT measurements with current injections of 0.5A

  • Current-Voltage relationship in a leaky epitheliaNa+Cl-Na+Cl-~100 cm2

  • Structure of the Tight JunctionFarquhar and Palade, J. Cell. Biol. 1963. 17:375-412.

  • Tight Junctions in leaky epithelia i.e. small intestineIn leaky epithelia up to 95% of ion transport in through Paracellular pathway

  • Claudins - The Paracellular Ion channels22-kDa proteins cloned in 1998, Furuse et al. J. Cell. Biol.Claudin from the Latin word "claudere" (to close) Mutation of Claudin external loop charges modifies selectivity of paracellular permeability (Colegio and Anderson, 2002, Am. J. Physiol)

  • CateninsE-CadherinCa2+Ca2+ZO-1ActinOccludinClaudinsAdapted from Johnson and Quay, 2005JAM-1

  • The Resistance and ionic selectivity of Tight Junctions will vary by claudin compositionFuruse and Tsukita, Trends in Cell Biology, 2006

  • Tight Junctions are strands with aqueous pores that connect cellsFuruse and Tsukita, Trends in Cell Biology, 2006

  • Furuse and Tsukita, Trends in Cell Biology, 2006Tight Junctions can also act like a sieve and size select

  • Studying the Tight Junction

  • ISC and The Ussing Technique

  • What is ISC?What does it mean?What information does it tell you?

    The Ussing Technique and Methods for measuring RT

  • What is short-circuit current? -What is Short-circuit Current

  • What is short-circuit current? - Well it is not long!!-Anonymous RSMWhat is Short-circuit Current-From November GI team East meeting in Boston, MA.

  • Short-circuit Current

  • When an epithelium is bathed in identical solutionsThe transepithelial p.d. is maintained at 0mVNo net transfer of passive ionsi.e. there is no driving force for paracellular transport

  • Na+Cl-145mM NaCl145mM NaClShort-circuit Current (Isc)00VTE = V1-V2 = 0mV

  • The ions that are actively transported will continue to flow.The current running through the short circuit will be the resultant of all the net transport processes.

  • Na+Cl-145mM NaCl145mM NaClShort-circuit Current (Isc)00VTE = V1-V2 = 0mV

  • Cl-145mM NaCl145mM NaClShort-circuit Current (Isc) + lubiprosotone-0VTE = V1-V2 = -ve mV

  • Cl-145mM NaCl145mM NaClShort-circuit Current (Isc) + lubiprosotone00VTE = V1-V2 = 0 mV

  • Cl-145mM NaCl145mM NaClShort-circuit Current (Isc) + lubiprosotone00VTE = V1-V2 = 0 mVISCAmplifier

  • ISC is an unphysiological measurementIt is forcing a p.d. to 0mV that would not normally be at 0mVISC is shorting the paracellular pathway which would normally conduct most of the ions

  • ISC is measuring the sum of all the net transport processesNot what would happen in vivo

    Many German epithelial physiologists dont use ISC. They will measure VT and RT to determine ion movement and epithelial function.

  • Open Circuit Vs Short-Circuit

  • Grahammer et al., J Biol. Chem. 2001. 276:42268-42275. Methods for measuring RTOpen-circuit VT measurements with current injections of 0.5A

  • Lubiprostone induced recovery of bandelettes de fermeture function in ischemic porcine ileum. Moeser et al., 2007, AJP 292:G647-656.