Download - Mac Gregor Paracellular La 03192007
•MacGregor, Just go over the basics. No-one wants to hear your theories on paracellular permeability, keep them to yourself.
-Anonymous RSM
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 RSM
•MacGregor, pure basics would be good, definitions, (how to measure TER, etc), not so much detail so that we’re all going to want to set up our own electrophysiology labs in our basements or anything.
-Anonymous RSM
Requests 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 space
Introduction to the Epithelial Physiology
•Since the epithelia of choice for many years was frog skin, with negligible fluxes through intercellular space.
•Frog skin RT~40,000 Ω•cm2
•Seal 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 Diamond
Ultrastructural Geometry in backwards fluid transporting epithelia
“Many fluid transporting epithelia posses dead-end long and narrow channels”
•Epithelia like the small intestine or gallbladder that exhibit little or no ISC and have a very low RT
•These were thought to be too fragile and had been damaged while mounting in Ussing chamber
•However, “leaky” epithelia did show a robust ability to transport water and solutes
Introduction to the Epithelial Physiology
•Electrical resistance across leaky epithelia is much lower ~ 4 - 80 Ω•cm2
•Eventually concluded that leakiness was not due to damage and was property of large intercellular permeation (shunt) route (~1970)
Introduction to the Epithelial Physiology
•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 Ω•cm2, and “leaky” epithelia, Ω•cm2.
Introduction to “Leaky” Epithelia
•Boulpaep revised definitions of tight and leaky epithelia.
•Basically “tight” epithelia Ω•cm2, and “leaky” epithelia, Ω•cm2.
Revised concepts of “Tight” and “Leaky” Epithelia
• Research in intercellular movement surfaced 1962-1965.
•It did not gain recognition until early 1970’s
•During 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 osmolarity
Introduction to “Leaky” Epithelia
•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 TJ’s 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
Devor et al., J Gen. Physiol. 1999. 113:743-760.
Methods for measuring RT
Short-circuit current Isc measurements with voltage deflections of ± 2mV.
MacVinish et al., Br. J. Pharmacol. 2007. e-pub ahead of print
Methods for measuring RT
Short-circuit current Isc measurements with a voltage deflection of 1mV.
Grahammer et al., J Biol. Chem. 2001. 276:42268-42275.
Methods for measuring RT
Open-circuit VT measurements with current injections of 0.5µA
Current-Voltage relationship in a leaky epithelia
-80 -60 -40 -20 20 40 60 80
-250
-200
-150
-100
-50
50
100
150
200
250
145/145
- (V1-V2), mV
- I, A
Na+
Cl-
Na+ Cl-
~100Ω •cm2
Tight Junctions in leaky epithelia i.e. small intestine
In leaky epithelia up to 95% of ion transport in through Paracellular pathway
Claudins - The Paracellular Ion channels
• 22-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)
Paracellular
space
Catenins
E-Cadherin
Ca2+
Ca2+ZO-1
Actin
Occludin
Claudins
Adapted from Johnson and Quay, 2005
JAM-1
The Resistance and ionic selectivity of Tight Junctions will vary by claudin composition
Furuse and Tsukita, Trends in Cell Biology, 2006
Tight Junctions are strands with aqueous pores that connect cells
Furuse and Tsukita, Trends in Cell Biology, 2006
Furuse and Tsukita, Trends in Cell Biology, 2006
Tight Junctions can also act like a sieve and size select
•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? - Well it is not long!!
-Anonymous RSM
What is Short-circuit Current
-From November GI team East meeting in Boston, MA.
•When an epithelium is bathed in identical solutions
•The transepithelial p.d. is maintained at 0mV
•No net transfer of passive ions
•i.e. there is no driving force for paracellular transport
•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.
Cl-
145mM NaCl
145mM NaCl
Short-circuit Current (Isc) + lubiprosotone
0 0
VTE = V1-V2 = 0 mV
ISC
Amplifier
• ISC is an unphysiological measurement
•It is forcing a p.d. to 0mV that would not normally be at 0mV
•ISC is shorting the paracellular pathway which would normally conduct most of the ions
• ISC is measuring the sum of all the net transport processes
•Not what would happen in vivo
•Many German epithelial physiologists don’t use ISC. They will measure VT and RT to determine ion movement and epithelial function.
Grahammer et al., J Biol. Chem. 2001. 276:42268-42275.
Methods for measuring RT
Open-circuit VT measurements with current injections of 0.5µA