Download - Mac Gregor Paracellular La 03192007

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 RSM


•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


•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

The Intercellular Pathway

Intercellular Pathway

Intracellular Pathway

•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.


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”

Jared Diamond

Pulitzer Prize Winning Author

•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


•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)


Electrical Resistances of Various Epithelia

Powell, 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 Ω•cm2, and “leaky” epithelia, Ω•cm2.


Introduction to Leaky Epithelia

1982

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

Intracellular and Intercellular routes

Powell, DW. 1981.Am. J. Physiol. 4: G275-G288.

• 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.


•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.


•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.


• 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


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

1970

The Paracellular Pathway


1970


1970

Emile 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 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.


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



ss

s

GR

V

IG

1

,

Paracellular fluxVm

Na+

Cl-

+ -

Na+ Cl-

+ 0

Simplified Ussing Chamber Technique

ss

s

GR

V

IG

1

,

Measure ΔI

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


Short-circuit current Isc measurements with a voltage deflection of 1mV.

Grahammer et al., J Biol. Chem. 2001. 276:42268-42275.


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

Structure of the Tight Junction

Farquhar and Palade, J. Cell. Biol. 1963. 17:375-412.

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



Tight Junctions can also act like a sieve and size select


SBAT RRRR

111

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 RSM

What is Short-circuit Current

-From November GI team East meeting in Boston, MA.

Short-circuit Current

•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

Na+ Cl-

145mM NaCl

145mM NaCl

Short-circuit Current (Isc)

0 0

VTE = 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 NaCl

145mM NaCl

Short-circuit Current (Isc)

0 0

VTE = V1-V2 = 0mV

Cl-

145mM NaCl

145mM NaCl

Short-circuit Current (Isc) + lubiprosotone

- 0

VTE = V1-V2 = -ve mV

Cl-

145mM NaCl

145mM NaCl


0 0

VTE = V1-V2 = 0 mV

Cl-

145mM NaCl

145mM NaCl


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.

Open Circuit Vs Short-Circuit

Grahammer et al., J Biol. Chem. 2001. 276:42268-42275.


Open-circuit VT measurements with current injections of 0.5µA

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

Download - Mac Gregor Paracellular La 03192007

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