Epithelial Transport I: Salts and Water

Experimental approach to studying epithelial tissues

The frog skin was the first salt-absorbing epithelium to be well understood, because it has simple structure, large size, survives and functions in vitro.

We will follow its development as an experimental model of salt absorbing tissues. It is an especially useful model of the distal tubule of the kidney, a ferociously difficult tissue to study because of its small size.

The regulatory challenge of life in fresh water.

H2O (osmotic)

NaCl (down concentration gradient)

Shown below are the expected passive flows of water and salt for a frog in fresh water, where the NaCl concentration is <1 mM, while body fluids are about 150 mM NaCl.

What the frog’s skin does for the frog: restores NaCl lost by diffusion and through urine.

NaCl absorption against concentration gradient

H2O (dilute urine)

NaCl recovery from urine in kidney and bladder

How frogs wake up

The isolated frog skin preparation

This method was pioneered by Hans Ussing in 1947

The isolated skin maintains a trans-epithelial potential of about 50 mV blood side positive, for many hours if bathed in frog saline on both sides.

2. To determine whether Na+, Cl- or both are actively transported,

First eliminate all external driving forces - chemical and electrical.

No chemical forces: identical solutions both sides

No electrical forces: pass opposing current large enough to make transepithelial potential = zero; this is called short-circuiting

Then measure net fluxes of Na+ and Cl- using radioisotopes, with the tissue under short circuit. A known specific activity of isotope is added to one side; the other side is counted at intervals of time.

Blood side Pond side

Unidirectional fluxes of 36Cl- are equal; there is no net flux.

Unidirectional fluxes of 22Na+ or 24Na+ are not equal; there is an inward net flux.

In frog skin, the short-circuit current is a close measure of the net Na+ fluxRemember, this is the current that was needed to balance the tissue’s active current.

This adds to the evidence that Na+ is at least by far the major actively transported ion.

(If there were significant net fluxes of more than one ion, the short-circuit current would approximate the algebraic sum of the net ionic fluxes.)

What we know at this point:Na+ is actively transported; Cl- could be following passively down its electrochemical gradient.

Next question: Where is the active step for Na+ transport located, the apical or basolateral membrane?

Microelectrodes locate the active step

Voltage

Microelectrode track through tissue

0

+50

Electrode advanced

Pond sideBlood side

Conclusions from microelectrode experiments

• 1. Na+ entry across the apical membrane from the pond occurs down both a chemical and an electrical gradient (remember, [Na+] inside cells is lower than outside).

• 2. Na+ exit across the basolateral membrane into the frog occurs against both an electrical and a chemical gradient

Specific inhibitory drugs identify the basolateral and apical steps.

Ouabain is a highly specific inhibitor of Na+K+ ATPase.

Amiloride is a specific inhibitor of Na+ channels at a concentration of 0.1 mM.

Both drugs were first identified because of their diuretic action - they increase urine volume flow.

Is the Na+/K+ ATPase responsible for the active step?

Applied to the blood side of the tissue, ouabain abolishes the open-circuit transepithelial potential, the short-circuit current, and causes the unidirectional fluxes of Na+ to become equal.

Applied to the apical side, it has no effect.

Conclusion: Na+/K+ pump = Na+/K+ ATPase

How does Na+ cross the apical membrane?

Amiloride applied to the apical side abolishes all signs of Na+ transport.

Applied to the blood side, it has no effect.

Conclusion: At least with frog saline on the pond side, essentially all Na+ uptake is by way of the amiloride-sensitive Na+ channels (ENaC).

K+ leak

Na/K pump

POND BLOOD

Tight junctions

Amiloride-sensitive Na+

channels

A Problem: the Na+ concentration of actual pond water water is less than 1 mEq/l. At this concentration, Na+ cannot go downhill into the cell across the apical membrane without some extra energetic help.

The Solution: add some extra voltage to the apical membrane, by inserting V-type H+ ATPase there. The V-ATPase pumps H+ from cytoplasm to pond, making the cytoplasm more electronegative relative to the pond side.

K+ leak

Na/K pump

POND BLOOD

Tight junctions

Amiloride-sensitive Na+

channels

V-ATPase

H+

With the V-ATPase in place, Na+ uptake through Ena channels is still passive - the effect of the V-ATPase is to make the electrochemical gradient seen by Na+ to be in the right direction for Na+ uptake. The extrusion of H+ from the cell also assists in the elimination of NH3, a product of protein metabolism.

H+

NH3

NH4+

The H+ secretion traps diffusible NH3 as impermeant NH4

+

How is electroneutrality maintained?

1. Primary Na+ active transport sets up a blood-side-positive transepithelial potential.

2. Cl- diffuses down its electrochemical gradient from pond side to blood side.

What route does the Cl- take through the tissue?

Cl-

This is called the paracellular route (as opposed to the transcellular route taken by Na+)