Epithelial Transport I: Salts and Water

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Epithelial Transport I Salts and Water
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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.
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The regulatory challenge of life in fresh water.
Shown below are the expected passive flows of
water and salt for a frog in fresh water, where
the NaCl concentration is lt1 mM, while body
fluids are about 150 mM NaCl.
NaCl (down concentration gradient)
H2O (osmotic)
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What the frogs skin does for the frog restores
NaCl lost by diffusion and through urine.
NaCl absorption against concentration gradient
NaCl recovery from urine in kidney and bladder

H2O (dilute urine)
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How frogs wake up
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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.
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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
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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 22Na or 24Na are
not equal there is an inward net flux.
Unidirectional fluxes of 36Cl- are equal
there is no net flux.
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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
tissues 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.)
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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?
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Microelectrodes locate the active step
Pond side
Blood side
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Voltage
Electrode advanced
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Microelectrode track through tissue
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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

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Specific inhibitory drugs identify the
basolateral and apical steps.
Ouabain is a highly specific inhibitor of NaK
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.
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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
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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).
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Tight junctions
POND
BLOOD
Na/K pump
Amiloride-sensitive Na channels
K leak
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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.
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Tight junctions
POND
BLOOD
Amiloride-sensitive Na channels
Na/K pump
H
V-ATPase
K leak
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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.
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The H secretion traps diffusible NH3 as
impermeant NH4
NH3
NH4
H
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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.
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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)