Acta Physiol 2011, 202, 435–464
REVIEW
Reconciling the Krogh and Ussing interpretations of
epithelial chloride transport – presenting a novel hypothesis
for the physiological significance of the passive cellular
chloride uptake
Erik Hviid Larsen
Department of Biology, University of Copenhagen, Copenhagen Ø, Denmark
Received 15 September 2010,
revision requested 17 October
2010,
final revision received 17
November 2010,
accepted 8 December 2010
Correspondence: E. H. Larsen,
Department of Biology, August
Krogh Building, Universitetsparken
13, DK-2100 Copenhagen Ø,
Denmark.
E-mail: [email protected]
Abstract
In 1937, August Krogh discovered a powerful active Cl) uptake mechanism
in frog skin. After WWII, Hans Ussing continued the studies on the isolated
skin and discovered the passive nature of the chloride uptake. The review
concludes that the two modes of transport are associated with a minority cell
type denoted as the c-type mitochondria-rich (MR) cell, which is highly
specialized for epithelial Cl) uptake whether the frog is in the pond of low
[NaCl] or the skin is isolated and studied by Ussing chamber technique. One
type of apical Cl) channels of the c-MR cell is activated by binding of Cl) to
an external binding site and by membrane depolarization. This results in a
tight coupling of the uptake of Na+ by principal cells and Cl) by MR cells.
Another type of Cl) channels (probably CFTR) is involved in isotonic fluid
uptake. It is suggested that the Cl) channels serve passive uptake of Cl) from
the thin epidermal film of fluid produced by mucosal glands. The hypothesis
is evaluated by discussing the turnover of water and ions of the epidermal
surface fluid under terrestrial conditions. The apical Cl) channels close when
the electrodiffusion force is outwardly directed as it is when the animal is in
the pond. With the passive fluxes eliminated, the Cl) flux is governed by
active transport and evidence is discussed that this is brought about by an
exchange of cellular HCO3) with Cl) of the outside bath driven by an apical
H+ V-ATPase.
Keywords amphibian epidermal surface fluid, Cl)/HCO3) exchange,
epithelial Cl) channels, mitochondria-rich cells, patch clamp, proton
V-ATPase.
The aim of this review article is to discuss a unifying
description of two sets of framework on mechanisms of
ion transport across epithelia. They have their origin in
Copenhagen from the Zoophysiological Laboratory by
work of August Krogh and his student Hans Ussing
respectively. Directing the attention to the dependence
of ion transport on energy metabolism in epithelia, they
have had a significant impact on the way we consider
ion regulation in animals and man, the methods by
which we study epithelial ion transport and the interpretation of the functional organization of transporting
epithelia (Andreoli 1999, Reuss 2001, Larsen 2002,
Kirschner 2004, Evans 2008, Palmer & Andersen 2008,
Hillyard et al. 2009). First, I shall consider specific
experiments by Krogh and Ussing and then proceed by
discussing more recent studies including our own
investigations, which have led to the hypothesis that
Krogh and Ussing’s findings constitute two different
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Functions of mitochondria-rich cells in anuran skin
Æ E H Larsen
functions of a specific type of mitochondria-rich (MR)
cells of the epithelium: the one with the uptake of Cl)
coupled to cellular energy metabolism (Krogh’s observation) and the other with the uptake of Cl) governed
by electrodiffusion (Ussing’s observation). It is a hallmark of our synthesis that the mode of operation of the
MR cell is controlled by the external driving force for
the Cl) flux providing a straightforward explanation for
the apparently conflicting observations of the early
studies. It will be shown that the regulation of the
passive Cl) conductance enables tight coupling of the
inward flow of Na+ through principal cells and Cl)
through MR cells. With reference to this ‘Ussing mode’
of epidermal transport function, I advance and discuss a
new hypothesis that the passive uptake of Cl) driven by
the active uptake of Na+ serves regulation of ion
concentrations and volume of the thin surface fluid
produced by the subepidermal mucous glands. A
reversible closure of the apical Cl) channels occurs
when electrodiffusion would be outwardly directed.
This ‘Krogh mode’ of epidermal transport function
prevails in aquatic environments of low ion concentrations. With the passive fluxes eliminated, the Cl) flux is
governed by the active mechanism and evidence is
discussed that this is brought about by an exchange of
cellular HCO3) with Cl) of the outside bath driven by
an apical H+ V-ATPase.
The early studies on the mechanism of
epithelial chloride transport
August Krogh: active transport of chloride
By the end of the 1930s, Krogh initiated investigations
of the concepts of active and passive transport of ions
both in experimental studies in his own laboratory and
in monographs (Krogh 1939, 1946). The first experimental paper (Krogh 1937) dealt with two major
themes of osmoregulation: that is, the existence of
active transport in osmoregulatory epithelia, and the
significance of active and passive transport for the ion
balance in freshwater animals respectively. Krogh
designed a chamber that allowed for the collection of
samples both from the urine and the bath in which the
frog was submersed. Stirring of the bath was obtained
by the animal’s breathing movements (Fig. 1a). By
titrating samples of the bath taken at intervals of hours,
Krogh discovered a powerful Cl) uptake mechanism in
frog skin (Fig. 1b). For investigating whether Cl) or
Na+, or both ions, are submitted to active transport, the
analysis was extended by ion substitution protocols
resulting in the following observations (Krogh 1938): (i)
Na+ and Cl) were absorbed together from a diluted
solution of NaCl; (ii) from solutions of KCl, Cl) and K+
were also absorbed together, but the uptake was limited
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Acta Physiol 2011, 202, 435–464
(a)
(b)
Figure 1 Krogh’s investigation of Cl) uptake by the frog
(Krogh 1937). (a) The experimental chamber designed for
measuring rates of cutaneous Cl) uptake, urine production
and urinary Cl) loss. (b) Uptake of Cl) from the bath containing an initial Cl) concentration of 1/100 of that of Ringer’s
solution with Na+ and K+ as cations and glucose added for
obtaining an osmotic concentration similar to that of the
Ringer’s solution. At about 7 h, the bath was replaced with a
NaCl solution that was diluted ·500 with respect to [Cl)] as
compared to Ringer’s and with glucose added to maintain an
osmotic pressure similar to that of Ringer’s solution. The
frog was kept in running distilled water for 3 weeks prior to
the experiment.
to a small amount of the two ions; (iii) little or no Ca2+
was taken up from CaCl2 solutions, but a limited
amount of Cl) was taken up in exchange of HCO3);
and (iv) Cl) and Na+ were taken up by independent
mechanisms. From these observations, Krogh concluded
that anurans submersed in solutions of low ion concentration, Cl) is the ion always actively absorbed. The
above findings were confirmed and extended by in vivo
studies on other anuran species (Jørgensen et al. 1954,
Garcia-Romeu et al. 1969, Mullen & Alvarado 1976).
The strength of the discovered transport mechanisms
is illustrated by the electrochemical work performed by
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E H Larsen
the frog. Krogh observed the uptake of NaCl down to
a concentration of about 10 lm. Following salt depletion, the concentration of extracellular Cl) and Na+ is
about 80 and 100 mm, respectively (Krogh 1937,
Jensen et al. 2003) from which an electrochemical
work, D~
lNaCl ¼ 42 kJ mol1 NaCl transported into the
animal can be calculated. The link between energy
metabolism and uphill uptake of ions will be discussed
later in the study.
Æ Functions of mitochondria-rich cells in anuran skin
(a)
Hans Ussing: passive transport of chloride
In 1951, Ussing and Zerahn developed the short-circuit
technique for the study of active ion transport by the
isolated frog skin. The skin was mounted between two
chambers containing identical Ringer’s solutions, and
the transepithelial potential was short-circuited to 0 mV
with an external circuit for bringing all solutes in
thermodynamic equilibrium across the skin (Fig. 2a).
Ussing and Zerahn showed that the short-circuit current
is carried by the net flux of Na+ calculated as the
difference between the unidirectional Na+ fluxes estimated by 24Na+. In the short-circuited skin, the transepithelial transport work is zero. Thus, with the sodium
ions being transported away from thermodynamic
equilibrium, the study provided the first unequivocal
demonstration of active Na+ transport. Subsequently,
applying the isotope tracer technique for measuring the
unidirectional Cl) fluxes, Ussing and co-workers discovered the passive nature of Cl) transport in the
isolated frog skin. Taking advantage of the variation of
the transepithelial potential among the skin preparations, the experiments were performed at open-circuit
conditions with Ringer’s solution on the inside and 1/10
Ringer’s solution on the outside. The results in Fig. 2b
indicate agreement between the observed flux ratio and
the flux ratio predicted for electrodiffusion (KoefoedJohnsen et al. 1952a).
Functional organization of anuran skin
epithelium
Figure 3 shows a micrograph of the anuran skin with
the heterocellular, multilayered absorbing epithelium
and the subepidermal secreting mucous glands. Note the
relatively large secretory area as compared to the
absorptive epidermis. The functional interplay between
these two units is discussed in a separate section below
on the role of the Cl) channels of the epidermal
epithelium.
Since the publication of the above seminal papers,
numerous studies have provided a fairly detailed
description of the functional organization of the
anuran skin epithelium, which will be outlined in this
section. Subsequently, in chronological order, I shall
(b)
Figure 2 Ussing and his coworkers’ investigation of ion
transport by the isolated frog skin (Ussing & Zerahn 1951,
Koefoed-Johnsen et al. 1952a). (a) With the setup for shortcircuiting the isolated skin preparation exposed bilaterally to
Ringer’s solution it was shown that the short-circuit current
was carried by a net flux of Na+ directed from the outside to
the inside of the skin. (b) Analysis of unidirectional Cl) fluxes
through frog skin. The graph shows the calculated flux ratios of
transport by electrodiffusion (Ussing 1949) plotted against
those observed. Efflux (Mout) was measured with 36Cl) and
influx (Min) was determined as the sum of the efflux and the net
flux with the latter obtained by Cl) titration of the outside
bath. Skins of nine frogs were used with two data set obtained
from each skin.
discuss the experimental evidence regarding the pathways for chloride uptake. The first model of the Na+transporting cell was configured with a Na+-selective
outward-facing membrane and Na+/K+ pumps in an
inward-facing K+-selective membrane. It was supposed
to be located in the bottom of the multilayered
epithelium and assumed permeable to Cl) (KoefoedJohnsen & Ussing 1958). Ussing & Windhager (1964)
discovered paracellular transport of, e.g. Cl), SO42)
and Na+, and based upon a microelectrode study of the
intraepithelial electrical potential profile, they hypothesized that all cells beneath the dead cornified layer
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437
Functions of mitochondria-rich cells in anuran skin
Apical membrane
of first living cell layer
Principal cell
Gland duct
compartment
Æ E H Larsen
Acta Physiol 2011, 202, 435–464
Cornified layer
MR cell
20 µm
Lumen of
mucosal
gland
Basal lamina
Acinar epithelium
Iridiophore
constitute a functional syncytium. A similar conclusion
was arrived at in an ultrastructural analysis of the skin,
which included a characterization of the cell junctions
of the epithelium (Farquhar & Palade 1964, 1965).
The new model implied that the outward-facing Na+selective membrane would be the apical plasma membrane of the outermost living cell layer. This notion
was verified by morphological studies of the volume
response of cells of this layer to perturbations of the
active Na+ transport (Voûte & Ussing 1968), and by
estimating the diffusion distance of Na+ between the
outside bath and the Na+-selective barrier (Fuchs et al.
1972). The hypothesis of a functional syncytium was
verified by X-ray microprobe analysis of the intraepithelial redistributions of small diffusible ions in
response to perturbations of the transepithelial Na+
flux (Rick et al. 1978). As a further extension of the
classical frog skin model, the Na+/K+ pumps were
found to be located in all the plasma membranes lining
the lateral intercellular spaces with no expression
neither on the apical membrane of the outermost layer
of transporting cells nor on the inward-facing plasma
membrane of the germinativum cells (Farquhar &
Palade 1966, Mills et al. 1977). The localization of the
Na+/K+ pumps to the plasma membranes lining the
lateral intercellular space applies to transporting epithelia in general (Stirling 1972, Mills & Ernst 1975,
Ernst & Mills 1977, Mills & DiBona 1978, DiBona &
Mills 1979, Kashgarian et al. 1985, Pihakaski-Maunsbach et al. 2003, Grosell et al. 2005).
The present model takes into account that the
amphibian skin is a heterocellular epithelium with
principal cells and flask-shaped MR cells (Budtz &
Larsen 1973, Whitear 1975, Spies 1997). The apical
438
Figure 3 Cellular organization of anuran
skin (Rana esculenta). The skin contains
two ion and water transporting units. The
multilayered, heterocellular epidermis is
an absorbing epithelium with principal
cells and mitochondria-rich (MR) cells.
The acinar epithelium of the subepidermal mucous glands is secretory. Courtesy
of Dr. Åse Jespersen.
plasma membrane of the MR cell faces the subcorneal
space with the cell body between the Na+-transporting
principal cells (cf. Fig. 4a). The MR cell morphology
varies; typically they are flask shaped with the neck
located between the outermost principal cells with
which they form tight junctions. With a density
between 103 and 105 cells cm)2 and an apical membrane area of 4–10 lm2 (disregarding membrane infoldings), their total membrane area constitutes a small
fraction of the total epidermal surface area (Ehrenfeld
et al. 1976, Willumsen & Larsen 1986, Larsen et al.
1987). Figure 4b shows a model of the functional
organization of the heterocellular epithelium with
identified transport systems of the plasma membranes.
Considering the structural complexity of the multicellular skin epithelium (e.g. Fig. 3), this is a simplifying
presentation focusing on the transport systems of
significance for the discussion below. The sodiumtransporting principal cell compartment displays amiloride-sensitive apical Na+ channels (Lindemann & Van
Driessche 1976, Palmer 1992, Palmer & Garty 1997)
and basolateral K+ channels with an ouabain-sensitive,
rheogenic Na+/K+ P-ATPase in the basolateral membrane (Skou 1965, Nagel 1980, Nielsen 1982a). The
other important function of the skin, the uptake of
water reviewed by Jørgensen (1997), is regulated by the
insertion of aquaporins into the apical plasma membrane of the principal cells by arginine vasotocin (AVT)
stimulation (Hasegawa et al. 2003, Suzuki & Tanaka
2009, Ogushi et al. 2010a). Amphibians have two
AVT-stimulated aquaporin isoforms (homologs of
AQP-h2 and AQP-h3) that were first characterized in
the tree frog Hyla japonica. Water is transported across
the basolateral plasma membrane via a constitutively
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E H Larsen
Æ Functions of mitochondria-rich cells in anuran skin
(a)
(b)
(c)
Figure 4 The heterocellular anuran skin epithelium. (a) Immunolocalization of mitochondria-rich cells. MoAb, Monoclonal
antibody; hCK, human cytokeratin. This minority cell type is typically flask shaped with the apical membrane just below the dead
cornified layer (Spies 1997). (b) Model of the functional organization of the skin epithelium. Above: the large Na+-transporting
syncytial compartment. It should be noted that the Na+/K+ pumps are located exclusively in the plasma membranes lining the lateral
intercellular spaces. Below: The c-type mitochondria-rich cell specialized for Cl) uptake. PKA, protein kinase A; c.s., catalytic
subunit; CA, carbonic anhydrase. (c) Models of the acid secreting a-type mitochondria-rich cell (above) and the base secreting b-type
mitochondria-rich cell (below).
expressed aquaporin that was characterized as AQPh3BL (Akabane et al. 2007). Other transport systems
have been discovered, like apical K+ channels that add
another function to the skin, that is, active K+ secretion
in animals in a positive K+ balance (Nagel & Hirschmann 1980, Van Driessche & Zeiske 1980, Frazier &
Vanatta 1981, Nielsen 1984). The apical plasma
membrane is also configured with a non-selective
Ca2+-sensitive cation channel of unknown function
(Van Driessche & Zeiske 1985). There is evidence that
the cell water volume of the principal cell compartment
is regulated by the activity of basolateral NaK2Cl cotransporters and parallel Cl) channels as indicated in
Fig. 4b (Ferreira & Ferreira 1981, Ussing 1982, 1985,
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Functions of mitochondria-rich cells in anuran skin
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Dörge et al. 1985). The Na+/H+ exchange mechanism
and the Cl)/HCO3) exchange mechanism serve intracellular pH regulation. They control the [H+]-dependent open/close kinetics of the apical Na+ channels and
the basolateral K+ channels, which constitutes an
important element of the molecular mechanism coordinating the activity of the two cation channels (Harvey
et al. 1988) denoted as ‘cross talk’(Schultz 1992).
Studies of the regulation of the transport systems of
the principal cell compartment were reviewed recently
(Hillyard et al. 2009).
The MR cell model depicted in Fig. 4b is of the ctype and has Cl) uptake as its major function (Larsen
1991). There is compelling evidence for another type of
MR cells that serves regulation of the acid balance
(Ehrenfeld & Garcia-Romeu 1977, Duranti et al. 1986,
Harvey 1992, Ehrenfeld & Klein 1997). A similar cell
type was first discovered in studies on distal renal
epithelia together with a base secreting cell, denoted as
the a- and b-type intercalated cell respectively (Steinmetz 1974, 1986, Gluck et al. 1982, Stetson et al.
1985, Madsen et al. 1991). They are shown in Fig. 4c
and discussed briefly below. It is the configuration of
transport systems in the apical membrane of the c-type
MR cell, which is of the major concern here. This
membrane contains amiloride blockable sodium channels, chloride channels (at least two types as indicated)
together with a proton pump and a Cl)/HCO3)
exchange mechanism. Of special notice is the chloride
channel that is depicted as being controlled by the
apical membrane potential and external Cl), the latter
via a hypothetical chloride-binding site facing the
outside bath. The experimental evidence for the apical
transport systems shown in Fig. 4b is presented in the
sections below, which also contain discussions of their
specific functions. Throughout, this is done by reference to quantitative experimental analyses that have
enabled us to reveal a most interesting interplay
between the cell types and their plasma membrane
mechanisms.
Concentration dependence of the passive
chloride permeability
Early studies showed that frog skin (Rana temporaria)
becomes tight to Cl) when this ion is removed from the
outside bathing solution (Koefoed-Johnsen & Ussing
1958). In an in vivo study on Rana pipiens Kirschner
(1970) observed a strong dependence of the transepithelial potential difference (VT) on the major anion of
the bathing solution, see Fig. 5. With the poorly
permeating SO42) in the bath, VT increased substantially with increasing [Na2SO4]bath as indicated by the
O-symbol. This is caused by the [Na+]bath-dependent
increase in the active Na+ uptake as predicted by the
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Acta Physiol 2011, 202, 435–464
Figure 5 Dependence of transepithelial potential difference on
external Na+ concentration in salt-depleted Rana pipiens.
Measurements were performed first with SO42) and then with
Cl) as the accompanying anion. Unlike the sign convention
used in the other figures of the review article, the transepithelial
potential difference is here indicated relative to bath. Mean
and SEM. Redrawn from Kirschner (1970).
Ussing–Zerahn circuit analysis (Ussing & Zerahn
1951). With NaCl in the bath, a similar increase was
observed in the lower range of concentrations (left-hand
side of Fig. 5). However for [NaCl]bath > 5–10 mm, VT
decreased with increasing [NaCl]bath as indicated by the
h-symbol. This was taken as an indirect demonstration
of an activation of the passive Cl) permeability (PCl) for
[Cl)]o > 5–10 mm. The control of PCl by Cl) of the
outside bath was verified in detailed studies on the skin
of R. temporaria (Kristensen 1982), and Bufo bufo
(Harck & Larsen 1986). These studies also showed that
in the PCl-activated state, the epithelium exhibits a high
conductive permeability to all anions tested according
to the following ranking of permeabilities, SCN) : Br) :
Cl) : I) = 1.7 : 1.3 : 1 : 0.7–0.8. Interestingly, external
Cl) and Br), but not the other anions, can activate the
chloride permeability. Based upon these observations, it
was hypothesized that the activation of anion channels
is because of the binding of chloride to a membrane site
with a high but selective affinity for Cl) (and Br)), and
that the flux of anions in open channels is associated
with a translocation site of poor anion selectivity
(Kristensen 1982).
Quantitative description of the concentration dependence
With the purpose of illustrating quantitative relationships, the experiments on the skin of B. bufo shall be
considered in more detail. In the absence of active Na+
transport, the outward current (VT = )80 mV, outside
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E H Larsen
Æ Functions of mitochondria-rich cells in anuran skin
(a)
Figure 6 Concentration dependence of
halide fluxes in toad skin exposed to
external solutions with the impermeant
K+ as cation (Harck & Larsen 1986). (a)
The dependence of the influx of Cl) on the
external concentration of Cl) (gluconate
substitution). The isotope flux (red symbols) was measured simultaneously with
recording of the clamping current (blue
symbols). The left-hand Y-axes is scaled
by the Faraday for indicating that Cl) is
carrying the clamping current
(VT = )80 mV). (b) The dependence of
the rate constant for the Cl) influx on
[Cl)]o. Since the driving force is constant
in
the rate constant, kCl ¼ JCl
=½Cl o is a
measure of the chloride permeability, PCl.
(c) The simultaneously measured I) influx
as a function of [Cl)]o. (d) Dependence of
the clamping current on external halide
ion. Only Cl) and Br) carry large currents. The iodide current is just a little
larger than the leak current estimated
with gluconate in the external solution.
(b)
(c)
(d)
bath negative) is carried by an influx of Cl) in the
interval, 1.45 mm £ [Cl)]o £ 110 mm. The relationship
between the influx of Cl) and [Cl)]o is sigmoid rather
than linear or hyperbolic (Fig. 6a). Figure 6b shows that
the rate constant of the Cl) influx (kCl ) is a continuous
function of [Cl)]o with strong permeability activation in
the lower range of concentrations ([Cl)]o £ 20 mm),
which explains Kirschner’s observations reproduced in
Fig. 5. ‘Self-inhibition’ is seen at larger external chloride
concentrations (cf. Fig. 6b), which causes the apparent
in
‘saturation’ of the JCl
½Cl o relationship (Fig. 6a). In
these experiments, the outside bath contained 3 mm I)
whereas the I) influx, JIin , was traced by 125I). Figure 6c
shows that the [Cl)]o-dependence of JIin is similar to the
PCl ½Cl o relationship, providing the evidence for the
two halide ions passing the same population of [Cl)]oactivated anion channels. The ratio of the rate constants
of the two halide ions, kI =kCl , did not indicate strong
Cl) : I) selectivity; it varied in a non-systematic way
between 0.73 0.05 and 0.60 0.06 within the range
of external [Cl)] studied. This is contrary to the finding
that if all external Cl) is substituted by I) at constant
electric driving force, the iodide current is just a little
larger than the leak current measured with gluconate as
the major anion outside (Fig. 6d). Considering the
demonstrated poor Cl) : I) selectivity of open channels,
the very small iodide current seen in Fig. 6d would be
compatible with the hypothesis of an external binding
site of high binding specificity for chloride when
compared with that of iodide.
The external regulatory chloride-binding site
Kristensen’s hypothesis of a regulatory binding site
facing the outside bath (Kristensen 1982) resembles that
forwarded the following year by Kostyuk et al. (1983),
who suggested that a calcium channel in a mollusc
neurone has two ion-selecting filters, an external one
that binds divalent cations in a highly specific manner
and an ion-selecting filter in the channel pore determin-
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Functions of mitochondria-rich cells in anuran skin
Æ E H Larsen
ing the selectivity for different divalent cations. In
subsequent studies, Hess & Tsien (1984) and Friel &
Tsien (1989) presented a multi-ion pore model of the
Ca2+ channel, which accounted for the earlier findings
without the need of an external binding site. Therefore,
the hypothesis of a regulatory Cl)-binding site outside
the chloride channel pore needs further qualification;
the anion selectivity of the amphibian channel,
Br) : Cl) : I) = 1.3 : 1 : 0.7 is different from the
sequence of free diffusion coefficients of these ions in
water, DBr : DCl : DI = 1.02 : 1 : 1.01 (0.1 m K+ solutions, 25 C; Robinson & Stokes 1970), which provides
the evidence that the ions interact with sites in the
channel pore. It was further shown that for
[Cl)]o ‡ 5 mm, the unidirectional Cl) and I) fluxes
obey the flux-ratio criterion with a flux-ratio exponent
of unity from which it could be concluded that the
independence principle holds for the flow of the two
anions (Harck & Larsen 1986), which also applies to
the unidirectional Cl) fluxes in frog skin for
[Cl)]o ‡ 12 mm (Koefoed-Johnsen et al. 1952a, Kristensen 1983). Therefore, multi-ion pores exhibiting
single filing (Hodgkin & Keynes 1955) can be excluded.
This conclusion was further supported by the finding
that the Cl) : I) selectivity in the epithelial anion
channel is independent of the mole fraction and total
concentration of the two halide ions (Harck & Larsen
1986), which is in agreement with a channel pore
holding no more than one ion at a time (Hille &
Schwartz 1978), and contrary to the multiple ion
occupancy pore exhibiting anomalous mole fraction
effect. Thus, the hypothesis of an external regulatory
binding site of high Cl) specificity is the most plausible
explanation for the S-shaped increase in the Cl) influx
with increasing [Cl)]o shown in Fig. 6a.
Voltage dependence of the passive chloride
permeability
For understanding the physiology of the anuran skin
epithelium, it is equally important to realize that its
passive chloride permeability is controlled by voltage
(Larsen & Kristensen 1978). Thus, with Ringer’s
solution on the outside, the chloride currents of the
skin of B. bufo are activated by transepithelial hyperpolarization and deactivated when VT is reversed. That
is, the chloride channels are shut when the electric
driving force on chloride is outwardly directed. As is
the case for the above-mentioned concentration dependence of PCl, the voltage dependence also seems to be a
general feature of anurans: R. temporaria and Rana
esculenta (Kristensen 1983), Bufo viridis (Katz &
Larsen 1984), Bufo marinus (Larsen et al. 1987,
Lacaz-Vieira & Procopio 1988) and Hyla arborea
(Katz et al. 2000). According to the last mentioned
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Acta Physiol 2011, 202, 435–464
study, the skin of Xenopus laevis constitutes an
exception.
Quantitative description of the voltage dependence
Transepithelial chloride currents were characterized by
Larsen & Rasmussen (1982), and examples are shown
in Fig. 7. Shifting VT from 0 to )80 mV (outside bath
negative) slowly activated the chloride current from
)30 to )80 lA cm)2, whereas a step in the opposite
direction, to +80 mV, resulted in current deactivation
(Fig. 7a). The currents decayed towards their prior
steady-state level when VT was returned to the
holding value of +50 mV showing that the channel
activations are reversible (Fig. 7b). Substituting outside Cl) with gluconate (Fig. 7c) significantly reduced
the current at all potentials and eliminated the timedependent activations. The half-time (t½) of Cl)
current activation decreases with increasing hyperpolarizing voltage step and is usually ranging from 50 to
10 s (Fig. 7d). The dynamic behaviour of the chloride
current results in an inverse S-shaped relationship
between the steady-state chloride conductance (G) and
the transepithelial potential difference (VT) and a
strongly rectified steady-state ICl–VT relationship with
large outward currents in the physiological range of
VT carried by an inward flux of Cl), cf. Fig. 7e,f.
Typically, the chloride conductance is maximally
activated in the range of )80 mV < VT < )100 mV.
By controlling PCl and by driving Cl) through the
activated channels, VT has a dual function in passive
chloride uptake.
Role of mitochondria-rich cells
In toad skin (B. bufo), the amplitude of the Cl) current
activation recorded at )100 mV was found to be
linearly correlated with the density of MR cells with a
slope of )2.6 nA per MR cell (Willumsen & Larsen
1986), which conformed to the hypothesis that MR cells
is the site of the ‘chloride shunt’ as suggested for frog
skin: R. esculenta (Voûte & Meier 1978, Kristensen
1981) and R. pipiens (Foskett & Ussing 1986). Similarly, the net inward flux of chloride in the skin of B.
marinus was correlated with the density of MR cells
(Devuyst et al. 1990). However, a correlation between
the chloride flux and the density of MR cells was not
observed in all studies (Nagel & Dörge 1990, Rozman
et al. 2000), which has so far received no satisfactory
explanation. Further evidence for the hypothesis that
MR cells constitute the Cl) pathway was obtained by
measuring the volume of single MR cells in situ by
Spring’s method of video-enhanced quantitative microscopy (Spring & Hope 1979). It was shown that
individual MR cells undergo a reversible volume
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Acta Physiol 2011, 202, 435–464
E H Larsen
Æ Functions of mitochondria-rich cells in anuran skin
Figure 7 Dependence of the chloride permeability on transepithelial potential difference, VT. (a) The Cl) current response to step
change of VT from a holding value of 0 mV to +80 and )80 mV and back (sign refers to outside bath). In the physiological region
(VT < 0 mV), chloride channels are reversibly activated allowing Cl) to flow from the outer solution into the serosal bath. (b)
Dependence of the Cl) current on the transepithelial potential difference, which was pulsed from a holding potential of +50 mV to
)100 mV in steps of +25 mV. (c) With gluconate outside the slow time-dependent activations are eliminated. (d) The half-time (t½) of
the Cl)-current activation decreases with increasing the activating voltage step (numerically increasing VT). Generally, the gating is
slow with half-times in the order of 10–50 s. (e) The steady-state currents are strongly rectified with large components in the
physiological range of VT. In this region, the current, I, is carried by an inward flow of Cl). (f) The gating of chloride channels results
in an ‘inverse S-shaped’ conductance–voltage relationship of the epithelium. The conductance, G, saturated (all channels open) in the
range, )120 < VT < )80 mV. The line is the best fit to the Boltzmann distribution: G ¼ ðGmax Gmin Þ=f1 exp½ðVT V0 Þ=DVg þ Gmin ,
where Gmax = 1025 21 lS cm)2, Gmin = 89 17 lS cm)2, V0 = )32 1.4 mV, DV = 10.5 1.2 mV, R2 = 0.998 and errors
given for each of the free parameters. Modified from Willumsen et al. (2002).
increase in response to a reversible activation of the
chloride current (Foskett & Ussing 1986, Larsen et al.
1987).
Whole-cell patch-clamp studies of single isolated
mitochondria-rich cells
A free suspension of MR cells can be obtained by
trypsin treatment of the isolated epithelium (Larsen &
Harvey 1994). Whole-cell currents carried by chloride
were obtained with 100 lm amiloride and 5 mm Ba2+
added to the bath, and 10 mm Cs+ in the pipette
solution. Generally, the chloride conductance increased
with depolarizing voltage as would be expected if the
voltage-activated Cl) channels are located in the apical
plasma membrane. (It is noted that a transcellular
hyperpolarization is associated with a depolarization of
the apical and a hyperpolarization of the basolateral
plasma membrane). The power density spectrum of
stationary chloride current fluctuations recorded at a
membrane potential where the chloride conductance
was half-maximally activated (open probability,
pO = 0.5) could be fitted by a single Lorentzian component, see Fig. 8. The unitary chloride conductance,
cCl, is given by:
cCl ¼
r2
;
ICl ð1 pO ÞðVm ECl Þ
where r2 ¼
pS0 fC
:
2
ð1Þ
2
Here, r is the variance of current fluctuations about the
mean current, ICl, Vm is the plasma membrane potential
and ECl the chloride equilibrium potential. The corner
frequency, fC, and the low-frequency asymptote, S0, of
the power density spectrum were determined by curve
fitting as explained in the legend of Fig. 8. For six
spectra, we obtained the following values (mean SEM), S0 = 14.6 1.3 pA2 Hz)1 and fC = 34 2.6 Hz
from which the unitary conductance of, cCl = 250 18 pS, was calculated.
The above results are compatible with the hypothesis
that the passive chloride uptake is governed by voltagecontrolled Cl) channels in the apical membrane of MR
cells. However, comparisons between macroscopic and
whole-cell currents revealed quantitative problems that
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443
Functions of mitochondria-rich cells in anuran skin
Æ E H Larsen
Figure 8 Power density spectrum of whole-cell ICl of a mitochondria-rich cell. The stationary fluctuations were recorded
with 120 mm Cl) in both pipette and bath and Vp = 25 mV
where the open probability of Cl) channels is, pO = 0.5. The
line is the best fit of the Lorentzian function, S(f) = S0[1 + f2/
fC2], with S0 = 11.7 0.5 pA2 Hz)1 and fC = 41.3 2.7 Hz.
Since the open probability was known, the spectral parameters
could be used for estimating the Cl) current through open
channels and the single-channel conductance, cf. Eq. 1. Modified from Larsen & Harvey (1994).
have not yet been solved. Specifically, the corner
frequency of the power density spectrum of about
35 Hz corresponds to a time constant of the fluctuations
of 4.5 ms. This is a very small number compared with
the slow (seconds) activation of the transcellular Cl)
current. Further, the Cl) current rectification in wholecell mode was nearly instantaneous, with only few cells
exhibiting current activations with a long-time constant,
and still other cells exhibiting no voltage activation at
all. One possibility is that we are dealing with two
different time-dependent processes: (i) a slow activation
governed by the insertion of Cl) channels in the plasma
membrane requiring an intact sub-membrane domain,
which is vulnerable to the invasive whole-cell configuration; and (ii) stationary fluctuations because of fast
gating of channels residing in the plasma membrane.
Acta Physiol 2011, 202, 435–464
et al. 1989), and by transcellular current recordings in
intact epithelia and in single MR cells (Willumsen et al.
1992, Larsen et al. 2003). Like mammalian CFTR, it is
activated by b-adrenergic receptor agonists and exogenous cAMP. Furthermore, the human and the amphibian channel share anion selectivity sequence as probed
by the cAMP-activated current response to external
anion substitutions. Disappointingly, only 3% of the
sealed patches displayed the predicted large-conductance Cl) channel; an example is shown in Fig. 9b. It is
characterized by a noisy open state of different conductance levels with stepwise open–close transitions. The
lower panel histogram with Gaussian fits indicates coexpression of three small 10-pS channels and one big
246-pS channel.
It is useful to analyse the problem in quantitative
terms. The relationship between the fully activated ICl
and the density of MR cells indicated that the Cl)
current flowing through each cell is, on average
)2.6 nA corresponding to a transcellular conductance,
GT 26 nS per cell (Willumsen & Larsen 1986). With
the purpose of illustrating the problem, we assume that
the area of the apical membrane is 6 lm2 (disregarding
microplicae) giving a specific transcellular conductance
of 4.5 nS lm)2. Thus, the specific conductance of the
apical membrane must be larger than 4.5 nS lm)2
depending on the voltage divider ratio of the apical and
basolateral plasma membranes. As the diameter of the
tip of the patch pipette is about 1 lm, the patched
membrane would display a conductance that is
>3.5 nS. With an estimated single-channel conductance
of 250 pS and an open probability less than unity,
significantly more than 14 big channels would be
expected in the patched membrane, which may not be
resolved easily. As a result of this difficulty, there are
significant residual uncertainties about its molecular
phenotype, which may not be clarified before the
channel gene is sequenced and expressed in a model cell
system.
Single-channel studies
Application of the patch clamp technique in the cell
attached- and inside-out configuration allowed us to
record currents in single channels of the apical membrane of MR cells (Sørensen & Larsen 1996). With the
single-channel conductance and the current-voltage
relationship as criteria, several types of Cl)-selective
channels were observed. A small linear channel with a
conductance of about 9 pS was observed in 26% of 179
sealed patches, see Fig. 9a. The activity of this channel
was independent of membrane potential and it occurred
more frequently in cells pre-treated with forskolin when
compared with non-treated control cells. Probably, this
is the channel that has been studied by macroscopic
noise analysis in Ussing-chamber experiments (De Wolf
444
Transcellular chloride currents of single mitochondria-rich
cells
As neither the whole MR cell currents nor the estimated
single-channel activity accounted quantitatively for the
macroscopic chloride currents of the intact epithelium, I
decided to take advantage of a method originally
introduced for the study of the electrophysiology of
retinal rod cells (Yau et al. 1981), in which the tip of the
cell is sucked into a pipette of appropriate geometry.
With this method, our standard voltage clamp protocols
could be applied to single MR cells for testing directly
the hypothesis that the macroscopic dynamic chloride
current is the summation of discrete currents generated
by individual MR cells; see Fig. 10.
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Acta Physiol 2011, 202, 435–464
E H Larsen
(a)
Æ Functions of mitochondria-rich cells in anuran skin
Figure 9 Single-channel recordings of apical membrane patches of c-type MR cells. (a) The small CFTR-like channel in an
excised inside out patch. First, channel activity was recorded
with 125 mm in pipette and bath (left hand panel and blue
symbols). The full line is the linear regression with slope,
c = 8.4 pS. Next, the activity was recorded with the Cl) concentration of the bath reduced to 25 mm (gluconate substitution, free-[Ca2+] = 100 nm, right-hand panel, red symbols).
[K+]bath = 0, [Na+]bath = 10 mm and [NMDG+]bath = 140 mm
for both conditions. The Cl) equilibrium potential is indicated
for [Cl)]bath = 25 mm. GHK: Fit of the Goldman–Hodgkin–
Katz electrodiffusion equation to the experimental data,
PCl = 2.1 · 10)14 cm3 s)1 from which the single-channel conductance with 125 mm Cl) on both sides can be calculated,
c125/125 = 10.0 pS. (b) Upper panel: The predicted large-conductance Cl) channel in a cell attached patch with 125 mm Cl)
in pipette and bath, )Vp = 20 mV. Note the noisy open state
and the stepwise open–close transitions. Lower panel: All point
histogram analysis with Gaussian fits showing that a giant
channel with conductance, cCl = 246 pS, was co-expressed
with three small channels each of a conductance, cCl = 10 pS.
(b)
Representative experiments are shown in Fig. 11.
With Ringer’s solutions in pipette and bath, the pipette
potential, VP, was pulsed from a positive holding value
of +50 to )100 mV (Fig. 11a). Prior to the voltage
pulse, the holding current was 2.3 nA (VP = +50 mV),
and when VP was stepped to )100 mV, ‘instantaneously’ the current reversed to )4.6 nA. This ohmic
current is assumed to flow in the seal between the cell
and the glass wall of the pipette with an associated leak
conductance of 46 nS. Subsequently, the pipette current
was slowly activated, and with a half-time of 37 s, the
current approached a new steady state of )5.7 nA
revealing a dynamic component of )1.1 nA. Upon
Figure 10 Summary of method for studying transcellular
currents of a single polarized mitochondria-rich cell. Upper left:
Mitochondria-rich cell and principal cell prepared by trypsin
treatment of whole epithelium isolated by collagenase according to Larsen & Harvey (1994). During the separation of cells,
the bath is nominally Ca2+-free, however, with no chelator
added for maintaining sufficient enzyme activity. Lower left:
Recording mode with the neck of the mitochondria-rich cell in
the tip of a low-resistance ‘patch pipette’. Right: Currents are
recorded at low gain (0.5–1 mV pA)1) by the Axon 200B
amplifier and digitized at a rate usually between 20 and 50 Hz.
Rf: feedback resistor of preamplifier, ip: pipette current, Vc:
clamping voltage, Vo: output voltage of preamplifier.
return of VP to +50 mV, the current returned to +3.2 nA
followed by a fast deactivation to +2.5 nA, which is a
little above the pre-pulse value of +2.3 nA. The VP pulse
was repeated twice resulting in current responses
resembling the initial response, not exactly so but
reasonably well. In Fig. 11b,c are shown results
obtained from two MR cells that generated significantly
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445
Functions of mitochondria-rich cells in anuran skin
(a)
(b)
Æ E H Larsen
(c)
Figure 11 Examples of transcellular current activations
obtained by shifting the pipette potential from a holding
potential were the apical chloride channels are closed to
)100 mV. The dynamic Cl) currents are indicated by vertical
arrows to distinguish them from the instantaneous linear
ohmic response to voltage clamping. (a) and (b) Unpublished
recordings, however, included in the statistical material
published by Larsen et al. (2001). (c) Unpublished recording
of 18 July 2003.
larger current activations, i.e. )4.1 and )6.5 nA
respectively. Table 1 collects the amplitudes of the
current activations in single-cell recordings together
with the average chloride currents obtained in epithelial
preparations in which MR cell counting was performed.
The table also lists chloride currents sampled above
individual MR cells by a self-referencing microprobe
(Foskett & Ussing 1986). In the bottom row of Table 1
Acta Physiol 2011, 202, 435–464
is shown two experiments with the MR cell body in the
pipette and the tip of the neck in bath. Here, the leak
current component was estimated by perfusing the
apical plasma membrane with a Cl)-free solution
(gluconate substitution). Taken together, the single-cell
currents of Table 1 obtained by different methods
provide compelling evidence that the chloride current
of the intact epithelium is the sum of single-cell currents
generated by the population of MR cells of the
epithelium. This was confirmed by comparing the
steady-state ICl–V relationship of the intact epithelium
(Fig. 7e) with that of single MR cells (Fig. 12a, b). Both
the intact epithelium and the isolated MR cells display
strong outward current rectification associated with
large inward Cl) fluxes for V < 0 mV. Independent of
recording mode, the inward currents at steady state are
vanishingly small. In the example given in Fig. 12c, the
transcellular conductance saturated for VP £ –75 mV,
which usually is the case also for the chloride conductance of the intact epithelium (Fig. 7f).
In several of the single MR cells examined, the
chloride current activation was significantly faster than
that of the intact epithelium; compare Fig. 7a,d with
Figs 11b, 11c and 12d. Much slower activations similar
to those of intact epithelia were also observed as
exemplified in Fig. 11a and detailed earlier (Larsen
et al. 2001, Willumsen et al. 2002). Therefore, the longtime constants of current activations characterizing a
majority of intact epithelia are also observed in single
MR cells. This indicates that slow activations are not
caused by electrical coupling of MR cells to the principal
Table 1 Fully activated transcellular Cl) currents of MR cells, ICl, and the associated transcellular conductance, GCl
Method
Slope of ICl–DMRC relationship
Self-referencing probe
DICl/DDMRC
Following salt depletion
Single MR cell voltage clamp
Non-perfused
Non-perfused
Perfused
Perfused
Cell body in pipette: Itotal–Igluconate
ICl (nA per cell)
VT = )100 mV
GCl
(nS per cell)
Remarks
Ref.
)2.6
r = 0.96, N = 12
)1.2
N = 15
)2.0
N=5
26
MR cells in situ B. bufo
(1)
12
(2)
20
MR cells in situ R. pipiens
Salt acclimated frogs
MR cells in situ B. bufo
)2.9 0.5
N = 16
)2.1 0.7
N=5
)8.0 1.5
N = 10
)4.5 0.9
N = 10
)2.6, )5.5
N=2
29 5
Trypsin isolation B. bufo
(4)
21 7
Pronase isolation B. bufo
(4)
80 15
Trypsin isolation B. bufo
(4)
45 9
Trypsin isolation B. bufo
(5)
26, 55
Trypsin isolation B. bufo
Aqua dist acclimated
(5)
(3)
(1) Willumsen & Larsen (1986), (2) Foskett & Ussing (1986), (3) Budtz et al. (1995), (4) Larsen et al. (2001), (5) Larsen et al.
(2003).
446
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Acta Physiol 2011, 202, 435–464
Figure 12 Voltage-activated Cl) currents
of a single mitochondria-rich cell. (a)
Family of currents obtained from a holding potential of +50 mV. (b) Steady-state
I–V relationship of the dynamic component (Idynamic) recorded in (a). (c) Steadystate conductance–voltage relationship of
the mitochondria-rich cell, G = Idynamic/
Vp. (d) Similar to the dynamic current of
the intact epithelium (e.g. Fig. 7), the halftime (t½) of current activation decreases
with V in the hyperpolarizing region of
the current–voltage relationship. (Larsen
et al. 2001).
E H Larsen
Æ Functions of mitochondria-rich cells in anuran skin
(a)
(c)
(b)
(d)
cells. We have no satisfactory explanation for the above
differences in time constants among MR cells. Two
more aspects of the time-course of the transcellular
currents should be mentioned. The current activation in
Fig. 11a is preceded by a brief current decrease. The MR
cell recordings in Fig. 11b,c show no such initial current
response. Similar types of time-course have been
observed in experiments on intact toad (B. bufo,
B. marinus) and frog (R. esculenta) skin epithelium
(Larsen & Rasmussen 1982, Kristensen 1983, Larsen
et al. 1987). Computer-assisted analysis of a mathematical model of the epithelium enabled us to predict how
ion redistributions between extra- and intracellular
water affect the time-course of slowly gated currents.
For reproducing the rectified Cl) current, the apical
plasma membrane’s PCl was assumed to be voltage and
time dependent according to the simple scheme proposed for the K+ permeability of excitable membranes
(Hodgkin & Huxley 1952). The analysis showed that
the response in Fig. 11a may reflect that the gated
current is superimposed on an early positive Na+
current, which decays as the cellular Na+ pool is being
depleted (Larsen & Rasmussen 1982). However, as the
model was underdetermined, we could choose other sets
of equally plausible input variables that resulted in a
computed current similar to Fig. 11b,c. The ‘hump’ seen
on the decay of the positive current during channel
deactivation at positive potentials (e.g. Fig. 12a) might
be because of a slow depletion of the cellular Cl) and K+
pools governed by a relatively small K+ permeability of
the basal plasma membrane (Larsen & Rasmussen
1985). Although the above straightforward explanations conform to pool sizes and ion permeabilities of
MR cells, it has not been excluded that the initial
current decrease preceding activation and the ‘hump’
during deactivation are caused by a more complicated
kinetics of the apical PCl than assumed in our modelling.
In support of this notion, we could show that the
kinetics of current activation depends on the holding
voltage prior to the activating voltage step, which is
fundamentally different from the dynamic cation currents of excitable cells (Larsen & Rasmussen 1982).
Finally, it should be mentioned that the Cl) permeability
is not always fully deactivated in the range 0 < VT <
50 mV, which applies to single cells as well as to intact
epithelia. As the voltage-activated PCl resides in the
apical membrane, a simple explanation would be
that the voltage divider ratio of the outward- and
the inward-facing plasma membrane is submitted to
variation.
Apical PNa of principal cells controls the apical
PCl of mitochondria-rich cells
Our finding that membrane potential controls the apical
chloride permeability of the MR cell has an interesting
implication, which throws light on its physiological
significance. The question that shall be discussed here is
whether the active sodium current generated by the
principal cells controls the dynamic Cl) permeability of
the MR cells, PMRC
, if the transepithelial potential
Cl
difference is allowed to develop, i.e. at open-circuit
conditions. Under these conditions, one would expect
that the apical membrane of MR cells becomes depolarized by the current loop resulting from the active Na+
current through the principal cells. Owing to the small
size of MR cells, this is not easy to test experimentally,
but it can be analysed by a mathematical model of the
epithelium comprising a large principal cell compartment, numerous MR cells of a physiological density and
tight junctions (Larsen & Rasmussen 1985). In the
computations discussed here, all independent variables
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447
Functions of mitochondria-rich cells in anuran skin
Æ E H Larsen
were chosen to simulate measured intracellular ion
concentrations, cell volumes, membrane potentials and
membrane conductances as discussed in Larsen (1991).
The set of equations was solved numerically for
different apical Na+ permeabilities of the principal cells
PCell
(PPCell
Na ). There is evidence that AVT stimulates PNa ,
only, leaving the apical PNa of MR cells unaffected
(Kristensen 1981). All other independent variables of
principal and MR cells (including MR cell density) were
kept constant and similar to those of the non-stimulated
cells. Therefore, changes of currents through MR cells
would be because of the electrical coupling between the
principal cell compartment and the MR cells via
transepithelial (external) current loops. The range of
the Na+ permeability of the principal cells indicated on
the x-axis of Fig. 13 corresponds to estimated values in
the non-stimulated and fully stimulated epithelium. (For
reasons explained in the legend of Fig. 13, the maximal
flux of the Na+/K+ pump was increased in the upper
range of the physiological values of PPCell
Na ). As can be
seen by the green-coloured graph of Fig. 13, the model
predicts that the apical chloride permeability of MR
cells increases several fold by stimulating the apical
sodium ion permeability of the principal cells. As a
result, the inward fluxes of the two ions shown by the
red- and blue-coloured graphs, respectively, increase
about 30 times. Except for the small permeabilities, the
two fluxes are about the same. Literally speaking, it
looks like the two ions are transported by the same
transport system, but they are not; the sodium ions are
flowing through the large syncytial principal cell compartment, whereas the chloride ions are flowing through
6 · 104 MR cells cm)2.
We do not know much about the nature of the
voltage control of the apical chloride permeability of
the c-type MR cell. However, the above computations
show that, at open-circuit conditions, activation of the
apical Cl) permeability of the MR cell is brought about
by stimulation of the apical Na+ permeability of the
principal cell compartment. The evolution of this
mechanism has converged to a solution where the range
of the apical PCl of the c-MR cell is being covered when
the apical PNa of the principal cell compartment is going
from its inactivated to its fully activated state.
Role of apical chloride channels of the c-type
MR cell: regulation of osmotic concentration
and volume of the epidermal surface fluid of
amphibians on land
The adaptation of amphibians to a terrestrial environment was associated with the development of a cornified epidermal cell layer and subepidermal exocrine
glands (Fig. 3) derived from the epidermis (Noble
1931). The secretion of the subepidermal mucous
448
Acta Physiol 2011, 202, 435–464
Figure 13 Analysis of the epithelial model of the heterocellular anuran skin epithelium. Open-circuit conditions simulating
a stimulation of the apical Na+ permeability of the principal
+
cells,PPCell
Na .Upper graphs: The Na permeability activation
reproduces the well known numerical increase in the transepithelial potential difference, which is predicted to depolarize
the apical plasma membrane of the mitochondria-rich cells by
the associated current loop. This illustrates the effective
external electric coupling between the two cell types. Lower
graphs: The above depolarization of mitochondria-rich cells
activates the apical Cl) permeability where PMRC
Cl;max ¼
1:5 103 cm s1 . At low PPCell
and similarly low PMRC
(leftNa
Cl
hand side of the graph), the very small and constant active Na+
uptake by MR cells is large relative to the other ion fluxes
whereas the loop current is carried predominantly by the back
flux of Na+ through the Na+-selective tight junctions. FollowMRC
ing the activation of PPCell
by
Na and associated activation of PCl
external electrical coupling, the loop current is carried by Cl)
through the mitochondria-rich cells. This results in Na+- and
Cl) fluxes through the principal cell compartment and the MR
cells, respectively, of about similar magnitude. Unpublished
computations by the mathematical model presented in Larsen
& Rasmussen (1985) with the independent variables listed in
6
table 5 of Larsen (1991). However, for PPCell
cm s1 ,
Na 3 10
the maximal flux of the Na+/K+ pump of principal cells was
increased from its standard value of 0.7 to 1.4 nmol cm)2 s)1.
Thereby, the computed [Na+]c was kept within its physiological
range both in the weakly stimulated state (<10 mm) and near
its fully stimulated state (20–28 mm), cf. Nielsen (1982b).
glands keeps the skin moist in terrestrial habitats.
Maintenance of a moist skin surface prevents desiccation of the epidermal cells and is of importance for skin
respiration (Krogh 1904). The surface film protects
against bacterial infection and entry of moulds, and as
the secretion is viscous, it makes the animal slippery and
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Acta Physiol 2011, 202, 435–464
E H Larsen
difficult for potential predators to seize it (Lillywhite &
Licht 1975, Stebbins & Cohen 1995). As an additional
function, glandular secretion has been associated with
evaporative cooling as observed in bullfrog R. catesbeiana (Lillywhite 1971) and in Australian and Indian tree
frogs, Litoria caeulea (Brattstrom 1970) and Polypedates maculatus (Lillywhite et al. 1998) respectively.
Lillywhite (1971) showed that in bullfrog, the activity
of the glands increases synchronously with body temperature in the range of 20–28 C. This was achieved
predominantly by increasing the frequency of discharge
without significant effects on the amount of fluid per
discharge or the number of active glands, which was
about 1.6 · 103 cm)2. Generally, however, amphibian
thermoregulation by evaporative cooling is considered
far less important than behavioural thermoregulation
(Toledo & Jared 1993, Hillman et al. 2009). Gland
secretion is controlled by a number of hormones via badrenergic, a1-adrenergic, muscarinic M1/M3 and prostaglandin E2/E4 receptors, and is driven by secondary
active Cl) transport as explained below (KoefoedJohnsen et al. 1952b, Mills 1985, Mills et al. 1985,
Bjerregaard & Nielsen 1987, Andersen et al. 1994,
Nielsen & Nielsen 1999, Sørensen & Larsen 1999,
Gudme et al. 2000, Sørensen et al. 2001). The selective
pressure driving the evolution of the above skin adaptations is the spending of longer periods of time outside
the aquatic environment and expanded niche exploration. In the following, I shall discuss the hypothesis that
the adaptations include the evolution of an epidermal
MR cell type specialized for passive chloride uptake.
Turnover of ions and water of the epidermal surface fluid
Irrespective of the function of the gland secretion,
because of the short duct (Fig. 3) the fluid emerging on
the skin surface is rich in diffusible electrolytes for
becoming near-isosmotic to the body fluids by stimulation (Campbell et al. 1967, Watlington & Huf 1971,
Lang et al. 1975, Bjerregaard & Nielsen 1987). Unless
the ions are reabsorbed during the unavoidable evaporation of water, the ion concentrations of the surface
fluid would increase rapidly and dramatically. Water
loss by evaporation may be compensated for partly by a
flow of water from a wet substrate driven by capillary
forces in the sculptured epidermis of especially terrestrial species of Bufonidae (Lillywhite & Licht 1974,
Castillo & Orce 1997). However, this mechanism
cannot prevent loss of body electrolytes by gland
secretion. For illustrating the problem, Campbell et al.
(1967) measured the rate of Na+ and Cl) secretion in R.
pipiens sitting in moist air. Non-treated animals
secreted about 1.2 nmol s)1 of both Na+ and Cl),
which increased to 9 (Na+) and 6 (Cl)) nmol s)1 per
animal, respectively, following adrenaline injection. The
Æ Functions of mitochondria-rich cells in anuran skin
low secretion rate would result in an ion loss of 0.5%
per hour of the extracellular Na+ and Cl) pools,
whereas the adrenaline-injected frogs would lose about
3% of their extracellular Na+ and Cl) per hour. It is
conceivable that a function of the voltage-activated
apical chloride channels of the MR cells is to enable Cl)
to follow passively an active reabsorption of Na+ by the
principal cells, which would be necessary for regulating
the concentration of these ions in the fluid covering the
body surface and avoiding loss of ions to the surroundings. One way of justifying this notion is to see whether
the epidermal transport mechanisms would be able to
maintain the surface fluid isotonic at high rates of
evaporative water loss. Carey (1978) measured the
evaporative water loss (ewl) in the montane B. boreas
boreas and the lowland B. boreas halophilus kept in a
temperature-regulated chamber flowed through with
dry air. The rate of ewl increased by a factor of about
3.5 when the ambient temperature was increased from
10 to 30 C with insignificant differences between the
two subspecies of similar body-mass range; at three
different temperatures she obtained the following average rates of ewl (mg g)1 h)1): 10.7 (10 C), 24.5
(20 C) and 36.6 (30 C). With a mean body mass of
34.5 g and a surface area (A) to mass (M) relationship
of B. boreas of, A = M0.56 (Mullen & Alvarado 1976),
the above numbers can be recalculated to provide areaspecific water loss rates (nl cm)2 s)1): 0.408 (10 C),
0.936 (20 C) and 1.34 (30 C). With NaCl concentration of the mucous gland secretion of 100 mm, the
maintenance of a constant concentration of Na+ and
Cl) in the surface fluid required the following reabsorption fluxes of NaCl (pmol cm)2 s)1): 41 (10 C), 94
(20 C) and 140 (30 C) respectively. They are all
within the measured capacity of the epidermal transport
systems (cf. e.g. Fig. 13). With the estimated maximal
capacity of the transport systems, the conclusion above
seems to hold even if ewl is not uniformly distributed
over the body surface. As it was assumed that all of the
evaporated water was derived from the secreted fluid
with no contribution from an epidermal efflux of H2O,
the example above corresponds to about the maximal
load of the transport mechanisms. The relative contribution of secreted water and a transepidermal water
flux to ewl was discussed by Toledo & Jared (1993).
For answering this specific question, it seems that
quantitative information is not available. In a separate
section below, I shall attack the problem by considering
the fact that the transepithelial water flow is solute
coupled.
Little is known about the recovery of the potassium
ions. The potassium concentration of the gland secretion of frog skin is 8–12 times that of plasma (Campbell
et al. 1967, Watlington & Huf 1971). This is because
of active K+ secretion by the acinar cells with no
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Functions of mitochondria-rich cells in anuran skin
Æ E H Larsen
contribution from the epidermal principal cells, which
was proven by demonstrating the absence of active K+
secretion in hormone-stimulated, gland-free preparations of the isolated epithelium (Sørensen et al. 2001).
This conforms to the general scheme of vertebrate
exocrine glands, i.e. the [K+] is several times larger in
the primary secretion than in the extracellular fluid
(Thaysen & Thorn 1954, Thaysen et al. 1954, Schwartz
& Thaysen 1956, Young & Van Lennep 1978, Petersen
& Findlay 1987, Cook & Young 1989, Tan et al. 1992,
Nakamoto et al. 2008, Palk et al. 2010). The functional
Acta Physiol 2011, 202, 435–464
organization of the acinar epithelium is discussed in a
separate section below. The above-mentioned secretion
of K+ by the frog skin gland would result in a loss of
20% of the extracellular K+ pool per hour, probably
leading to a redistribution of K+ between the extra- and
intracellular water. In an early study, Levi & Ussing
(1949) obtained evidence for active K+ uptake by the
isolated frog skin. The a-type MR cell of mammalian
cortical collecting tubule reabsorbs K+ actively. This is
accomplished by a series arrangement of an apical K+/
H+ P ATPase and a basolateral K+ channel stimulated
by K+ depletion (Doucet & Marsy 1987, Garg &
Narang 1988). The question of active K+ uptake by
anuran skin epithelium seems to have been neglected.
Reabsorption of NaCl is regulated by small unilateral
osmotic concentration perturbations
Figure 14 Voltage dependence of the Na+ current (the amiloride sensitive current) through the apical membrane of the
principal cells of Rana temporaria skin. The apical membrane
potential was measured with a microelectrode in a principal
cell whereas VT was changed in pre-programmed voltage steps.
During the control period (blue symbols), the skin was bathed
with frog Ringer’s solution on both sides (227 mOsm). The red
symbols indicated as, +20 mOsm, was obtained with 20 mm
DMSO added to the outside solution. The full lines are best fit
of the Goldman–Hodgkin–Katz current equation (Hodgkin &
Katz 1949) to the experimental data with PPCell
Na and associated
1
[Na+]c as free parameters. PPCell
(control)
Na ¼ 9:1 2:4 cm s
and 13.4 2.7 cm s)1, P < 0.001 (N = 9 preparations). Thus,
the small inwardly directed osmotic gradient stimulated the
apical membrane’s Na+ permeability. Adapted from Brodin &
Nielsen (1993).
Brodin & Nielsen (1993) discovered that the sodium
permeability of the apical plasma membrane of the
principal cells, PPCell
Na , is sensitive to small osmotic
concentration differences across the epithelium, see
Fig. 14. This response of PPCell
is relatively fast (minNa
utes). Although a small unilateral increase in outside
osmolarity (po) significantly affected the rate of Na+
uptake, a bilateral increase keeping the osmolarity of
the serosal fluid (pserosa) equal to that of the outside
solution (po = pserosa) had no sustained effect. The PPCell
Na
stimulation was elicited if, po > pserosa, no matter
whether this was produced by increasing the osmotic
concentration of the outside solution or by diluting that
of the serosal solution. By reversing the osmotic
gradient (po < pserosa), PPCell
Na decreased as demonstrated
by the results listed in Table 2, which also documents
the expected changes of the apical membrane potential
and the fractional resistance of this membrane, fRa,
relative to the total transcellular resistance of the
principal cell compartment. Finally, the authors showed
active
Table 2 Sodium permeability of the apical plasma membrane of principal cells (PPCell
Na ) and active sodium transport (ISC ; JNa )
of frog skin (Rana temporaria) exposed to Ringer’s solution on the outside (control, second column) and a hypertonic Ringer’s
solution outside (third column), and hypertonic Ringer’s solution on the serosal side (sixth column) respectively
6
PPCell
cm s1 Þ
Na ð10
Va (mV)
fRa
ISC (lA cm)2)
active
JNa
ðpmol cm2 s1 Þ
Control
+20 mOsm
apical
9
)75
0.80
39
404
13
)70
0.72
52
539
2
6
0.06
13
135
3
6
0.05
14
145
p
Control
+20 mOsm
basolateral
<0.001
<0.01
<0.05
<0.01
9
)78
0.83
30
311
5
)86
0.93
18
187
2
4
0.03
4
41
1
9
0.01
4
41
p
<0.02
<0.02
<0.05
<0.01
The hypertonicity was produced by 20 mm DMSO. Osmolality of the Ringer’s solution, 227 mOsm. Data from Brodin & Nielsen
(1993).
active
Va = apical membrane potential, fRa = fractional resistance of apical membrane,JNa
¼ ISC =F, where F is the Faraday.
450
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E H Larsen
that similar effects on the Na+ transport were obtained
by adding 20 mOsm of NaCl, sucrose, KCl or DMSO to
the solution bathing the outside of the preparation. In
other words, it is the osmotic concentration gradient,
which regulates PPCell
(by an unknown mechanism)
Na
independent of how the gradient is established. Thus,
this regulation of the apical Na+ permeability has
nothing to do with the well-described downregulation
of PPCell
following a fast increase of [Na+]o, which
Na
explains the ‘recline’ response of the short-circuit
current and the saturation kinetics of the transepithelial
Na+ influx (Fuchs et al. 1977). This mechanism prevents swelling and bursting of the cells following a
sudden increase in the external Na+ concentration, e.g.
from the [Na+] of freshwater to that of isotonic Ringer’s
solution (Larsen 1978). It should also be emphasized
that the regulation of the apical PNa by the abovediscussed small transepithelial osmotic concentration
gradient is different from the opening of tight junctions
by much larger unilateral outside osmotic concentrations (Ussing & Windhager 1964). The increased
conductive permeability of the skin thus produced was
non-selective, paracellular and had no significant effect
on the short-circuit current (PPCell
Na ).
The results described by Brodin & Nielsen (1993)
would constitute a paradox if considered as a mechanism regulating the epidermal or the extracellular Na+
concentration. However, considering the importance of
maintaining an isotonic concentration of the epidermal
surface fluid, the observed changes in the flux of Na+
and the associated changes in the flux of Cl), e.g.
Fig. 13, resolve the paradox as this type of regulation
would enable an effective control of the ion content of
the small volume of epidermal surface fluid.
Reabsorption of isotonic fluid
Another function of the chloride channels of the MR
cell is associated with solute-coupled isotonic fluid
uptake. Recent studies of the skin epithelium of B. bufo
indicated that b-adrenergic receptor stimulation activates the CFTR like apical Cl) channel of MR cells, the
ENaC of principal cells and the water permeability of
the skin (Nielsen & Larsen 2007). The latter-mentioned
activation occurs by the insertion of AQP water
channels in the apical plasma membrane of the principal
cells (Ogushi et al. 2010b). Our simultaneous measurements of ion and water fluxes showed that the activation of the above-mentioned three different membrane
pathways results in near-isotonic fluid absorption. We
provided evidence that the water uptake predominantly
is translateral and driven by slightly elevated ion
concentrations in the maze of lateral intercellular spaces
(Larsen et al. 2007, Larsen et al. 2009). Continuing the
above line of reasoning, the function of solute coupled
Æ Functions of mitochondria-rich cells in anuran skin
isotonic fluid uptake by the anuran skin epithelium
might be associated with the regulation of the volume
(height) of the epidermal surface fluid produced by the
subepidermal mucosal glands under conditions of minimal water loss by evaporation.
The secretion-reabsorption hypothesis for anuran skin
The proposed interplay between the subepidermal
glands and the epidermal cells is summarized in
Fig. 15. The secretion by the glands is accomplished
by a single-cell type of the acinar epithelium (see
Fig. 15a). Two cell types participate in the reabsorption: the principal cells transport Na+ actively, and the
c-MR cells transport Cl) by electrodiffusion (detailed in
Fig. 4b). The secreting acinar epithelium and the
absorbing epidermal epithelium, respectively, constitute
a functional unit, which serves formation of a thin film
of fluid on the body surface and the regulation of its
volume, osmotic concentration and ionic composition
(Fig. 15b).
The acinar epithelial cells are electrically coupled via
gap junctions (Sørensen & Larsen 1999) with at least
three ion channel types in the luminal membrane
(Fig. 15a). The secretion of ions is brought about by
the NaK2Cl co-transporter in the basolateral membrane
and the amphibian CFTR in the luminal plasma
membrane fuelled by ATP hydrolysis at the lateral
Na+/K+ pumps (Mills et al. 1985, Bjerregaard & Nielsen 1987, Sørensen & Larsen 1998). As indicated, the
active K+ secretion is accomplished by the lateral Na+/
K+ pumps in series with maxi-K+ channels, which are
co-expressed with CFTR in the luminal plasma membrane (Sørensen et al. 2001). The luminal plasma
membrane also expresses an AQP-x5 like aquaporin
as identified by immunofluorescence labelling of the
subepidermal glands of Bufo woodhouseii by Stanley
Hillyard (Hillyard et al. 2009). The pathway for transepithelial water secretion shall not be discussed here.
Different hypotheses are discussed by Ussing et al.
(1996) and Fischbarg (2010). The supposedly different
roles of the receptor pathways in regulating the activity
of the submucosal glands, cf. Fig. 15a, are far from
clear.
The transport pathways of the epidermal cells have
been studied in great detail as discussed in the above
sections. An essential feature regarding their regulation is that they are directly controlled by the
chemical composition of the external solution. Thus,
a small transepithelial osmotic concentration gradient
controls the activity of ENaC (Fig. 14, Table 2),
whereas the Cl) permeability is controlled by the
external Cl) concentration and, notably, by the
activity of ENaC via the external current loop and
apical membrane potential (Figs 6, 7 and 13). These
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Functions of mitochondria-rich cells in anuran skin
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Acta Physiol 2011, 202, 435–464
(a)
(b)
Figure 15 The secretion-reabsorption hypothesis of anuran skin. (a) The functional organization of the exocrine mucous gland of
anuran skin. Adapted from Sørensen & Larsen (1999). (b) Model of the interplay between the secretory mucous glands and the
absorptive epidermis. Two c-type MR cells are shown in grey. The water accompanying the secreted ions is either evaporated or
reabsorbed by the epithelium (ewl; evaporative water loss). The ions are reabsorbed by the functional syncytium of principal cells
(Na+) and by the c-MR cell (Cl)). The coloured symbols indicate the Cl) channels of the acinar cells (CFTR), the Na+ channels
(ENaC) of the principal cells and Cl) channels of the MR cells (two types are discussed in the text). Note the localization of pumps
along the lateral intercellular spaces in both the absorbing epithelium and in the exocrine gland, cf. Mills et al. (1977) and DiBona
& Mills (1979) respectively. In agreement with experimental analysis (Sørensen & Larsen 1999), the acinar cells are depicted as
being electrically coupled. Also the principal cells of the epidermis are electrically coupled whereby they constitute a functional
syncytium, which excludes the MR cells. The relative thickness of the different layers is not drawn to scale. For example, the
thickness of the surface fluid may not be much different from that of the cornified layer.
interesting discoveries find their logical explanation by
the hypothesis advanced here that the epidermal
transport systems serve the regulation of the volume
and composition of the isotonic surface fluid when the
animal is on land.
With an isotonic epidermal surface fluid, a net water
flow across the epidermal epithelium has to be solute
coupled, that is, water is being absorbed owing to the
activity of the lateral Na+/K+ pumps as discussed in
detail in Larsen et al. (2009), see Fig. 15b. Evaporation
of water inevitably results in a hypertonic surface fluid.
However, water absorption still prevails provided the
surface fluid’s osmotic concentration does not exceed
452
the epithelium’s capacity for uphill water transport. In
the terrestrial B. bufo, the isoproterenol stimulated
water flow stopped when the outside osmotic concentration was raised by DCrev = 28.9 3.9 mOsm, n = 5
(Nielsen & Larsen 2007), whereas in the semi-aquatic
R. esculenta, DCrev = 15.5 3.0 mOsm (n = 9) in
AVT-stimulated preparations (Nielsen 1997). By
increasing the outside osmotic concentration by more
than DCrev, the water flow across the epidermis reversed
and became outwardly directed (loc. cit.). In vivo this
means that now ewl is derived from two components:
(i) water secreted by the glands, and (ii) the outward
flow of water across the skin driven by the osmotic
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E H Larsen
mechanism just discussed. With the lateral intercellular
space as the osmotic coupling compartment, the capacity
for uphill water transport is determined by the maximal
osmotic concentration of the lateral space, which is
given by the ratio of the pump flux of Na+ through the
pump;lm
lateral plasma membranes, JNa
, and the Na+ (leak)
permeabilities of tight junction, PtjNa , and interspace
basement membrane (basal lamina), Pibm
Na respectively.
Generally, it can be shown (Weinstein & Stephenson
1981, Larsen et al. 2000b), as good approximation
DCrev is given by the following equation, which does not
contain the water permeability of the epithelium:
DCrev ¼
pump;lm
rtj JNa
PtjNa þ Pibm
Na
;
JV ¼ 0;
ð2Þ
where rtj is the tight junction reflection coefficient,
which is near one for a tight epithelium. Equation 2
predicts that DCrev is linearly dependent on the rate of
active Na+ transport, which has been verified in
experiments with frog skin (Nielsen 1997). As
tj
ibm
Pibm
Na >>PNa , a small change in PNa has a significant
effect on DCrev. Studies on other epithelia have shown
that DCrev spans a very large range from 75 to
200 mOsm in vertebrate small intestine (Parsons &
Wingate 1958, Naftalin & Tripathi 1986, Larsen et al.
2000a) to 12 mOsm in kidney proximal tubule of the
rat (Green et al. 1991). In future studies, it is of special
interest to see whether DCrev or Pibm
Na reflects the type of
preferred habitat of different amphibian species.
Sensing of ion composition and concentration of potential
hydration sources on land
The above considered functions of the Cl)-transporting
MR cells of anuran skin may be of relevance also in
respect to the novel physiological concept developed by
Hillyard and his associates. They have provided evidence
that the abdominal seat patch of terrestrial anurans,
specialized for ‘cutaneous drinking’ (McClanahan &
Baldwin 1969, Christensen 1974), can detect water
available for rehydration (Hoff & Hillyard 1993, Nagai
et al. 1999, Hillyard et al. 2007a,b). They have found
that the seat patch region of Bufonidae, which contains
MR cells, is particularly rich in mucous glands (Hillyard
et al. 2009). This would indicate that glandular secretion and ion reabsorption involving MR cells are
functionally coupled for allowing the epidermal cells to
sense the ion composition and concentrations of
potential hydration sources.
The mechanism of active chloride transport
In the aquatic environment, amphibians exploit the
above cutaneous mechanism of active sodium ion
Æ Functions of mitochondria-rich cells in anuran skin
transport. The active chloride uptake is meeting the
additional energy requirement of ionic regulation in
freshwater of low ionic strength. Krogh (1937) pointed
out that he was unable to detect cutaneous Cl) uptake
unless the frog was starved and sprayed by distilled
water for several weeks. He suggested that frogs
normally obtain ions from the food they eat. Studies
tracing the Cl) fluxes by radioactive isotopes reported
active cutaneous uptake of Cl) in non-starving animals
(Zadunaisky & Fisch 1964, Bruus et al. 1976, Rotunno
et al. 1978). Owing to lack of systematic studies, the
relative significance of dietary and cutaneous Cl) uptake
for maintaining whole body NaCl balance in freshwater
is unknown.
Chloride/bicarbonate exchange across the apical
membrane of mitochondria-rich cells
When exposed on the outside to a low concentration of
chloride, the isolated skin displays a saturating influx of
this ion that reaches half-maximum saturation in a
concentration range of no more than 0.1–0.5 mm
(Alvarado et al. 1975, Bruus et al. 1976, Ehrenfeld &
Garcia-Romeu 1978). The detailed study by Ehrenfeld
& Garcia-Romeu (1978) on the isolated skin of
R. esculenta showed that the net excretory flux of base
and the unidirectional influx of Cl) are linearly correlated, and that both fluxes are stimulated by raising
external concentration of chloride. Both fluxes saturated for [Cl)]o 2 mm. Ehrenfeld and Garcia-Romeu
obtained further evidence for coupling via a common
membrane transport system by showing that the net
excretion of base is significantly reduced (open-circuit)
or eliminated (short-circuit) when external Cl) is
replaced by SO42), and that both fluxes are reduced
by the carbonic anhydrase inhibitor acetazolamide. The
authors concluded that the skin displays a ‘saturable
transport system in which chloride absorption and base
excretion are coupled’ (loc. cit.). This is in agreement
with the earlier in vivo studies by Garcia-Romeu et al.
(1969) and with Krogh’s original idea: ‘The CO2
produced by metabolism and excreted through the skin
or gills is probably sufficient to serve in exchange for
Cl) absorbed’ (Krogh 1938). Further support for a
carrier-mediated, electroneutral uptake of Cl) was
obtained by the following approaches. First, in the
range 100 £ [Cl)]o £ 2000 lm, the radioactive 36Cl)
influx was significantly reduced by increasing the
concentration of ordinary chloride, that is, cis side
interaction between the two isotopes was demonstrated
(Jensen et al. 2002), cf. Stein (1967). Secondly, the
unidirectional Cl) influx measured under open-circuit
conditions was not changed significantly by shortcircuiting the skin (Kristensen 1972, Ehrenfeld &
Garcia-Romeu 1978). Further to this argument, in the
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Functions of mitochondria-rich cells in anuran skin
Æ E H Larsen
skin of B. bufo, the Cl) influx was unaffected by shifting
the open-circuit potential by as much as 135 mV by
adding amiloride to the outside solution (Jensen et al.
2003). A histochemical study of frog skin showed that
the carbonic anhydrase is selectively localized to the
MR cells (Rosen & Friedly 1973), and an immunohistochemical study of B. viridis indicated the existence of
a band-3-related protein in the apical membrane of this
cell type (Katz & Gabbay 1993). Taken together, the
above investigations provide the evidence for the apical
Cl)/HCO3) exchanger of MR cells as shown in Fig. 4b,
which would be the major pathway for Cl) uptake from
low concentrations of its salt.
Although the scheme of Fig. 4b provides a logical
interpretation of the above anion flux data, it is not an
exhaustive description of anion transport across the
epithelium at low external chloride concentration. In
R. esculenta, the rate of net base secretion was about
50% of the unidirectional Cl) influx (Ehrenfeld &
Garcia-Romeu 1978). Thus, in this species under the
given conditions, the exchange mechanism may not
operate with a 1 Cl) : 1 HCO3) stoichiometry. A likely
explanation for this is that the transport system operates
as a Cl) : Cl) exchange mechanism as well, which is
consistent with large Cl) : Cl) exchange fluxes in
B. bufo (Kristensen & Larsen 1978, Jensen et al.
2002) and R. esculenta (Kristensen 1983). Another
possibility would be that a simultaneous H+ efflux
masked a constant fraction of the base efflux, which
shall be discussed in the paragraph below. In R. esculenta, the chloride efflux (but not the influx, cf. above) is
dependent on the transepithelial potential difference as
expected for electrodiffusion (Kristensen 1972, Ehrenfeld & Garcia-Romeu 1978). This was not a consistent
finding in B. bufo (Jensen et al. 2002). An obvious
reason would be that the efflux contains a paracellular
component of varying magnitude, but it remains to be
proven experimentally.
The proton pump of anuran skin epithelium
It has long been known that the anuran skin decreases
the pH of the outside bath whereas the serosal side
may become alkalinized (Huf et al. 1951, Fleming
1957). Early studies also showed that external acidification takes place at transepithelial thermodynamic
equilibrium, proceeds in the absence of external Na+,
is reduced by inhibitors of carbonic anhydrase, and is
sensitive to external pO2 (Emilio et al. 1970, Emilio &
Menano 1975, Machen & Erlij 1975). These characteristics indicated that the skin displays an apical
proton pump like the distal epithelia of vertebrate
kidney (Steinmetz 1974, Al-Awqati et al. 1977, Steinmetz & Andersen 1982). In frog skin (R. ridibunda
and R. temporaria), the rate of proton secretion was
454
Acta Physiol 2011, 202, 435–464
independent of the anion of the external bath (Emilio
et al. 1970, Machen & Erlij 1975), whereas proton
secretion in B. bufo was reduced to below the
detection level when external SO42) was replaced by
Cl) (Emilio et al. 1970). This latter observation is
compatible with the model of Fig. 4b with an apical
Cl)/HCO3) exchange mechanism in parallel with a
proton pump, which would mask the proton efflux in
the presence of external Cl). In the skin of frogs, a
Cl)/HCO3) exchanger is also located in the basolateral
membrane (Duranti et al. 1986) similar to the a-type
intercalated MR cells of renal epithelia (Steinmetz
1986), which would make the external acidification
more or less independent of external Cl). A cell
configuration with the proton pump in the apical
membrane and anion exchangers in the apical as well
as in the basolateral membrane would also account for
the apparent Cl)/HCO3) stoichiometry being below
unity in frog skin as reported by Ehrenfeld & GarciaRomeu (1978).
Localization and identification of the proton pump
The first attempts to localize the proton pump to cell
type applied double-barrelled H+-sensitive microelectrode for scanning the horizontal pH profile of the
unstirred layer above the transparent isolated epithelium exposed on the outside to a Cl)-free bath. The
studies reported pH gradients above visually identified
MR cells, which were reduced by proton-pump inhibitors (Harvey 1992) or abolished by replacing external
gluconate with chloride (Larsen et al. 1992).
Harvey (1992) applied the whole-cell patch-clamp
method to single MR cells of frog skin stimulated by
aldosterone. The whole-cell configuration was obtained
by perforating the membrane patch in cell-attached
mode with the ionophore amphotericin B in the patch
pipette. Membrane currents of Na+, K+ and Cl) were
abolished by specific channel inhibitors and ion substitutions. Proton current fluctuations, which were induced by increasing pCO2 in the serosal bath, could be
fitted by a single Lorentzian function. This was taken to
indicate that the fluctuations are generated by a
homogeneous population of spontaneously fluctuating
proton-pump channels from which Harvey calculated
the number of proton pumps to be 600 per MR cell.
From the estimated apical membrane area and assuming
that the MR cells occupy about 10% of the skin area, he
arrived at a total outward H+ current of the polarized
cell of )12 lA cm)2. This was close to the proton efflux
in intact frog skin of 97 pmol cm)2 s)1, which would
carry an outward current of )9 lA cm)2.
In toad skin epithelium with a low density of MR
cells, [H+] gradients were evaluated by the diffusion
equation for radial diffusion from a point source
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E H Larsen
Æ Functions of mitochondria-rich cells in anuran skin
Figure 16 Theoretical variations in horizontal concentration profile obtained at
relatively vertical distances (y) above a
skin epithelium containing 122 500 point
sources. Below the graphs, the position of
four-point sources is indicated. See text
for more detailed explanation. From Jensen et al. (1997).
(Larsen et al. 1992). The H+ flux of a single MR cell
was estimated to be 60 attomol s)1. Assuming a
similar single-cell flux in skins with an average density
of 6 · 104 MR cells cm)2, the above estimate turned
out to be fairly small, 3 pmol cm)2 s)1, in contrast with
a macroscopic proton efflux of 9 pmol cm)2 s)1. The
reason is most likely because of a too simplified
theoretical treatment of the measured proton concentration gradient. Thus, in a subsequent study, we
realized that even in preparations with a low density
of MR cells, a local gradient cannot be assumed to
reflect the flux of protons from the MR cell in focus.
This is because protons diffuse horizontally above the
epithelium so that any local pH gradient becomes
dissipated both by vertical and horizontal diffusion
(Jensen et al. 1997). This ‘point-source problem’ is
illustrated in Fig. 16, which depicts the theoretical
proton concentration profile above a population of
point sources distributed evenly in a quadrangular grid
located in the x–z plane. That is, the x–z plane
corresponds to the surface of the skin. The x–z plane
contained 122 500 point sources corresponding to a
density of 6 · 104 MR cells cm)2 of the epithelium
mounted in a 2-cm2 mini Ussing chamber. As expected
and shown by the theoretical graphs of Fig. 16, with the
H+-sensitive microelectrode very close to the x–z plane,
i.e. at y = 0.01, individual point sources can be
resolved. However, when the tip of the pH-sensitive
microelectrode is moved vertically away from the skin
surface (increasing y), individual point sources are no
more discernable. At the given average density of MR
cells of toad skin, the individual point-source profiles
vanish at the outer surface of the cornified cell layer
where the profile approaches that of diffusion from a
‘plate source’ (y > 0.1). We developed the associated
equations for evaluating the flux of protons based upon
mapping of the pH profile in the unstirred layer. An
example is given in Fig. 17a. The fit of the theoretical
equation, which takes into account diffusion of free
protons as well as of proton-loaded buffer molecules
(Tris), resulted in a flux of 12.6 pmol cm)2 s)1 with an
average of 8.5 2.4 pmol cm)2 s)1 for 17 preparations, see Fig. 17b (Jensen et al. 1997). Further results
obtained by this method shall be discussed in the
section, ‘functions of the proton pump’, where also the
above-mentioned significant difference in proton secretion fluxes of frog and toad skin shall be commented
upon.
By immunocytochemical labelling and laser scanning
confocal microscopy, it was verified that the apical
proton pump of MR cells of the frog skin is an H+ VATPase (Ehrenfeld & Klein 1997, Klein et al. 1997). By
specific immunostaining and fluorescent microscopy,
this conclusion was generalized to the MR cells of toad
skin (Jensen et al. 2003). Members of this ion motive
ATPase family have a wide distribution among eukaryotes where they together with the Na+/K+ P-ATPase
energize extracellular ionic and osmotic homeostasis
(Harvey & Wieczorek 1997, Nelson & Harvey 1999,
Beyenbach & Wieczorek 2006). The H+ V-ATPase of
anuran skin is rheogenic with the secretion of one
proton being associated with the movement of one
positive charge in the outward direction (Harvey &
Ehrenfeld 1988, Jensen et al. 1997).
Functions of the proton pump
Three different functions of the proton pump have been
proposed:
(1) Energizing proton secretion in animals in a positive
acid balance (Harvey 1992).
(2) Energizing the passive uptake of sodium ions from
pond water (Ehrenfeld et al. 1985).
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Acta Physiol 2011, 202, 435–464
(a)
Figure 17 Resolution of the proton concentration gradient above the isolated
skin epithelium of Bufo bufo exposed to a
Cl)-free external solution. (a) The external pH profile resolved in vertical steps of
50 lm. The zero position is at the outer
border of the cornified cell layer. The
upper recording is the electrode potential
of the reference barrel of the doublebarrelled pH-sensitive microelectrode.
The lower trace is the difference between
the pH-sensitive signal and the signal of
the reference barrel. Arrows indicate
downward and upward movements of
the microelectrode tip respectively. (b)
Example of a [H+]-profile obtained by the
method in (a). The full line is the best fit
of the integrated flux equation for diffusion of protons in the unstirred layer
with a proton buffer. Data from Jensen
et al. (1997).
(b)
(3) Energizing the active uptake of chloride (Larsen
1991).
(1) The first of these has received experimental support
in studies of acid loaded R. esculenta providing the
evidence for an a-type MR cell in anuran skin
epithelium as depicted in Fig. 4c (upper panel).
Investigations by Harvey (1992) indicated that
acute stimulation of cutaneous proton secretion is
because of the insertion of H+ pumps into the
apical membrane by exocytosis from a cytosolic
pool. In the same study, it was found that longterm metabolic acidosis increases the density of
MR cells with an increased apical membrane
surface area of this cell type. This treatment
together with stimulation by aldosterone resulted
in quite impressive proton fluxes in the order of
100 pmol cm)2 s)1. Electroneutral transport at
open circuit was accomplished by an inward active
Na+ flux preferentially through the principal cell
compartment. However, as MR cells also display
the apical amiloride-sensitive Na+-selective channel
and the basolateral ouabain inhibitable Na+/K+ PATPase, they are configured for the active uptake
of Na+ as well (Larsen et al. 1987, Harvey 1992,
Rick 1992). In all three studies, the active Na+
uptake by MR cells was suggested to be of
relatively minor importance. Its physiological significance is unknown.
(2) It has been suggested that the rheogenic H+ pump
by hyperpolarizing the apical membranes of MR
456
and principal cells (at open circuit) allows for
cellular Na+ uptake at an external [Na+] £ 2 mm
(Ehrenfeld et al. 1985, Ehrenfeld & Klein 1997).
This interesting hypothesis has aroused much
attention, but is difficult to verify experimentally.
It is possible, however, to consider some quantitative implications of the hypothesis. It is unlikely
that proton pumping is driving the cell potential to
a value that is numerically larger than the K+equilibrium potential, EK, across the basolateral
membrane. Otherwise, potassium ions flow from
the interstitial fluid into the cell both via basolateral K+ channels and Na+/K+ pumps. It follows that
the above function of the proton pump would be
manifest only for VT > 0 mV, i.e. with the outside
of the skin being relatively positive. At the physiological serosal-[K+] of 2.0 mm, we measured an
intracellular [K+] of 150 8 mm of the principal
cell compartment in toad skin epithelium, corresponding to EK = )106 2 mV (Larsen et al.
1992). This conforms well with an intracellular
[K+] of principal cells of frog skin of 153 mm (Rick
et al. 1978), and a basolateral membrane potential
of )108 2 mV (Nagel 1976) or )101 1 mV
(Larsen et al. 1992) in skins of relatively low or
eliminated apical Na+ permeability respectively.
Thus, with an intracellular [Na+] of, e.g. 3–10 mm,
and VT 0 mV (where the apical membrane
potential is close to the above EK), the passive flux
of Na+ across the apical plasma membrane would
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Acta Physiol 2011, 202, 435–464
E H Larsen
reverse at an external concentration of 35–118 lm
Na Na
(20 C, activity-coefficient ratio, fcell
=fo ¼ 0:76).
This indicates that even at an external [Na+] less
than that of most lakes and creeks, a relatively
large K+ conductance of the basolateral membrane
is sufficient for establishing a driving force for the
net flux of Na+ from the environment into the cell.
This conclusion is in agreement with results
reported by Harvey & Kernan (1984), who measured an inwardly directed electrochemical gradient for Na+ across the apical membrane at an
external Na+ concentration of 100 lm and an
apical membrane potential of )101 mV. The
additional lesson from the above calculation is
that the uptake of Na+ and Cl) from 10 lm of the
salt (Krogh 1937) would not be possible unless
proton pumping reverses the transepithelial potential difference, that is, hyperpolarizes the apical
plasma membrane to a value below )106 mV,
in casu, between )120 and )145 mV. It is within
this very low range of concentrations, the above
hypothesis would be of biological significance and
should be tested.
(3) Finally, I shall discuss the hypothesis that ATP
hydrolysis at a proton-pump ATPase supplies the
metabolic energy for the active chloride uptake
discovered by August Krogh. Much of the experimental evidence for this hypothesis has been
gained in studies on toad skin epithelium. By
considering the reversible elimination of the acidification of the external solution following transient substitution of chloride for a non-permeating
anion (Emilio et al. 1970, Larsen 1991, Larsen
et al. 1992, Jensen et al. 1997), toad skin seems to
exhibit a density of the c-type MR cell (Fig. 4b)
that exceeds significantly the density of other MR
cell types. Because of the depicted coupling of
proton and bicarbonate effluxes via the cellular
carbonic anhydrase, our hypothesis predicts the
active chloride uptake to be rheogenic: one negative charge carried by Cl) is being moved in the
inward direction across both the apical and the
basolateral plasma membrane per cycle of Cl)/
HCO3) exchange driven by the apical H+ VATPase (cf. Fig. 4b). This point has been verified
in studies on active cutaneous Cl) uptake for three
different anuran species (Zadunaisky et al. 1963,
Bruus et al. 1976, Berman et al. 1987). This is
important, because it distinguishes the rheogenic
active uptake of Cl) by the c-type MR cell from the
non-rheogenic active uptake of Cl) by the b-type
MR cell, which displays proton pumps in the
basolateral membrane, cf. Fig. 4c (lower panel). It
is further predicted that in skins exposed to dilute
saline on the outside, the influx of Cl) and efflux of
Æ Functions of mitochondria-rich cells in anuran skin
Figure 18 Proton efflux and Cl) influx in isolated skin of Rana
esculenta. Active uptake of Cl) was stimulated by keeping the
frogs in a 50 lm NaCl solution for 2–3 weeks. The proton flux
was determined by pH-stat titration on skins exposed to a Cl)free external solution; mean SEM for N = 7 preparations.
The chloride influx was measured with 36Cl) and 3.3 mm Cl)
in the external solution, mean SEM for N = 9 preparations.
Data from Jensen et al. (2002).
H+ would be of similar numerical magnitude.
Shown in Fig. 18, this was verified in experiments
with starving frogs (R. esculenta) kept in 50 lm
NaCl for weeks prior to isolation of the skin.
Figure 18 further shows that the proton-pump
inhibitor, concanamycin A, reduces the uptake of
Cl) by about the same amount as the active proton
flux is being reduced. Thus, the fluxes of the two
ions are indirectly coupled in such a way that the
Cl) uptake is depending on the activity of the
apical proton pump in agreement with the scheme
shown in Fig. 4b (Jensen et al. 2002). Our hypothesis also handles the active bromide uptake (Krogh
1937) and the active iodide uptake (Harck &
Larsen 1986) from low external concentrations of
the ions as we could show that both of these ions
have similar capacity as chloride to reduce acidification of the apical unstirred layer of toad skin
epithelium (Jensen et al. 1997). Therefore, all three
halide ions share this mechanism of active transport by their ability to exchange with cellular
bicarbonate at the apical membrane of the MR cell.
The model outlined in Fig. 4b has an interesting
limitation; apparently, it cannot handle the specific
finding of active uptake of Cl) in selectively chloridedepleted frogs. Garcia-Romeu et al. (1969) observed
that Calyptocephalella gayi kept in Cl)-free solutions
that stimulated the epidermal Cl) uptake mechanism,
only, secreted base (bicarbonate) and took up chloride
with a stoichiometry of 1 : 1 with no other transcuta-
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457
Functions of mitochondria-rich cells in anuran skin
Æ E H Larsen
neous ion movements. We have previously suggested
(Jensen et al. 2003) that this non-rheogenic active Cl)
uptake is performed by the b-type MR cell as shown in
Fig. 4c (lower panel). This cell type, displaying the
proton pump in the basolateral membrane, is associated
with renal base secretion in animals submitted to
alkalosis (Stetson et al. 1985). Indirect evidence for a
similar type of MR cell in anuran skin comes from the
study by Vanatta & Frazier (1981) showing cutaneous
base secretion in base-loaded R. pipiens. The plasticity
of anuran MR cell functions indicated in their study and
in the study by Garcia-Romeu et al. (1969) would have
to be studied in detail for verifying the hypothesis of a btype MR cell in anuran epidermis.
The coupling of cellular energy metabolism and NaCl
uptake from diluted solutions
Any model of cutaneous ion transport has to account
for the uptake of NaCl from a concentration just above
10 lm (Krogh 1937). In this limit, the electrochemical
work was about 42 kJ mol)1 of NaCl transported, see
above (the method of calculation is discussed in Jensen
et al. 2003). The useful work of the Na+/K+ P-ATPase
of a stoichiometry of 3 Na+ per ATP split and a free
energy of ATP hydrolysis of )60 kJ mol)1 would be
)20 kJ mol)1 Na+ transported. A similar calculation for
the H+ V-ATPase of a stoichiometry of 2 H+ per ATP
hydrolysed (Kibak et al. 1992) gives )30 kJ per Cl)
transported. Therefore, the model depicted in Fig. 4b
should be able to provide sufficient metabolic energy for
the cutaneous NaCl uptake in Krogh’s study.
The above calculation assumes that the Cl)/HCO3)
exchange is electroneutral. The experimental justification for this was presented above. However, quite
recent interesting discoveries raise the question, whether
the non-rheogenic mode of operation applies to all
possible environmental conditions. Thus, a molecularbiological study of osmoregulation in marine teleosts
identified members of the SLC26 anion exchange gene
family, which operate with a Cl)/nHCO3) stoichiometry (Grosell et al. 2009). The authors pointed out that
anion exchange fluxes (in small intestine) via an apical
rheogenic exchanger (n > 1) become accelerated if the
apical membrane potential is hyperpolarized by the
associated H+ V-ATPase (mucosal fluid relatively
positive). They also suggested a role of rheogenic anion
exchangers for chloride uptake by freshwater animals.
For illustrating the gain in electrochemical work
performance of a rheogenic anion exchanger in parallel
with the H+ V-ATPase, assume n = 2; as a result of the
coupling of proton pumping and Cl) uptake via the
carbonic anhydrase, it follows that 2 H+ are pumped
out of the cell for each Cl) transported into the animal.
Thus, the available useful work would be )60 kJ mol)1
458
Acta Physiol 2011, 202, 435–464
rather than )30 kJ mol)1 of Cl) transported. Provided
the Cl) affinity of the external anion-binding site of the
anion exchanger is sufficiently high, as it is in zebrafish
(Danio rerio) acclimated to 35 lm NaCl (Boisen et al.
2003), this would serve osmoregulation at ion concentrations <10 lm NaCl.
Concluding remarks: the interrelationship of
active and passive chloride uptake
The conclusion from the above experiments in which
the external chloride concentration and transepithelial
potential difference were varied is that the c-type MR
cells is permeable to passive chloride transport only for
an inward flux of the ion. If the driving force is reversed,
as it is when the frog is in the pond water of low NaCl
concentration, the active uptake of Cl) takes over. This
important point is convincingly demonstrated by depictin
ing the ratio of unidirectional steady-state Cl) fluxes, JCl
out
and JCl
, as a function of the driving force on chloride
ions, zCl · (VT ) ECl), according to the following set of
equations (Ussing 1949):
Figure 19 Flux-ratio analysis of Cl) transport across toad
skin. Unidirectional Cl) fluxes were determined by 36Cl) in
paired half skins. The straight line was calculated by Eq. 3 with
the sign of the transepithelial electrical potential difference
being that of the bath on the cornified side of the epithelium.
The graph shows that the experimental flux ratio follows the
theoretical line for electrodiffusion if the driving force,
zCl · (VT ) ECl), is in the inward direction at elevated external
[Cl)], where the apical Cl) channels of the c-MR cells are
activated (right-hand side). If zCl · (VT ) ECl) is in the outward direction (left hand side), the apical Cl) channels are
closed and active Cl) transport fuelled by the apical H+
V-ATPase and Cl) : Cl) exchange diffusion become the
dominating modes of Cl) transport. Data from Willumsen &
Larsen (1986) and Jensen et al. (2002).
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Acta Physiol 2011, 202, 435–464
in
JCl
zCl F ðVT ECl Þ
;
¼
exp
out
RT
JCl
ECl
R T fo ½Cl o
¼
:
ln
zCl F fi ½Cl i
E H Larsen
ð3Þ
VT is measured with serosal bath (subscript, i) grounded
(as above), the activity coefficients are indicated by f,
and F, R and T have their usual meanings. In Fig. 19 the
flux ratio is depicted on a logarithmic scale with the
driving force in mV on the x-axis so that the theoretical
flux ratio depicts a straight line of slope, 10)3 · F/
(R · T) = 0.0396 mV)1 (20 C). For the driving force
acting on the chloride ions being inwardly directed, the
chloride channels are open allowing for a flow of
chloride from the outer bath to the interstitial fluid. This
is illustrated on the right-hand side of the diagram
where the agreement is good between experimental and
theoretical flux ratios. When the driving force is
reversed, either by depolarizing the transepithelial
potential difference (resulting in hyperpolarization of
the apical plasma membrane of the MR cells) or by
reducing the external chloride concentration, the experimental flux ratio deviates significantly from the ratio
predicted for electrodiffusion. Here, on the left-hand
site of the diagram, the small active flux overrules the
passive flux. The regulation by membrane potential and
external chloride of the apical chloride permeability of
the MR cell secures the chloride uptake from the
environment independent of the direction of the external driving force. The synthesis of experimental results
presented in Fig. 19 covers Krogh’s as well as Ussing’s
findings and shows that the two reported transport
modes belong to a common continuum of physiological
states.
Conflict of interest
There is no conflict of interest.
Our laboratory has been supported by grants from the Danish
Natural Science Research Council, The Carlsberg Foundation,
Alfred Benzon Foundation and the Novo Nordisk Foundation.
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